CARACTERIZAÇÃO DA MACIEZ DA CARNE POR ANÁLISES … · Tese apresentada para a obtenção do título de ... quartos de crianças judias do campo de extermínio nazista de Auschwitz)

UNIVERSIDADE FEDERAL DE GOIÁS

ESCOLA DE VETERINÁRIA

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIA ANIMAL

CARACTERIZAÇÃO DA MACIEZ DA CARNE POR ANÁLISES

PROTEÔMICAS E MOLECULARES

Leonardo Guimarães de Oliveira

Orientador: Prof. Dr. João Teodoro Padua

GOIÂNIA

2016

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LEONARDO GUIMARÃES DE OLIVEIRA

CARACTERIZAÇÃO DA MACIEZ DA CARNE POR ANÁLISES

PROTEÔMICAS E MOLECULARES

Tese apresentada para a obtenção do título de

Doutor em Ciência Animal junto à Escola de

Veterinária e Zootecnia da Universidade

Federal de Goiás

Área de Concentração:

Produção Animal

Linha de Pesquisa:

Metabolismo nutricional, alimentação e

forragicultura na produção animal

Orientador:

Prof. Dr. João Teodoro Padua - EVZ/UFG

Comitê de Orientação:

Prof. Dr. Reginaldo Nassar Ferreira - ICB/UFG

Prof. Dr. Cláudio Ulhôa Magnabosco – EMBRAPA

GOIÂNIA

2016

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Dedicatória

Dedico aos meus pais Orci e Ângela, meus irmãos

e à minha esposa Fabiana.

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Epígrafe

“Amanhã eu fico triste...

Amanhã!

Hoje não.

Hoje eu fico alegre!

E todos os dias,

por mais amargos que sejam,

eu digo:

Amanhã eu fico triste,

hoje não!”

(Poema encontrado na parede de um dos

quartos de crianças judias do campo de

extermínio nazista de Auschwitz).

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Agradecimentos

Agradeço a Deus por estar ao meu lado em todos os momentos da minha vida, que juntamente

aos meus, ajudou a superar todos os obstáculos que a vida apresenta.

Aos meus pais Orci e Ângela e aos meus irmãos Wellington, Cristiane e meus familiares pelo

apoio e sábios conselhos, nunca me deixando desistir dos meus sonhos.

À minha esposa Fabiana Barbosa por existir na minha vida e fazer meus dias mais felizes.

Ao professor Dr. João Teodoro Padua, pela orientação, conselho e apoio neste trabalho.

Ao professor Dr. Reginaldo Nassar Ferreira, pela amizade e por todos os passos da minha

caminhada científica, pela oportunidade da primeira pesquisa no meu primeiro ano da graduação, e

desde então sempre auxiliando nesta caminhada, nas horas certas e nas horas mais incertas, sempre

presente.

Aos professores Clayton Luiz e Alexandre Bailão por todos os ensinamentos e ajuda no

decorrer deste trabalho, sempre prontamente dispostos a conversas.

A professora Célia Maria por disponibilizar o laboratório de biologia molecular José Salum

para a condução das análises.

Ao professor Emmanuel Arnhold pelas valiosas conversas e ensinamentos.

Ao professor Aldi França pelas inúmeras oportunidades de aprendizado e ensinamentos

passados.

Ao professor José Henrique Stringhini por todas as oportunidades e amizade.

A todos os professores do Departamento de Zootecnia e da Escola de Veterinária e Zootecnia

da UFG.

Ao professor Dr. Cláudio Ulhoa Magnabosco, pela oportunidade e fundamental ajuda no

desenvolver da pesquisa.

Ao professor Dr. Pedro Veiga pela grande oportunidade, pela motivação e por toda ajuda.

Aos professores Steven Lonergan e Elisabeth Huff-Lonergan pela grande oportunidade e por

me acolher tão bem em seu laboratório.

Aos amigos da Iowa State University pelo companheirismo e acolhimento.

À família Portes pela grande amizade.

A todos da família Barbosa, Sr. Heriberto, Sra. Ivani, José Humberto, Erika Fukushima, Davi,

Lais, Emílio, Cláudia, Eduardo e Henrique.

Aos amigos Reginaldo Jacovetti e Sérgio Ferreira, pela amizade e pela ajuda nas horas boas e

principalmente nas horas de dificuldade.

Aos amigos da pós graduação, Marcondes Dias, Kíria Karolline, Tiago Pereira, Hugo Jayme,

Tayrone Prado, Gustavo Feliciano, Marcela Luzia, Barbara Lemos, Flavia Martins, Juliana Macedo,

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Leticia Castro, Ludmilla Brunes, Ligia Moreira, Silvia, Adesvaldo, Leandro, Jean, Eduardo Rodolfo

e José Tiago por toda ajuda e todos os bons momentos de convivência.

Aos amigos da veterinária pela incrível amizade para todas as horas.

Aos amigos do laboratório de biologia molecular pela ajuda e companherismo, em especial ao

André Luiz pela grande ajuda neste processo.

À EMBRAPA por disponibilizar e viabilizar a participação nesta pesquisa.

À Guaporé Agropecuária e a Fazenda Barreiro pelas instalações e animais.

À Coordenação e todos os professores da Escola de Veterinária e Zootecnia, que contribuíram

para a minha formação.

Aos funcionários da secretaria de Pós Graduação em Ciência Animal, e do Departamento de

Zootecnia, pelo suporte nas nossas necessidades acadêmicas.

A todos aqueles que me apoiaram nesta caminhada.

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Sumário

CAPITULO 1 – CONSIDERAÇÕES INICIAIS ........................................................................ 18

1 INTRODUÇÃO ............................................................................................................................. 18

Objetivos gerais ................................................................................................................................. 19

Objetivos específicos ......................................................................................................................... 19

2 REVISÃO DE LITERATURA ...................................................................................................... 20

2.1 Estrutura muscular ....................................................................................................................... 20

2.2 Maciez da carne ........................................................................................................................... 21

2.2.1 Sistema calpaína ........................................................................................................................ 22

2.3 Técnicas utilizadas para o estudo de proteínas ............................................................................ 24

2.4 Aplicação da proteômica na ciencia animal ................................................................................. 25

2.5 Purificação de proteínas ............................................................................................................... 26

3 Referências ...................................................................................................................................... 26

CAPITULO 2 - CALPASTATIN VARIANTS ACTIVITIES DURING BEEF AGING AND

DIFFERENTIAL POSTMORTEM PROTEOLYSIS IN TWO MUSCLES ..................................... 31

1. Introduction .................................................................................................................................... 32

2. Matherials and methods ................................................................................................................. 33

3. Results and discussion ................................................................................................................... 39

4. Conclusions .................................................................................................................................... 47

5. References ...................................................................................................................................... 48

FIGURES ........................................................................................................................................... 52

TABLES ............................................................................................................................................. 60

CAPITULO 3 – PURIFICATION AND CHARACTERIZATION OF ACTIVE PEAKS OF

CALPASTATIN FROM SWINE LONGÍSSIMUS DORSI MUSCLE. .............................................. 63

Abstract: ............................................................................................................................................. 63

INTRODUCTION ............................................................................................................................. 64

MATHERIALS AND METHODS .................................................................................................... 65

Calpastatin extraction ......................................................................................................................... 65

Calpastatin Activity ........................................................................................................................... 66

Calpastatin peaks purification ............................................................................................................ 67

SDS-PAGE and immunoblotting ....................................................................................................... 68

Two-Dimensional Difference in Gel Electrophoresis. ....................................................................... 69

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RESULTS AND DISCUSSION ........................................................................................................ 71

CONCLUSIONS ................................................................................................................................ 74

LITERATURE CITED ...................................................................................................................... 74

Tables and Figures ............................................................................................................................. 76

CAPITULO 4 – ANÁLISE PROTEÔMICA DO MÚSCULO “LONGÍSSIMUS DORSI” DE

BOVINOS DA RAÇA NELORE MOCHO DE DIFERENTES GRUPOS DE MACIEZ. ............... 83

INTRODUÇÃO ................................................................................................................................. 83

MATERIAL E MÉTODOS ............................................................................................................... 84

Coleta e preparo das amostras ............................................................................................................ 84

Extração proteica ............................................................................................................................... 85

Análise proteômica ............................................................................................................................ 85

Determinação da atividade da enzima superóxido dismutase ............................................................ 87

RESULTADOS E DISCUSSÃO ....................................................................................................... 87

CONCLUSÕES ............................................................................................................................... 103

AGRADECIMENTOS .................................................................................................................... 103

REFERÊNCIAS ............................................................................................................................... 104

Anexos ............................................................................................................................................. 110

CAPITULO 5 – CONSIDERAÇÕES FINAIS ................................................................................ 115

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LLISTA DE TABELAS

Figure 1– Estrutura do músculo estriado esquelético. ...................................................................... 20

Figure 2 Representative blot and immunodetection of calpastatin in sarcoplasmic fraction using

antibody anti Calpastatin (MA3-944). ............................................................................................... 53

Figure 3 Representative blot and immunodetection of calpain-1 in sarcoplasmic fraction using

antibody anti Calpain-1. ..................................................................................................................... 53

Figure 4 Representative blot and immunodetection of troponin-T in myofibrillar fraction using

antibody anti troponin. ....................................................................................................................... 54

Figure 5 Representative blot and immunodetection of desmin in myofibrillar fraction using antibody

anti desmin. ........................................................................................................................................ 55

Figure 6 A representative two-dimensional difference in gel electrophoresis showing different

expressed spots in Longissimus lumborum (LL) muscle.Circles represent total area of detected spot

and the different expressed spots are identified by numbers. ............................................................ 56

Figure 7 A) SyPro Ruby total protein stain and B) ProQ Diamond phosphoprotein stain to identify

phosphorylated proteins from: TB0- 40 µg of protein from sarcoplasmic fraction of Triceps brachii

day 0; TB1- 40 µg of protein from sarcoplasmic fraction of Triceps brachii day 1; ST0- 40 µg of

protein from sarcoplasmic fraction of Semitendinosus day 0; Pk1 - 30µL of calpastatin peak 1

extracted from Semitendinosus day 0; Pk2 - 30µL of calpastatin peak 2 extracted from

Semitendinosus day 0......................................................................................................................... 57

Figure 8 Western blot and immunodetection using antibody anti Calpastatin (MA3-944). TB0- 40 µg

of protein from sarcoplasmic fraction of Triceps brachii day 0; TB1- 40 µg of protein from

sarcoplasmic fraction of Triceps brachii day 1; ST0- 40 µg of protein from sarcoplasmic fraction of

Semitendinosus day 0; Pk1 - 30µL of calpastatin peak 1 extracted from Semitendinosus day 0; Pk2 -

30µL of calpastatin peak 2 extracted from Semitendinosus day 0. ................................................... 58

Figure 9 Two dimensional western blot and immunodetection using antibody anti Calpastatin (MA3-

944).of pooled fractions from the two peaks with calpastatin activity from Semitendinosus day 0. A)

Calpastatin peak 1. B) Calpastatin peak 2. ......................................................................................... 59

10 Figure 1 Calpastatin activity of eluted fractions (Arbitrary units) ............................................... 76

11 Figure 2 – Load check silver stained of initial and final step of calpastatin purification. Lane M)

Broad range molecular marker; 1) Pooled fractions of peak 1 activity of calpastatin from Q sepharose

column; 2) Pooled fractions of peak 2 activity of calpastatin from Q sepharose column; 3) Pooled

fractions of peak 1 activity of calpastatin from Phenil sepharose column; 4) Pooled fractions of peak

2 activity of calpastatin from DEAE CAPTO column. ...................................................................... 78

12 Figure 3 – Western blott stained by calpastatin antibody of purified calpastatin peak 1 (PK1) and

calpastatin peak 2 (PK2). ................................................................................................................... 79

13 Figure 4 – 2D DIGE of calpastatin peaks coomassie stained. A) Calpastatin peak 1; B) calpastatin

peak 2. Numbers of collected spots and sent to identification are presented in boxes. ..................... 80

14 Figure 5 – Representative calpastatin molecule aminoacid sequence from Sus scrofa gene: CAST;

713 Aminoacids; Mass (Da):77,124. Inhibitory domains are presented in closed boxes and peptides

identified are in uppercase and in yellow are from peak 1 and from peak 2 as identified by grey. .. 82

15 Figura 1 – Atividade da enzima SOD (% inibição da formação do íon super óxido) entre os grupos

baixo WBSF (Macio) e alto WBSF (Duro) ....................................................................................... 92

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LISTA DE FIGURAS

Table 1 Activityᶲ of calpastatin peak 1 (CAST 1), calpastatin peak 2 (CAST 2), Total calpastatin

(CAST total), calpain-1 and calpain-2 extracted from muscles Longissimus lumborum (LL) and

Triceps brachii (TB) during aging days of beef meat. ....................................................................... 60

Table 2 Calpastatin peak 1 (CAST 1), calpastatin peak 2 (CAST 2), total calpastatin (CAST total)

to calpain-1 activity ratios from muscles Longissimus lumborum (LL) and Triceps brachii (TB) at

day 0 postmortem. .............................................................................................................................. 60

Table 3 Abundance of immunoreactive bands from western blot of calpastatin (CAST), myofibrilar

and sarcoplasmic calpain-1, desmin and troponin-T during aging days of muscles Longissimus

lumborum (LL) and Triceps brachii (TB). ......................................................................................... 61

Table 4 Proteins identified in the spots of 2D-DIGE analysis of Longissimus lumborum (LL) and

Triceps brachii (TB). .......................................................................................................................... 62

5 Table 1 – Steps of purification of calpastatin peaks. ..................................................................... 77

6 Table 2 - Identified spots from calpastatin peaks gels. ................................................................. 81

7 Tabela 1 – Médias de força de cisalhamento transversal (WBSF) do músculo Longíssimos dorsi

dos animais selecionados para compor o grupo macio (M) e grupo duro (D). .................................. 87

8 Tabela 2 – Proteínas relacionadas à defesa celular, diferentemente expressas entre os grupos macio

(M) e duro (D). ................................................................................................................................... 89

9 Tabela 3 - Proteínas relacionadas a estrutura celular diferentemente expressas entre os grupos

macio (M) e duro (D). ........................................................................................................................ 95

10 Tabela 4 - Proteínas relacionadas ao metabolismo de carboidratos, diferentemente expressas entre

os grupos macio (M) e duro (D). ........................................................................................................ 97

11 Tabela 5 - Proteínas relacionadas ao metabolismo de proteínas, diferentemente expressas entre os

grupos macio (M) e duro (D). .......................................................................................................... 100

12 Tabela 6 - Proteínas relacionadas ao metabolismo de lipídeos, ligadas ao transporte de íons,

diferentemente expressas entre os grupos macio (M) e duro (D). ................................................... 102

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LISTA DE ABREVIATURAS

µM Mili mol

2D Segunda dimensão

2D-DIGE Diferença em gel de segunda dimensão

ADH Aldeido desidrogenase

ATBSα Sintetase de ATP alfa

ATP Trifosfato de adenosina

cAMP Monofosfato de adenosina cíclico

CAST Calpastatina

CAST1 Calpastatina eluída no primeiro pico da cromatografia

CAST2 Calpastatina eluída no segundo pico da cromatografia

Da Dalton

DNA Ácido desoxirribonucleico

EMBRAPA Empresa brasileira de pesquisa agropecuária

ERO Espécies reativas de oxigênio

HSP Proteína de choque térmico

ICB Instituto de Ciências Biológicas

IP Ponto isoelétrico

kDa Kilodaldon

LC-MS/MS Cromatografo líquido acoplado a dois espectrômetros

LL Longíssimus lumborum

M Mol

MALDI Disorção/ionização assistida por laser

MHC Miosina de cadeia pesada

MLC Miosina de cadeia leve

MW Peso molecular

MYOM Miomensina

NADPH Dinucleotídeo adenina nicotinamida fosfato

ONS Oxido nitroso sintase

PRX Peroxiredoxina

RNA Ácido Ribonucleico

RPM Rotações por minuto

SDS-PAGE Gel de poliacrilamida

SFGH Formilglutationa hidrolase

SIF Sistema de inspeção federal

SOD Enzima super óxido dismutase

ST Músculo semitendinoso

TB Triceps brachii

WBSF Força de cisalhamento Warner-Bratzler

αβCST Cristalinina alfa beta

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RESUMO

O perfil protéico de animais da raça nelore mocho de uma população segregante para a maciez da

carne de extremos valores de força de cisalhamento, grupo extremo baixo (M) e grupo extremo alto

(D), apresentou proteínas diferentemente expressas as quais houve maior abundância relativa de

proteínas do processo glicolítico no grupo M e proteínas do metabolismo oxidativo evidenciadas mais

expressas no grupo D, fato que provavelmente está correlacionado à maciez final da carne. Apenas

identificada no grupo D, a proteína citocromo c indica indução do processo apoptótico neste grupo

de animais. Proteínas estruturais foram identificadas no grupo M, indicando uma possível maior

proteólise. A calpastatina foi somente identificada no grupo D, esta proteína está altamente

relacionada com a maciez final da carne por ser inibidora natural das calpaínas. A separação de

calpastatina por cromatografia em coluna de troca iônica de dois músculos diferentes descreveu dois

picos de actividade inibitória de calpaínas: pico 1 (CAST1) e pico 2 (CAST2). Atividade de CAST 1

aumentada durante o período post mortem no Triceps brachii e não apresentou diferença entre os dias

em Longissimus dorsi e por outro lado, a atividade total de calpastatina e CAST 2 diminuiu durante

o envelhecimento post mortem. A banda de 115 kDa da calpastatina diminuiu sua intensidade durante

o envelhecimento post mortem em ambos os músculos com mais de 70% da alteração ocorrendo no

primeiro dia. As proteínas mitocondriais da subunidade ATP sintase beta aumentaram e a Succinil-

CoA ligase diminuiu após o envelhecimento e a Adenilato quinase isoenzima diminuiu no dia 7. Os

picos de calpastatina apresentavam faixas fosforiladas fracas e apresentavam manchas em IP e MW

diferentes do que 2D-SDS-PAGE. Durante o processo de purificação dos picos de calpastatina a

actividade por mg de proteína aumentou mas perdeu metade da atividade total apresentada durante a

primeira etapa de purificação. Para o pico 2 de calpastatina a actividade específica aumentou 139,8

vezes e no final deste processo permaneceu 36% da atividade total. A calpastatina purificada foi

identificada no gel de segunda dimensão com um peso molecular semelhante ao western blot. Spots

do pico 1 da calpastatina e dois do pico 2 da calpastatina foram identificados como peptídeos

pertencentes à molécula da calpastatina. Sequêcia de peptídeos identificados em Spot a partir do pico

1 purificado como parte do domínio inibidor III e IV e do terminal C e do pico 2 purificado uma

sequêcia de peptideos identificados como parte do domínio inibidor I, II e III. Estes resultados levam-

nos a crer que ambos os picos, neste caso, são produtos de degradação da molécula intacta e,

provavelmente, os pequenos peptideos são quebrados durante o processo. Os resultados do presente

estudo mostram que é possível a purificação de formas distintas de calpastatina activa, contudo a

forma intacta de calpastatina não estava presente nesta purificação. A presença de peptídeos não foi

conclusiva para determinar a origem ea composição de cada pico ativo.

Palavras chave: Calpastatina; coluna de troca iônica; immunobloting; purificação; SDS-PAGE.

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ABSTRACT

The protein profile of hornless Nellore cattle from a segregating population for meat tenderness of

extremes shear force, low extreme group (M) and extreme high (D) group, showed differentially

expressed proteins. They had greater relative abundance of proteins of the glycolytic process in group

M and proteins of the oxidative metabolism evidenced more expressed in group D, fact that probably

is correlated to the final tenderness of the meat. Only identified in group D, the cytochrome c protein

indicates induction of the apoptotic process in this group of animals. Structural proteins were

identified in group M, indicating a possible greater proteolysis. Calpastatin was only identified in

group D, this protein is highly related to the final meat tenderness because it is a natural inhibitor of

the calpain. Separation of calpastatin by ion exchange column chromatography of two different

muscles described two peaks of calpain inhibitory activity: peak 1 (CAST1) and peak 2 (CAST2).

CAST 1 activity increased during the post-mortem period in Triceps brachii and showed no

difference between days in Longissimus dorsi and on the other hand, total activity of calpastatin and

CAST 2 decreased during post-mortem aging. The 115 kDa band of calpastatin decreased its intensity

during post-mortem aging in both muscles with more than 70% of the change occurring on the first

day. The mitochondrial ATP synthase beta subunit proteins increased and Succinyl CoA ligase

decreased after aging and Adenylate kinase isoenzyme decreased on day 7. Calpastatin peaks had

weak phosphorylated bands and had different IP and MW patches than 2D-SDS -PAGE. During the

purification process of the calpastatin peaks the activity per mg of protein increased but lost half of

the total activity presented during the first purification step. For peak 2 of calpastatin the specific

activity increased 139.8 times and at the end of this process 36% of the total activity remained.

Purified calpastatin was identified on the second-size gel having a molecular weight similar to the

western blot. Spots from calpastatin peak 1 and two from calpastatin peak 2 were identified as

peptides belonging to the calpastatin molecule. Sequence of peptides identified in Spot from purified

peak 1 as part of the inhibitory domain III and IV and of the C-terminus and from the purified peak

2 a sequence of peptides identified as part of the inhibitory domain I, II and III. These results lead us

to believe that both peaks, in this case, are degradation products of the intact molecule, and probably

the small peptides are broken down during the process. The results of the present study show that

purification of distinct forms of active calpastatin is possible, however, the intact form of calpastatin

was not present in this purification. The presence of peptides was not conclusive to determine the

origin and composition of each active peak.

Key words: Calpastatin; immunoblotting; ionic change column; purification; SDS-PAGE.

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CAPITULO 1 – CONSIDERAÇÕES INICIAIS

1 INTRODUÇÃO

A bovinocultura de corte apresenta uma participação muito significativa no PIB Brasileiro com

mais de R$ 185,40 bilhões no ano de 2015 e um rebanho bovino de mais de 212,3 milhões de

animais(1).

Dentre os bovinos destinados à produção de carne no Brasil, a grande maioria é composta por

animais da sub espécie Bos indicus, principalmente da raça Nelore, tendo como característica animais

mais rústicos, em relação a animais Bos taurus mas com características de carne com menor maciez

(2,3).

A maciez da carne é uma das características organolépticas de grande importância e está

relacionada diretamente com a satisfação do consumidor (4). Uma das formas de sua medida é a

estimativa mecânica da força necessária para o cisalhamento de uma seção transversal de carne (5) e

está estimativa está altamente correlacionada com testes diretos de percepção de maciez como a

avaliação por painel sensorial com equipe treinada. Consumidores estão dispostos a pagar um maior

preço por uma carne mais macia (6).

A maciez da carne é influenciada por vários fatores ante mortem como a raça, a idade ao abate,

tipo de alimentação, manejo pré abate e fatores post mortem como a atividade de enzimas proteolíticas

presentes no músculo, disponibilidade de energia pós mortem e velocidade de resfriamento da carcaça

(7,8).

Diferenças consideráveis na maciez da carne podem ser explicadas pela herança genética e

segundo Alves et al.(2) um programa de melhoramento genético com seleção para maciez é uma

alternativa promissora para a produção de carne zebuína naturalmente macia.

Trabalhos envolvendo o melhoramento genético de bovinos têm sido desenvolvidos visando

selecionar indivíduos por suas características de interesse avaliadas no próprio indivíduo ou com base

na herdabilidade destas características. Alta herdabilidade e alta correlação negativa entre maciez da

carne e atividade da enzima Calpastatina em bovinos são relatadas na literatura (9).

A calpastatina é o inibidor natural das proteinases Ca+2 dependentes denominadas Calpainas.

As calpaínas são proteases presentes em todos vertebrados e atuam em várias funções no organismo

como no remodelamento do citoesqueleto e “turn over” protéico e são fundamentais para o

amaciamento da carne no período post mortem (10–12).

Vários são os sistemas que atuam na regulação da atividade das enzimas do sistema calpaína,

entre eles a fosforilação, proteínas de choque térmico, sistema do monofosfato de adenosina cíclico

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(cAMP), entre outros (13–16) e a regulação e interferência de cada sistema no processo metabólico

de amaciamento da carne necessita investigações científicas para ser elucidada.

A análise proteômica se apresenta como uma ferramenta para auxiliar na investigação dos

mecanismos metabólicos como os envolvidos na diferença fenotípica entre indivíduos. Ela se baseia

na identificação do perfil protéico da amostra analisada, permitindo analisar amostras de organismos

complexos quantificando a abundância de cada proteína identificada e assim permitindo a

comparação entre amostras (17,18).

Este trabalho traz informações sobre o perfil protéico do músculo Longíssimus dorsi de animais

da raça nelore mocho de uma população segregante para maciez da carne, particularidades da

atividade das enzimas do sistema calpaína e informações sobre o processo de purificação da

calpastatina.

Objetivos gerais

- Avaliar o perfil proteômico da carne e o sistema calpaína no período post mortem.

- Purificar e caracterizar a calpastatina.

- Avaliar o perfil proteômico dos extremos de força de cisalhamento do músculo

Longíssimus dorsi.

Objetivos específicos

- Determinar a atividade das enzimas m e µ-calpaína e de seu inibidor nos músculos

Longíssimus dorsi e Triceps brachii.

- Avaliar a diferença proteômica dos músculos Longíssimus dorsi e Triceps brachii ocorrida

entre o dia do abate e o sétimo dia após o abate.

- Purificar os picos do inibidor enzimático calpastatina por cromatografia de troca iônica

- Caracterizar os dois picos do inibidor enzimático calpastatina

- Avaliar a diferença proteômica do músculo Longíssimus dorsi de bovinos da raça Nelore

Mocho de extremos de força de cisalhamento.

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2 Revisão de literatura

2.1 Estrutura muscular

A carne é formada apartir da conversão do músculo estriado esquelético e a estrutura do está

representada na Figura 1.

Figure 1– Estrutura do músculo estriado esquelético.

Fonte: Adaptado de CHOI et al. (19)

O músculo é composto de 16 a 22% de proteínas e a estrutura mucular é composta pelas fibras

musculares (19). As fibras musculares são agrupadas em feixes por uma membrana (epimísio) e cada

fibra é envolvida também por outra membrana (endomísio). A fibra muscular é composta por

citoplasma diferenciado (sarcoplasma), e pelas miofibrilas.

As proteínas presentes no sarcoplsmaas (sarcoplasmáticas) constituem aproximadamente de

30 a 35 % da proteína total do musculo esquelético. São proteínas solúveis em água na sua maioria,

sendo facilmente separadas das proteínas miofibrilares por centrifugação. Entre elas estão todas as

enzimas da glicólise e a maior parte das enzimas da síntese de carboidratos e de proteínas (20).

As proteínas miofibrilares em sua maioria são insolúveis e são compostas principalmente pela

miosina, actina, proteína C, proteína M, tropomiosina, α-actina e β-actina. São proteínas que formam

os miofilamentos grossos e finos que constituem a miofibrila, e representam 52% a 56% das proteínas

musculares (20).

21

A Tropomiosina, troponina T, M-proteínas, α-actina, β-actina e C-proteínas compõe as

proteínas miofibrilares e são também proteínas que atuam na regulação do processo de contração

muscular, apresentando função de controle direto e indireto no complexo adenosina-trifosfato-actina-

miosina (20).

De acordo com o tipo de metabolismo, as fibras musculares podem ser classificadas em fibras

com metabolismo oxidativo e fibras com metabolismo oxidativo e este tipo de metabolismo foi

relacionado com a maciez da carne. Ouali & Talmant (21) demonstraram que fibras musculares com

o metabolismo predominante glicolítico apresentam maior atividade post mortem da calpaína

favorecendo a proteólise e a maciez. Este tipo de fibra possui em média maior conteúdo de glicogênio,

o que favorece o declínio do pH no período post mortem e está relacionada com uma maior retenção

de água o que resulta em maior suculência a carne (22).

2.2 Maciez da carne

Altamente influenciável por fatores ante mortem e post mortem, tem grande variação entre

animais, raças, músculos e cortes (23,24).

Fatores como idade ao abate, gênero, temperamento, cobertura de gordura subcutânea na

carcaça, quantidade de tecido conectivo, manejo pré abate, velocidade de resfriamento da carcaça e

tempo de maturação influenciam na maciez final da carne (10,25).

Raças zebuínas produzem em média carne menos macia quando comparadas com animais de

raças taurinas (26,27). A variação também é grande entre animais zebuínos (28).

A herdabilidade da caracteristica maciez da carne, tem sido relatadas na literatura como

moderada a alta. Avaliando a maciez por medidas de compressão pelo método de Warner-Bratzler

shear force (WBSF), Gregory et al. (29) reportaram valores de 0,29 de herdabilidade para esta medida

sendo próximo do valor de 0,34 encontrado por Splan et al. (30) e inferiores ao valor de 0,53

encontrados por Shackelford et al.(31). Utilizando painel sensorial para determinar a maciez Gregory

et al. (29) encontraram valor de 0,12 para herdabilidade da maciez da carne sendo inferior aos valores

encontrados por medidas mecânicas. Devido ao fato da herdabilidade da característica em questão ser

moderada a alta, torna-se possível e viável a seleção de animais com característica de carne mais

macia.

A carne de bovinos de origem européia é mais macia comparada à carne de animais de origem

zebuína, o que em parte é explicado pela maior concentração do inibidor enzimático calpastatina

presente no músculo dos animais zebuínos (32) cuja herdabilidade em bovinos foi estimada em 0,65

(31).

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2.2.1 Sistema calpaína

A maciez da carne está ligada principalmente à taxa e a extensão da proteólise miofibrilar de

proteínas chave na estrutura muscular, provocando o desarranjo e enfraquecimento desta estrutura. O

sistema enzimático das calpaínas é considerado o principal sistema envolvido no amaciamento da

carne no período post mortem e é composto por proteases de cisteína Ca2+ dependente (10,12,33–37).

Os principais membros deste sistema são a micro-calpaína (µ-calpaina) e a mili-calpaína (m-

calpaína), e seu inibidor calpastatina, inibidor natural e específico das referidas calpaínas (35).

Ocorrem em todas as células de vertebrados conservando 90% de homologia na sequência de

aminoácidos entre as espécies, localizadas exclusivamente no citoplasma celular associadas

primariamente, mas não sempre, às proteínas miofibrilares, membrana celular e outras proteínas

solúveis no citoplasma. Possuem o pH ótimo de 7,2 a 8,2 e receberam o nome de acordo com a

concentração molar de Ca2+ requerida para a sua atividade, sendo de 0,3 a 50,0 µM de cálcio para a

µ-calpaína e 0,4 a 0,8 M de cálcio para a m-calpaína (38).

A µ e m-calpaína são heterodímeros compostos por duas sub-unidades uma unidade maior com

peso molecular de aproximadamente 80 kDa e 28 kDa cada. A subunidade maior (sub-unidade de

80kDa), apresenta uma ligeira diferença entre a µ e a m- calpaína, sendo a sub-unidade grande da µ-

calpaína com aproximadamente 81.889 Da e da m-calpaína 79.900 Da (39,40). A sub-unidade menor

(sub-unidade de 28 kDa) é idêntica entre as duas calpaínas e codificada por um gene único,

desempenha um papel importante na regulação da atividade da molécula (41).

A sub-unidade de 28 kDa das calpaínas possui característica hidrofóbica por conter grande

quantidade de resíduos de glicina no domínio II. Dos 64 resíduos de aminoácidos, 40 são de glicina

e em uma sequência de 20 resíduos de glicina, sendo sugerido como provável local de ligação aos

fosfolipídeos juntamente ao domínio III da sub-unidade de 80 kDa, compõe a provável estrutura de

ligação às membranas (42).

Possuindo 4 domínios, a sub-unidade de 80 kDa é a unidade que apresenta a parte proteolítica

da molécula. No domínio II os resíduos de cisteína na posição 115 (µ-calpaína) ou 105 (m-calpaína),

histidina na posição 272 (µ-calpaína) ou 262 (m-calpaína) e asparagina na posição 296 (µ-calpaína)

ou 286 (m-calpaína), formam a tríade catalítica característica das proteases de cisteína (35). O

domínio IV está relacionado à dimerização da molécula, sendo o local da ligação com a sub-unidade

de 28kDa.

A ativação de ambas as enzimas pela ligação com o Ca2+ é predita pela cristalografia da m-

calpaína (até o presente momento, não se tem evidenciada a estrutura cristalográfica da µ-calpaína,

23

portanto a ativação é predita pela estrutura cristalográfica da m-calpaína), onde o Ca2+ se liga no

domínio IV da sub-unidade de 80kDa e no domínio II da sub-unidade de 28 kDa, provocando uma

pequena mudança conformacional aproximando os resíduos que compõe a tríade proteolítica de

cisteína tornando ativo o sítio catalítico da molécula (43).

A ativação da µ e m-calpaína também leva a uma autoproteólise e este evento diminui a

necessidade da quantidade de Ca2+ requerida para atingir a metade da atividade máxima da µ-calpaína

de 3 a 50 µM para 0,5 a 2,0 µM de Ca2+ e de 400 a 800 µM para 50 a 150 µM de Ca2+ para a m-

calpaína. Também reduz o requerimento de Ca2+ para ligar a calpastatina de 40 µM para 0.042 µM

de Ca2+ da µ-calpaína e de 250 a 500 µM para 25µM de Ca2+ da m-calpaína (35).

A autólise da sub-unidade de 80 kDa da µ-calpaína ocorre pela remoção de 14 resíduos de

aminoácidos da parte NH2-terminal, produzindo um produto intermediário de 78 kDa seguido pela

remoção de mais 12 resíduos de aminoácidos produzindo um fragmento de 76 Kda. A autólise da

sub-unidade de 80 kDa da m-calpaína se inicia com a retirada de 9 resíduos de aminoácidos seguidos

da remoção de 10 resíduos para a produção do fragmento de 78 kDa (44,45).

A taxa e a extensão da autólise da µ-calpaína está diretamente relacionada ao enfraquecimento

da estrutura miofibrilar, parte chave no processo de amaciamento da carne no período post mortem

(46). Algumas proteínas específicas são substratos para as calpaínas e entre elas estão a desmina,

distrofina, filanina, miosina, nebulina, talina, titina, tropomiosina, troponina-T, troponina-I, tubulina,

proteína C, vimetina e vinculina, sendo todas proteínas que compõe o citoesqueleto (35,47). A

produção de fragmentos das proteínas estruturais pela proteólise post mortem está positivamente

relacionada com a extensão da proteólise (10).

Inibidor exclusivo das calpaínas, a calpastatina foi descoberta nos anos 70 durante o processo

de purificação das calpaínas (48). Um único gene codifica a calpastatina em bovinos (gene CAST

presente no cromossomo 7) produzindo várias isoformas variando de 17,5 a 84 kDa (49) por

transcrições alternativas e diversos promotores presentes em vários tecidos.

A isoforma de calpastatina predominante identificada no músculo esquelético de bovinos

possui por volta de 74 kDa e migra para uma posição entre 115 a 125 kDa em gel de poliacrilamida

(50,51). Possui o primeiro domínio L mais quatro domínios com capacidade inibitória. Por esta

característica uma molécula de calpastatina pode inibir até quatro moléculas de calpaína (35).

Além de inibidora, a calpastatina se constitui substrato e é quebrada pela ação das calpaínas,

mas mesmo fragmentada possui atividade inibitória. A fragmentação da calpastatina presente no

músculo esquelético é relacionada à quebra pela ação das calpaínas e os fragmentos gerados são

semelhantes aos fragmentos gerados pela degradação in vitro da calpastatina purificada do músculo

Longíssimus dorsi (50).

24

A regulação desse sistema acontece por várias vias como através a via do AMPc, fosforilação,

acetilação e proteínas de shock térmico (13,33,52–54). Todos estes mecanismos e a possível interação

entre eles nos indicam que vários são os caminhos a serem pesquisados para melhor entender o

processo de proteólise post mortem. Uma das ferramentas que vem sendo utilizada para estudar estes

mecanismos é proteômica, utilizando tecnologia como a eletroforese em gel de poliacrilamida, a

cromatografia líquida e equipamentos de espectometria de massas de alta tecnologia e sensibilidade

(55).

2.3 Técnicas utilizadas para o estudo de proteínas

A proteômica é o estudo feito para avaliar e caracterizar o perfil protéico de uma dada amostra.

Algumas técnicas adotadas no estudo da proteômica utilizam princípios como a eletroforese em gel

unidimensional e bidimensional, baseada na separação das proteínas pelo seu ponto isoelétrico e peso

molecular ou utilizando técnicas de cromatografia. Ambos os métodos necessitam da espectometria

de massas para identificar o perfil protéico (56,57).

O termo “proteomica” surgiu para descrever o conjunto de proteínas expressas pelo genoma.

A aplicação da técnica de eletroforese bidimensional em gel de poliacrilamida aliado a espectrometria

de massas em conjunto a bioinformática, foi primeiramente utilizada por professores da Universidade

de New South Wales – Sidney, Austrália, e professores da Universidade de Genebra, Suíça em uma

conferência em Siena – Itália, pelos professores PhD e pesquisadores Marc Wilkins e Keith Williams

em 1994 (17).

O genoma é o conjunto de genes de uma espécie, ou seja, todo o conjunto do DNA que ele

carrega em suas células (58). A genômica é o estudo do tamanho, da estrutura física e da seqüência

de informações contidas no DNA de um organismo. A proteômica dedica-se a estudar a soma total

de proteínas de uma célula do ponto de vista de suas funções individuais e como a interação de

proteínas específicas com outros componentes celulares afeta o funcionamento destas proteínas (59).

Devido à natureza dinâmica da produção protéica celular, fez-se muito importante a análise das

sequências de nucleotídeos, mas nem sempre há uma relação direta com níveis de proteínas expressas

e de sua atividade biológica. Por exemplo, as mudanças pós transducionais do RNA como a

glicosilação, fosforilação, acilação, ubiquitilação, hidroxilação, carboxilação entre outras

consequentes de diversos estímulos e condições, alteram a atividade. A proteômica representa, pelos

motivos expostos, uma das formas mais eficientes de investigar as funções e os processos metabólicos

das proteínas produzidas pelas células e para o estudo funcional dos genes de organismos complexos

(60).

25

2.4 Aplicação da proteômica na ciencia animal

Alterações bioquímicas durante todo o processo de transformação do músculo em carne e

durante o período de maturação da carne vêm sido elucidadas com a aplicação das técnicas

proteômicas. Todo o período pré o pós abate com as diversas condições de armazenagem

influenciarão no processo de maturação da carne ou amaciamento, processo este importante para a

incrementação deste atributo organoléptico (61).

A comparação entre animais com genes homozigotos e heterozigotos para genes com deleção

da miostatina, proteína responsável pela limitação do crescimento muscular, e um grupo controle com

a presença do gene da miostatina demonstrou alta expressão de miostatina quando comparado o perfil

proteômico dos animais controle com os animais homozigotos e heterozigotos para a deleção da

miostatina (62).

Mecanismos de controle do metabolismo glicolítico e o metabolismo oxidativo foram

encontrados em ovinos, juntamente com proteínas responsáveis pelo gasto de energia como a

glutationa-S-transferase-Pi. Proteínas responsáveis pelo transporte do ferro como a transferina

também foram evidenciadas induzidas com esta condição (63).

Na comparação do perfil proteômico entre animais no início da fase de engorda foram

encontradas diferenças como a expressividade da miosina de cadeia leve e a zinc finger 323, no

estágio final de acabamento e a triosefosfato isomerase e a succinato desidrogenase envolvidas no

metabolismo energético (19).

Durante o processo de tranformação do músculo em carne, no período entre 0 a 24 horas post-

mortem a ação das proteínas cofilina, lactoilglutationa liase, e mais 15 proteínas foram evidenciadas

por Jia et al. (64) e estes autores atriburam proteínas ligadas a proteção celular contra a morte celular

como as principais mudanças neste período post mortem.

Diferença proteômica entre bovinos sadios de raças diferentes foram encontradas comparando

animais da raça Chianina com animais da raça Holandesa (65). São raças selecionadas para produção

de leite (Holandesa) e produção de carne (Chianina). Os animais da raça holandesa tiveram uma maior

expressividade da argininosuccinato-liase, envolvida no ciclo da uréia, a acetil-CoA-acil-transferase-

1, envolvida na cadeia da β-oxidação e degradação do dos ácidos graxos, que também foi encontrada

a expressividade desta enzima em vacas holandesas em lactação por Xu & Wang (66).

Também foi observada maior expressividade da anexina-IV, sendo esta uma proteína de

membrana Ca+ dependente com a sua função fisiológica não totalmente elucidada, mas com um papel

na ligação entre as membranas celular e das vesículas de exocitose, diminuição da permeabilidade a

íons H+ e regulação da condutância de íons. Nos animais da raça Chianina foram mais expressas as

26

proteínas de cadeia-C, uma forma do fibrinogênio bovino, a galactose mutarotase, uma enzima

envolvida no metabolismo dos carboidratos, convertendo açúcares em galactose. A

fumarilacetoacetato hidrolase também foi mais expressa, esta proteína envolvida na síntese de

aminoácidos. Na via da gliconeogênese a frutose-1,6-bifosfatase é fundamental e foi mais expressa

em animais da raça Chianina juntamente com as enzimas da família das sulfotransferase, que são

envolvidas na atividade hepática de detoxificação em várias situações (65).

2.5 Purificação de proteínas

Um dos passos para o estudo mais detalhado de uma determinada proteína é a sua purificação

ou semi-purificação. A partir daí podemos caracterizar a estrutura e obter mais informações químicas,

bioquímicas e funcionais (67). Uma das técnicas utilizadas para a purificação protéica é a

cromatografia utilizando vários princípios como a cromatografia imunoafinidade. Esta técnica é

empregada para purificação das enzimas do sistema calpaína com sucesso (68), possibilitando o

estudo mais minucioso e detalhado das enzimas e sua atividade em várias situações in vivo e in vitro.

A técnica de eletroforese pode nos fornecer informações detalhadas sobre a estrutura e

modificações post mortem ocorridas (47,69). Informações interessantes podem ser retiradas

utilizando a técnica de western blotting e o uso de anti-corpos específicos para cada proteína desejada.

O uso destas técnicas conjugadas traz informações importantes, auxiliando na pesquisa com

proteínas.

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1. (Trabalho submetido à revista Meat Science)

CAPITULO 2 - Calpastatin variants activities during beef aging and differential postmortem

proteolysis in two muscles

Authors:

Leonardo Guimaraes de Oliveira

Department of Animal Production, Universidade Federal de Goias, Goiania, Brazil

Eduardo Francisquine Delgado

Department of Animal Science, ESALQ/Universidade de São Paulo, Piracicaba, Brazil

Edward M Steadham

Animal Science Department, Iowa State University, Ames, IA, United States of America

Elisabeth Huff-Lonergan


Steven M Lonergan


ABSTRACT:

The relative role played by the two peaks of calpastatin (CAST 1 and CAST 2) eluted during

chromatography has not been elucidated. The aim of this study was to verify the calpastatin variants

during meat ageing and possible relationship to calpain-1 autolysis as well as protein degradation.

The Longissimus lumborum (LL) and Triceps brachii (TB) muscles were obtained from six

crossbred steers and samples prepared from day 0, 1 and 7 postmortem.The drop of CAST total

during ageing was due to decrease of CAST 2 in both muscles. For CASTs and calpain-1 activities,

there were interactions between muscle and day PM. The CAST 2 to calpain-1 ratio at day 0 was

higher for TB, which presented lower calpain autolysis and myofibrillar protein degradation. Heat

shock 70 protein family were associated to tenderization during ageing, with greater differences in

LL. The calpastatin peaks protein profile and its function in the postmortem proteolytic process still

unclear and more investigation is necessary.

Key words: Triceps brachii, Longissimus lumborum, Calpain, proteome, chromatography,

proteolysis.

Highlights.

Calpastatin activity decrease during aging due decrease of peak 2 of calpastatin.

115 kDa calpastatin isoform is present in both calpastatin peaks.

Heat shock protein 70 probably is involved in meat ageing period.

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1. Introduction

Meat tenderization during postmortem aging is a result of weakening of the organized structure of

the myofibrils through enzymatic action largely governed by the calpain system (Koohmaraie,

1994; Huff-lonergan et al., 1996; Geesink et al., 2006; Ono and Sorimachi, 2012). Calpains are

calcium-dependent cysteine proteases with two ubiqitious isoforms, calpain-1 and calpain-2. Both

are composed of an 80 kDa subunit that contains the catalytic site. The 28 kDa subunit works as a

regulatory and stabilizing component of the heterodimer (Goll et al., 2003).

Meat from steaks with high calpastatin activity generally exhibit less postmortem proteolysis of

myofibrillar protein (Koohmaraie, 1994; S. M. Lonergan et al., 2001). Calpastatin is an endogenous

inhibitor of the ubiquitous calpains and acts as a suicide substrate for those proteinases (Doumit and

Koohmaraie, 1999). The degradation of calpastatin by calpain 1 and calpain-2 results in some

fragments that differ in their elution during liquid chromatography in the calpastatin purification

process (Mohan and Nixon, 1995). Experiments conducting calpastatin separation by anion

exchange column chromatography have reported two peaks of calpain inhibitory activity in rat

skeletal muscle (Pontremoli et al., 1992), bovine skeletal muscle (Geesink, Nonneman and

Koohmaraie, 1998; Monica Averna et al., 2001; Camou et al., 2007; Samanta et al., 2010; Cruzen

et al., 2015), porcine skeletal muscle (Cruzen et al., 2013) and salmon skeletal muscle (Gaarder,

Thomassen and Veiseth-Kent, 2011a). In addition, it has been reported that, there are differences in

observed postmortem proteolysis between the Triceps and Longissimus muscles that appear to be

related to calpastatin activity measured in the so-called peak 2 calpastatin (Cruzen et al., 2014).

Determining the exact origin, composition and the role of those calpastatin peaks during post

mortem aging is a challenge. Some possible explanations for the origin and composition of those

peaks could include: degradation products (Doumit and Koohmaraie, 1999), several transcripts

variants of calpastatin gene (Parr et al., 2004) and posttranslational modifications, including

phosphorylation (Pontremoli et al., 1992).

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Because of previous research by our lab and others, this study was designed to evaluate the activity

of calpain-1, calpain-2 and calpastatin peaks during post mortem aging of two beef muscles using

ion exchange chromatography, and to examine the changes of protein profile using proteomic tools.

2. Matherials and methods

2.1. Experiment 1

Six crossbred steers were slaughtered in pairs at the Iowa State University Meat Laboratory on 3

different days following standard humane procedures. The carcasses were not electrically

stimulated. Animals weighed 604.4 ± 44.7kg at the time of slaughter. Samples from each carcass

(approximately 0.3 kg center cut sections) from the Longissimus lumborum (LL), and Triceps

brachii (TB) were collected within 90 min post-exsanguination (day 1). Immediately after

collection, the samples were briefly placed on ice and immediately taken to the laboratory for

extraction, chromatographic separation and activity assays. The carcasses were held in a 1° C cooler

for 24 hours after slaughter. Muscle (LL and TB) samples were collected (approximately 1.0 kg)

from the chilled carcasses at 24 h postmortem. These samples were placed on ice and immediately

processed in the laboratory. Those samples were divided in two, one used for extraction, separation

and activity assays (day 1). The remainder of each muscle was vacuum packaged and stored for 6

days at 4 °C to complete 7 days post mortem (day 7) aging. The aged samples were then extracted

and analyzed after this period.

2.1.1.Extraction and calpain system activities

A transverse cut of muscle was taken and visible fat and connective tissue were removed. The

muscle was then finely minced using a knife. Two, samples, 5 grams each, were homogenized

immediately using a Polytron PT 3100 (Lucerne, Switzerland) in three 30 s bursts (with 30 s of

34

interval between bursts) in 3 volumes (w/v) of ice-cold extraction buffer (100 mM Tris–HCl, 10

mM EDTA, pH 8.3, 4 °C). Immediately before use, 0.1% 2-mercaptoethanol (2-MCE), 2 μM of E-

64, and 100 mg/L trypsin inhibitor were added to the buffer. The homogenate was centrifuged at

40,000 × g for 30 min at 4 °C, and the supernatant was filtered through cheesecloth and dialyzed in

40 volumes of TEM (40 mM Tris–HCl, 1 mM EDTA, pH 7.4, 0.1% MCE).

After dialysis, samples were centrifuged at 40,000 ×g for 30 min at 4 °C and the supernatant filtered

through cheesecloth. The filtered supernatant was loaded onto a 20 mL Q-Sepharose Fast Flow (GE

Healthcare Biosciences, Pittsburgh, PA) anion exchange column. Columns had been previously

equilibrated with TEM. After the sample was loaded, the column was washed with 250 mL TEM.

Calpastatin, μ-calpain, and calpain-2 were eluted using a linear gradient of 60 to 400 mM KCl in

TEM with a flow rate of 2.0 mL/min, fraction volume of 2.5 mL on an ÄKTA™ prime automated

liquid chromatography system (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The first peak

of calpastatin activity (CAST 1) was eluted between 60 to 90 mM KCl. The second peak of

calpastatin (CAST 2) was eluted between 120 to 180 mM KCl, followed by μ-calpain activity (180

to 240 mM KCl) and calpain-2 activity (300 to 400 mM KCl). An example of the elution peaks is

shown in Figure 1.

The caseinolytic method (Koohmaraie, 1990) with some modifications was used to determine the

identity of fractions containing CAST 1, CAST 2, calpain-1, and calpain-2 activity. The same assay

was used to quantify activity. One unit of μ-or calpain-2 activity was defined as the amount

required to catalyze an increase of 1 absorbance unit of the supernatant at 278 nm. One unit of

calpastatin activity was defined as the amount required to inhibit 1 unit of porcine lung calpain-2.

2.1.2 SDS-PAGE and immunoblotting

The same extraction protocol that was used to prepare samples for calpain/calpastatin extraction

(see Section 2.1.1) was used to extract samples for SDS-PAGE and immunoblotting. After

35

centrifugation of the sarcoplasmic fraction, the protein concentrations were determined using the

method described by Lowry, Rosebrough, Farr, & Randall, (1951) technique using premixed

reagents (Bio-Rad Laboratories, Hercules, CA). The protein concentration of each sample was

adjusted to 6.4 mg/mL and one mL was mixed with 0.5 mL of SDS sample buffer (30 mM Tris-

HC1, 3 mM EDTA, 3% [w/v] SDS, 30% [vol/vol] glycerol, and 30 pg of pyronin Y /mL, pH 8.0)

(Wang, 1982), 0.1 mL MCE and stored at -80 oC until analysis. The myofibrilar fraction was

obtained using the pellet following the protocol as described by Huff-Lonergan, Mitsuhashi,

Parrish, & Robson (1996). Protein concentrations were determined using the method described by

Lowry, Rosebrough, Farr, & Randall, (1951) technique using premixed reagents (Bio-Rad

Laboratories, Hercules, CA). Samples were adjusted to 6.4 mg/mL and one mL was mixed with 0.5

mL of SDS sample buffer (Wang, 1982), 0.1 mL MCE and stored at -80 oC until analysis. The

sarcoplasmic fraction was used to determine presence of calpastatin and calpain-1 autolysis. For

desmin and troponin-T degradation and calpain-1 autolysis the myofibril fraction was used.

An 8% polyacrylamide separating gel (acrylamide:N,N′-bis-methylene acrylamide = 100:1 [w/w],

0.1% [w/v] SDS, 0.05% [v/v] TEMED, 0.05% [w/v] ammonium persulfate, and 0.5 M Tris–HCl,

pH 8.8) was used to detect calpain-1 autolysis. Ten percent polyacrylamide separating gels were

used to determine calpastatin, desmin and troponin-T degradation. A 5% polyacrylamide gel

(acrylamide:N,N′-bis-ethylene acrylamide = 100:1 [w/w], 0.1% [w/v] SDS, 0.125% [v/v] TEMED,

0.075% [w/v] ammonium persulfate, and 0.125 M Tris–HCl, pH 6.8) was used for the stacking gel.

Gels (10 cm wide ×8 cm tall ×1.5 cm thick) were loaded with 40 μg protein per lane and run at a

constant 20 V on SE 260 Hoefer Mighty Small II (Hoefer, Inc., Holliston, MA) electrophoresis

units overnight.

Transfer of protein from SDS-PAGE gels to a PVDF membrane was performed as described by

Melody et al. (2004). Membranes were blocked for 1 h at room temperature in PBS with 0.1%

Tween-20 and 5% nonfat dry milk and were then incubated in primary antibody over-night at 4 °C.

Primary antibodies and dilutions were as follows: calpain-1, 1:10,000 (MA3-940, ThermoScientific,

36

Rockford, IL); calpastatin, 1:5,000 (MA3-944, Thermo Scientific, Rockford, IL); desmin 1:30,000

(Policlonal antibody anti desmin created in house by immunizing a rabbit with purified desmin in

1978 at Iowa State University and kindly provided by Dr. Ted Huiatt); and troponin-T, 1:40,000

(JLT-12; Sigma, St Louis, MO). Membranes were washed 3 times in PBS–Tween for 10 min each

at room temperature. The membranes incubated with anti-calpain-1, anti-calpastatin and anti-

troponin-T were then incubated in a goat anti-mouse horseradish peroxidase (No 2554, Sigma, St.

Louis, MO) secondary antibody at 1:10,000 dilution. Membranes incubated with anti-desmin were

incubated in a goat anti-rabbit horseradish peroxidase (No 31460, Sigma, St. Louis, MO) secondary

antibody at 1:60,000 dilution for 1 h at room temperature. After 3 additional 10 min washes in

PBS–Tween, blots were developed using Super Signal West Femto Maximum Sensitivity Substrate

(ThermoScientific, Rockford, IL) and imaged using a ChemiImager 5500 (Alpha Innotech, San

Leandro, CA) and Alpha Ease FC software (v 3.03 Alpha Innotech). Bands were quantified by

densitometry; the abundance of the bands were compared to a reference sample loaded on each gel.

2.1.3. Two-Dimensional Difference in Gel Electrophoresis.

Two-dimensional DIGE was used to determine the difference in a protein profile between day 0 and

7 in Longissimus and Triceps muscles from sarcoplasmic fraction as described by Anderson et al.

(2012) with some modifications. References used for each muscle was a specific pooled sample

obtained from samples of each animal in this experiment from day 0 and day 7 at equal amounts of

total protein. Preparation of the reference in this manner ensures that every protein in the

experiment is in the reference sample.

To label proteins of each individual sample, CyDyes 3 or 5 (GE Healthcare, Piscataway, NJ) were

used according manufacturer’s directions. The different dyes were alternated among day 0 and 7

samples. Reference samples were labeled using CyDye 2. Each gel was loaded using 15 μg of

labeled protein from the same animal and muscle at day 0 and 7, plus reference sample for a total of

37

45 μg of protein per gel. DeStreak Rehydration Solution (GE Healthcare, Piscataway, NJ)

containing 20 mM DTT was added to the labeled samples. An 11 cm immobilized pH gradient strip

(pH 4 to 7; GE Healthcare, Piscataway, NJ) placed on top of rehydration mix and the samples were

loaded on it passively during rehydration of the strip. The strip was left to rehydrate overnight at

room temperature in a sealed chamber. Isoelectric focusing was performed on an Ettan IPGphor

isoelectric focusing system (GE Healthcare) for a total of 14,500 V h.

After isoelectric focusing, strips were equilibrated using two sequential 15 min washes with

equilibration buffer (50 mMTris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 65 mM

DTT and 135 mM iodoacetamide. The equilibrated strips were loaded onto 12.5% SDS-PAGE gels,

using agarose as an overlay and run over-night at 80 V at 4 oC on Ettan DALT SIX system (GE

Healthcare) using 24 cm wide gels. Two 11 cm strips per gel were used. Gels were imaged using an

Ettan Imager (GE Healthcare) and images processed and analyzed using DeCyder 2D software (GE

Healthcare, version 6.5).

To identify the proteins in the spots that were differentially abundant (selected spots) between days

post mortem according to the DeCyder 2D software analyzer, unlabeled proteins were used. One

extract from each day of aging was selected and resolved in the first dimension using 13 cm

immobilized pH gradient strip (pH 4 to 7; GE Healthcare) and a 12,5 % SDS-PAGE in the second

dimension following the described previously protocol. Gels were stained with Colloidal Comassie

Blue Stain (1.7% ammonium sulfate, 30% methanol, 3% phosphoric acid, and 0.1% Coomassie G-

250) for 24 hours. Gels were destained in distilled and deionized water. All buffers and water used

in this process was filtered using Stericup Filter Unit, poresize 0.22 μm (Millipore Corp., Billireca,

MA) to minimize potential contamination.

Each selected spot identified in the gel was excised and sent to the Iowa State University Protein

Facility for identification. In-gel digestion (via trypsin) using Genomics Solution ProGest

(Chelmsford, MA) was done. Peptides were analyzed using Q Exactive Hybrid Quadrupole-

38

Orbitrap Mass Spectometer (LC-MS/MS) (Thermo Fisher Scientific, Waltham, MA). Spectra were

processed by MASCOT data-base search version 2.2.07 (MatrixScience, London, UK).

2.2 Experiment 2

2.2.1. Characterization of calpastatin peaks

For calpastatin purification, 45 minutes after exsanguination one sample of approximately 150

grams was taken of the Semitendinosus (ST) muscle from one commercial market steer at the Iowa

State University Meat laboratory. The animal was slaughtered following standard procedures and

not electrically stimulated. The sample was briefly placed on ice and immediately taken to the

laboratory for extraction of approximately 80 grams of muscle as described previously (Maddock et

al., 2005; Cruzen et al., 2014). A one mL sample (6.4 µg/µl) of the clarified dialysate was mixed

with 0.5 mL of SDS sample buffer (Wang, 1982), and 0.1 mL MCE. Fractionation on anion

exchange columns as described previously (Cruzen et al., 2014). Samples identified containing

calpastatin activity in peak 1 and peak 2 were not as concentrated as the dialysate, so the final

protein concentration in those samples varied, but the gel samples contained the same proportion of

sample buffer and MCE. Protein samples were stored at -80 oC until further analysis. The protocol

for SDS-PAGE and immunoblotting to characterize calpastatin peaks followed the protocol

described previously. The primary antibody anti calpastatin (MA3-944, Thermo Scientific,

Rockford, IL) was used in the dilution 1:5,000, and a goat anti-mouse horseradish peroxidase (No

2554, Sigma, St. Louis, MO) was used of a secondary antibody at 1:10,000 dilution.

2.2.2. Phosphoprotein and Total Protein Staining

Staining for phosphoproteins and total protein was conducted following resolution of protein on

12.5% SDS-PAGE gels. Loading was standardized based on protein content or calpastatin activity.

39

Gels were run at 20 V overnight, then stained using ProQ Diamond Phosphoprotein Stain

(Molecular Probes Inc., Eugene, OR), following the manufacturer’s protocol. Following imaging

for phosphoproteins gels were stained with SyPro Ruby Total Protein Stain (Molecular Probes Inc.,

Eugene, OR) following the manufacturer’s protocol. Gels were then imaged using a ChemiImager

5500 (Alpha Innotech, San Leandro, CA).

Immunoblotting of 2D gels was done to characterize calpastatin peaks from Semitendinosus muscle

using 80 µg of protein per strip and were, resolved using 7 cm immobilized pH gradient strip (pH 4

to 7; GE Healthcare) following protocol for rehydration as previously described. The isoelectric

focusing was performed on an Ettan IPGphor isoelectric focusing system (GE Healthcare) for a

total of 7,000 V h. Strips were loaded onto a 12.5 % SDS-PAGE gels, transferred to PVDF

membrane and imaged following the previously described protocol. The primary antibody anti

calpastatin (MA3-944, Thermo Scientific, Rockford, IL) was used in the dilution 1:5,000, and a

goat anti-mouse horseradish peroxidase (No 2554, Sigma, St. Louis, MO) was used of a secondary

antibody at 1:10,000 dilution.

2.3. Statistical analysis

Data from calpastatin peaks, calpain-1 and calpain-2 activity and abundance of bands from Western

blot were analysed using split plot design in a repeated measures arrangement. Animal as the whole

plot, muscle as the split plot and days post mortem were used as repeated measures. Statistical

analysis was performed using statistical software R (R Development Core Team, Vienna, AU). Gel

images from 2D-DIGE experiments were analyzed using Decider (version 6.5, GE Healthcare,

Piscataway, NJ). A P-value < 0.10 was considered statistically significant. Significant spots present

in more than 83% of the images were initially selected for identification.

3. Results and discussion

40

3.1. Experiment 1

3.1.1. Activity of CAST peak 1 and 2, calpain-1 and calpain-2.

In all extractions, the elution pattern of both peaks of calpastatin, calpain-1 and calpain-2 was

similar (Figure 1). The first peak of calpastatin was eluted in a range of 60 to 90 mM KCl followed

by second peak of calpastatin eluted in a range of 120 to 180 mM KCl, calpain-1 eluted in a range

of 185 to 260 mM KCl and calpain-2 eluted in a range of 330 to 400 mM KCl. This pattern of

elution that shows two peaks of calpastatin has been reported before (Pontremoli et al., 1992;

Salamino et al., 1994; Geesink, Nonneman and Koohmaraie, 1998; M Averna et al., 2001; Cruzen

et al., 2013). However, the exact characteristics of the calpain inhibitory species of those peaks are

not known.

Possible reasons for these two peaks include post translational modifications (potentially

phosphorylation) of calpastatin (Pontremoli et al., 1992; M Averna et al., 2001) that could change

the ionic charge of the molecule and modify its affinity for the column and thus its elution time.

Another possibility could be an alternative splicing of the gene (Geesink, Nonneman and

Koohmaraie, 1998; Gaarder, Thomassen and Veiseth-Kent, 2011b). Degradation of the calpastatin

molecule is another possibility for the separation of two peaks and could be an explaination for

some reports of lost activity during the purification process (Geesink, Nonneman and Koohmaraie,

1998).

Aging time post mortem resulted in an increase in CAST 1 from the Triceps brachii (TB) (P<0.05),

but had no significant influence on the CAST 1 from Longissimus lumborum (LL) (Table 1). On

the other hand, total CAST and CAST 2 activity decreased during post mortem aging in both

muscles. The drop in total CAST activity was mostly accounted for the CAST 2 decrease in activity

during aging. This could indicate that CAST 2 is the predominant form of CAST that is measured

during traditional assays.. This activity loss has been attributed to CAST degradation by calpain-1

and/or calpain-2 or other proteases (Doumit and Koohmaraie, 1999; De Tullio et al., 2000). The

41

hypothesis that post mortem CAST 2 degradation would result in CAST 1 was not corroborated by

activity level, unless it could be considered that CAST 1 inhibitory efficiency would be lower than

CAST 2 for calpain-2 (which was used as the positive control in the assays). It could also depend on

the phosphorylation status of the two fractions (Pontremoli et al., 1992; Salamino et al., 1994).

Calpain-1 activity was greater in LL than TB at day 0 and decreased more rapidly during post

mortem aging in LL (P<0.05). Calpain-2 activity had no change (P>0.05) during post mortem aging

or among muscles. This is consistent with previous observations (Koohmaraie et al., 1987). The

ratio of CAST 2 and CAST total to calpain-1 activity was greater in TB than in LL (Table 2). That

result may provide a partial explanation for the more rapid decline of calpain-1 activity in LL, since

greater CAST activity seems to spare calpain-1 from post mortem autolysis and activity loss

(Delgado et al., 2001), enabling the possibility of LL to exhibit more proteolysis and tenderness

than TB. Interaction between calpain and calpastatin is the most relevant mechanism involved in the

tenderization process during post mortem period mediated by cellular structural proteins (Melloni et

al., 2006). A similar difference between LL and TB in calpain-1 / calpastatin ratio was found by

(Cruzen et al., 2014).

3.1.2. SDS-PAGE and immunoblotting

In western blots for CAST bands between 115 kDa and 36 kDa were detected (Figure 2). Only the

bands presenting strong signal were analyzed. There was no detectable difference between muscles

for the 115 kDa band of CAST (Table 3), the size reported for intact calpastatin (Takano et al.,

1986; Nakamura et al., 1989; Cruzen et al., 2014). The 115 kDa band of CAST decreased in

intensity during post mortem aging in both muscles (P<0.05), with more than 70% of the change

occurring in the first day. At this same time, the abundance of 90 kDa band of CAST increased,

while there were no differences between day 0 and 7 for both muscles. At day 1, the 90 kDa band of

CAST was more abundant in the LL than in the TB. Although the 70 kDa band of CAST also

42

increased at day 1 for TB, differences in it were not detected for the LL between day 0 and 1.

Moreover, the abundance of the 90 kDa band of CAST was greater in TB than LL. Another

difference between muscles was observed for the 45 kDa band of CAST. The 45 kDa band of

CAST was more abundant in LL than TB over all days and did not change in abundance in the same

muscle over the aging period. Overall, the appearance of the two more intense bands of CAST

probably are result of degradation of the intact calpastatin, even though there was some differences

between muscles. A similar pattern of bands was found using the products of purified calpastatin

incubated with calpain-1 and calpain-2 (Doumit and Koohmaraie, 1999).

The autolysis of the 80 kDa band of calpain-1 in the sarcoplasmic fraction increased in both

muscles during post mortem aging. Autolysis decreases the requirement of free Ca2+ for calpain

activity from 3-50 µM to 0.5-2 µM Ca2+ (Goll et al., 2003) even though it also decreases its

stability at the higher ionic strengths found in post mortem muscle (Geesink and Koohmaraie,

1999b). Nonetheless, the autolysis is associated with activation of calpain and could provide

information about the proteolysis process in the muscle. The appearance of the 78 kDa autolyzed

calpain at day 1 was more pronounced in the LL compared to the TB, with a decrease at day 7 of

this band compared to day 1. In the TB, a decrease in the 78 kDa autolysis product of the catalytic

subunit was not detected between days 1 and 7 postmortem (Figure 3). The proportion of the

catalytic subunit present as the 76 kDa autolysis product increased at day 7 for both muscles with

greater amount in the LL. These changes in the sarcoplasmic fraction point to a slower rate of

autolysis in the TB, which might be associated to the greater calpastatin / calpain-1 ratio in that

muscle and perhaps a slower rate of protein degradation.

The calpain-1 is present in soluble in the sarcoplasmic fraction and during meat aging start to be

associated to myofibrils at the Z-line and A-band (Melody et al., 2004). The reason for appearance

of myofibril-bound calpain-1is not clear, and remains certain proteolytic activity during post

mortem aging period (Delgado et al., 2001). In the myofibrillar fraction, the decrease of the 80

kDa band of intact calpain-1 happened after 24 hours post mortem and in day 7 post mortem this

43

band was not abundant. The 78 kDa band of calpain-1 was increased in both TB and LL at day 1,

but it decreased at day 7 in LL; the 78 kDa band of autolyzed calpain-1 product was greater at day

7 compared to day 0 in TB. The abundance of 76 kDa band of autolyzed calpain-1 was increased at

day 7 in the myofibrillar fraction for both muscles. Therefore, autolysis of calpain-1 presents the

same pattern between sarcoplasmic and myofibrillar fractions. Similar autolysis progression in both

sarcoplasmic and myofibrillar fractions has been reported before (Melody et al., 2004; Rowe et al.,

2004).

Autolyzed bands of calpain-1across aging appear be more abundant in the myofibrillar fraction than

in sarcoplasmic fraction, suggesting that myofibril bound calpain-1 is less prone to continued

autolysis compared to the soluble enzyme present in sarcoplasmic fraction. Proteolysis in LL seems

to be more rapid than in the TB because at day 1 the 78 kDa band of calpain-1 is more abundant

than day 0 in the LL, which could mean more activity in LL early post mortem.

The 30 kDa band in myofibrillar fraction, a troponin T degradation product, is increased at day 7

compared to day 0 for LL, and was more abundant at day 7 in the TB than LL (P<0.05). In this

muscle there was no difference between days 1 and 7 (Figure 4). Those results suggest that

proteolysis occurred at a more rapid rate in the LL. The connection of postmortem tenderization of

LL to the appearance of 30 kDa band of troponin-T has been reported (Geesink and Koohmaraie,

1999a; S M Lonergan, Rowe, et al., 2001), and it probably could be related to proteolytic activity of

calpain-1. The current results together with the current literature suggests that variation in

calpastatin activity explains a portion of the differences in calpain activity and postmortem protein

degradation.

The degradation of intact band of desmin starts after 24 hours of aging in LL and this band is more

degraded in LL than TB at day 7 (Table 3). For the TB, no difference in the abundance of intact

desmin was detected (P<0.05) across days of aging. However, there was more abundance of bands

of degraded products of desmin (38 kDa and 35 kDa) at day 7 (Figure 5).

44

Similar degradation pattern of desmins and troponin-T degradation have been reported in aged beef

and purified myofibrils incubated with calpain-1 (Olson and Parrish, 1977; Huff-lonergan et al.,

1996). This degradation of desmin and troponin-T is highly related to beef tenderization (Huff-

Lonergan, Parrish and Robson, 1995; Geesink et al., 2006).

3.1.3. Two-Dimensional Difference in Gel Electrophoresis

A two-dimensional difference in gel electrophoresis gel (2D-DIGE) experiment showing spots that

were differentially abundant in Longissimus lumborum (LL) and Triceps brachii (TB) muscles in

sarcoplasmic fraction is summarized in figure 6. Coverage percentage of identified peptides from

intact protein and identification of proteins present in each picked spot is presented in table 4.

An interesting finding in this analysis is the greater amount of 70 kDa heat shock proteins (HSP) in

the sarcoplasmic fraction of aged beef. The HSP protein family is described as having an important

role in post mortem meat aging (Ouali et al., 2006; Carvalho et al., 2014). This protein family is

related to meat toughness and has been suggested to be a toughness marker due their cellular

protective function against apoptosis. During meat aging, the HSP 70 protein family acts in an anti

apoptotic function in the stress response, acting on the caspase-independent pathway and caspase

dependent pathway at both ways, upstream and downstream of caspase activation (Creagh,

Carmody and Cotter, 2000; Mayer and Bukau, 2005). A signal like cell damage for example,

induces the HSP 70 prevent the oligomerization of an apoptotic protease activating factor-1 (Apaf-

1), reventing maturation of caspase-9, this process could help the sarcomere maintenance and

organization (Beere, 2004; Ouali et al., 2006; Picard et al., 2010).

HSP 70 is normally located aggregated in cytoplasm in non stressed live tissue and after slaughter

attached to actin and α-actinin (Margulis and Welsh, 1991; Tupling et al., 2004). The presence of

more HSP 70 in day 7 sarcoplasmic fraction could be related to release of this protein from

myofibrillar fraction during post mortem aging probably due the proteolytic action. Under electrical

45

stimulation the relative abundance of HSP 70 bound to myofibrils is lower than non electrical

stimulated one and 24 hours after the slaughter, suggesting eletrical stimulation influences solubility

and fractionation of this protein (Bjarnadóttir et al., 2011).

Structural proteins decreased the relative abundance in day 7 compared to day 0 in both muscles in

sarcoplasmic fraction. This effect could occur by protease activity upon proteins like alpha4A chain

tubulin, desmin and alpha actin that are soluble in sarcoplasmic fraction and being degraded even in

the soluble fraction and the fragments are not detectable by the current used techinique.

Myosin light chain (MLC) could be released to sarcoplasmic fraction because of degradation of

proteins associated with MLC during post mortem aging (Anderson, Lonergan and Huff-Lonergan,

2012). In fact, Anderson et al. (2012) also demonstrated that incubation of myofibrils with calpain-

1 resulted in release of MLC from the myofibris. This could be related to the weakening of the

actomyosin and may be an important contributor to tenderization during aging time. The appearance

of MLC in the soluble fraction was related to the tenderization process and was negatively related to

tender meat 72 hours after slaughter but positively related at 14 days post mortem (Zapata, Zerby

and Wick, 2009).

The mitochondrial protein ATP synthase (subunit beta) was increased and Succinyl-CoA ligase

decreased in both muscles at day 7 compared to day 0. Adenylate kinase isoenzyme also decreased in

the LL at day 7 compared to day 0. The conversion of ADP to ATP is mediated to ATP synthase

present in the mitochondria and was found to be more abundant in aged pork meat (Bernevic et al.,

2011). Mitochondrial proteins found in the soluble fraction are related to the beginning of the

apoptotic process in early post mortem stages and those proteins may be related to the tenderization

process (Laville et al., 2009). Apoptosis has been suggested to be an early event related to the

tenderization process after slaughter, starting the cell protection machinery (Longo et al., 2015) and

ultimately affecting the tenderization process. Another interesting protein that was increased at day

7 in both muscles compared to day 0 is Prostaglandin reductase 2 (Table 4). This enzyme catalyzes

the NADPH dependent reduction of 15-keto-prostaglandin E2. Recently, 15-keto-prostaglandin E2

46

was proposed to trigger the translocation of the pro apoptotic protein Bax to mitochondria, thereby

inducing apoptosis (Yun-Chia Chang et al., 2012). This finding points to another protein in the

apoptotic pathway that changes during proteolysis post mortem.

3.2. Experiment 2

3.2.1. Phosphoprotein and Total Protein Staining

In the total protein stain assay, the Semitendinosus muscle (ST) showed the same pattern of bands

as the TB and proteins from pooled fractions that have active calpastatin from the ST ,CAST 1 and

CAST 2, are stained (Figure 7 A). CAST 1 had two bands a 115 kDa band and one upper band that

was approximately 125 kDa, while CAST 2 had only an approximately 125 kDa band. Both bands

are poorly phosphorylated (Figure 7 B). Although the CAST 1 pool had more total protein than

CAST 2 pool, they had a similar pattern of bands, with also the same phosphorylation pattern.

Obviously, CAST1 showed more phosphorylated bands which may be explained by greater amount

of total protein. Those differences are due to application of the same amount of eluted volume from

each pooled fractions from each calpastatin peak. This approach was taken because CAST 1 is

extremely unstable, and it was decided to minimize the manipulation of the samples, with methods

such as those for protein concentration.

Calpastatin immunoblots show CAST 2 had a band that migrated with an apparent molecular

weight greater than 115kDa and CAST 1 showed a 115kDa band (Figure 8). CAST 2 had a more

abundant 70 kDa band than CAST 1. This seems to be important considering the observation that

band was present in greater amount in TB at day 1 in experiment 1. This could corroborate that

calpastatin variants present in CAST 2 are more relevant to slowing down post mortem degradation.

In Semitendinous muscle from cattle, domain IV, 1xb exon and XL domain was found in a band

that was approximately 70 kDa and was attributed to a cleavage in the inhibitory domain II of the

47

calpastatin molecule. This cleavage generates two degradation products that migrate to similar

location in a polyacrylamide gel (Raynaud et al., 2005). Therefore the cleavage could form two

distinct products with different affinity for an anion exchange column thus generating the two

peaks.

The western blot results from Semitendinosus to detect CAST peaks fractions (Figure 8) were not

consistent with the explanation of the conversion of CAST 2 to CAST 1 occurring since some

fragments of the CAST 1 fraction may be even larger than what was found in CAST 2. Nonetheless,

a partial conversion should not be dismissed.

The two dimensional western blot of calpastatin peaks are presented in figure 9. The sample for the

first peak, CAST 1, showed more spots at different isoelectric points and molecular weight than

CAST 2 in a 2D immunoblot for calpastatin. Those blots make it difficult to consider that CAST 1

would be only a product of degradation of CAST 2, since there are high molecular weight spots at

acidic isoeletric regions in CAST 1 immunoblots. The reason for not identifying high molecular

weight spots in CAST 2 needs to be studied. Nonetheless, it could be more plausible to understand

those peaks as result of different transcripts variants associated with post-translation modifications,

which both may change the CAST proteolytic products that compose those peaks.

4. Conclusions

During the beef aging the decrease of total calpastatin activity is attributed to the decrease of

calpastatin peak 2 activity. Greater CAST2 to calpain-1 ratio at slaughter are related to lower

proteolytic rate and extension. The abundance proteins of heat shock 70 family in the sarcoplasmic

fraction of meat changes with post mortem aging process. There is consistent fractionation of two

forms of calpastatin in postmortem muscle and the changes in these forms could provide new clues

to shed light on the involvement of calpains in postmortem improvement of beef tenderness. The

48

exact composition of calpastatin peaks is still unclear and more investigation is necessary to

discover the composition and the function of each peak in the tenderization process.

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degradation of calpastatin by ??- and m-calpain in Ca2+-enriched human neuroblastoma

LAN-5 cells’, FEBS Letters, 475(1), pp. 17–21. doi: 10.1016/S0014-5793(00)01613-6.

53. Tupling, A. R., Gramolini, A. O., Duhamel, T. A., Kondo, H., Asahi, M., Tsuchiya, S. C.,

Borrelli, M. J., Lepock, J. R., Otsu, K., Hori, M., MacLennan, D. H. and Green, H. J. (2004)

‘HSP70 binds to the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+-ATPase

(SERCA1a) and prevents thermal inactivation’, Journal of Biological Chemistry, 279(50),

pp. 52382–52389. doi: 10.1074/jbc.M409336200.

54. Wang, K. (1982) ‘[23] Purification of titin and nebulin’, Methods in Enzymology, 85, pp.

264–274. doi: 10.1016/0076-6879(82)85025-8.

55. Yun-Chia Chang, E., Tsai, S. H., Shun, C. T., Hee, S. W., Chang, Y. C., Tsai, Y. C., Tsai, J.

S., Chen, H. J., Chou, J. W., Lin, S. Y. and Chuang, L. M. (2012) ‘Prostaglandin reductase

2 modulates ros-mediated cell death and tumor transformation of gastric cancer cells and is

associated with higher mortality in gastric cancer patients’, American Journal of Pathology.

doi: 10.1016/j.ajpath.2012.07.006.

56. Zapata, I., Zerby, H. N. and Wick, M. (2009) ‘Functional proteomic analysis predicts beef

tenderness and the tenderness differential’, Journal of Agricultural and Food Chemistry. doi:

10.1021/jf900041j.

52

FIGURES

Figure 1 - Example of elution of calpastatin peaks and calpain-1 and of calpain-2 during elution

using a Q-sepharose Fast Flow ion exchange column. The solid line represents inhibition of porcine

lung calpain-2 for calpastatin peak 1 (CAST 1), calpastatin peak 2 (CAST 2) and the caseinolitic

activity for calpain-1 and calpain-2. The dashed line represents the predicted concentration of KCl

(mM) at time of elution.

53

Figure 2 Representative blot and immunodetection of calpastatin in sarcoplasmic fraction using

antibody anti Calpastatin (MA3-944).

*Estimated molecular weight of bands.

#Molecular weight marker.

Figure 3 Representative blot and immunodetection of calpain-1 in sarcoplasmic fraction using

antibody anti Calpain-1.


54

Figure 4 Representative blot and immunodetection of troponin-T in myofibrillar fraction using

antibody anti troponin.


55

Figure 5 Representative blot and immunodetection of desmin in myofibrillar fraction using

antibody anti desmin.


56

Figure 6 A representative two-dimensional difference in gel electrophoresis showing different

expressed spots in Longissimus lumborum (LL) muscle.Circles represent total area of detected

spot and the different expressed spots are identified by numbers.

57

Figure 7 A) SyPro Ruby total protein stain and B) ProQ Diamond phosphoprotein stain to identify

phosphorylated proteins from: TB0- 40 µg of protein from sarcoplasmic fraction of Triceps brachii

day 0; TB1- 40 µg of protein from sarcoplasmic fraction of Triceps brachii day 1; ST0- 40 µg of

protein from sarcoplasmic fraction of Semitendinosus day 0; Pk1 - 30µL of calpastatin peak 1

extracted from Semitendinosus day 0; Pk2 - 30µL of calpastatin peak 2 extracted from

Semitendinosus day 0.

58

Figure 8 Western blot and immunodetection using antibody anti Calpastatin (MA3-944). TB0- 40

µg of protein from sarcoplasmic fraction of Triceps brachii day 0; TB1- 40 µg of protein from

sarcoplasmic fraction of Triceps brachii day 1; ST0- 40 µg of protein from sarcoplasmic fraction of

Semitendinosus day 0; Pk1 - 30µL of calpastatin peak 1 extracted from Semitendinosus day 0; Pk2

- 30µL of calpastatin peak 2 extracted from Semitendinosus day 0.

59

Figure 9 Two dimensional western blot and immunodetection using antibody anti Calpastatin

(MA3-944).of pooled fractions from the two peaks with calpastatin activity from Semitendinosus

day 0. A) Calpastatin peak 1. B) Calpastatin peak 2.

* Isoelectrical point # Molecular weigth

60

TABLES

Table 1 Activityᶲ of calpastatin peak 1 (CAST 1), calpastatin peak 2 (CAST 2), Total calpastatin

(CAST total), calpain-1 and calpain-2 extracted from muscles Longissimus lumborum (LL) and

Triceps brachii (TB) during aging days of beef meat.

Itemᶲ

Day 0 Day 1 Day 7

SEM

P-values

LL TB LL TB LL TB Muscle

(M) Day (D) M x D

CAST 1 0.93A* 0.82Y 0.96A 1.09XY 1.03A 1.30X 0.10 0.25 0.038 0.007

CAST 2 2.43A 2.98X 1.17B 1.31Y 0.07C 0.31Z 0.18 0.063 <0.001 <0.001

CAST total 3.22A 3.80X 2.12B 2.40Y 1.10C 1.61Y 0.23 0.065 <0.001 <0.001

calpain-1 1.06Aa 0.91Xb 0.13B 0.16Y 0.02B 0.03Z 0.04 0.312 <0.001 <0.001

calpain-2 1.27 1.30 1.13 1.11 1.21 1.16 0.07 0.821 0.092 0.195

* Means with different capital letters (within the muscle, (A and B to compare LL; X,Y and Z to

compare TB) and small letters (within the day) in the same row are significantly different.

ᶲActivity is reported as units per gram of tissue (Lonergan, Huff-Lonergan, Wiegand, & Kriese-

Anderson, 2001b)

Table 2 Calpastatin peak 1 (CAST 1), calpastatin peak 2 (CAST 2), total calpastatin (CAST total)

to calpain-1 activity ratios from muscles Longissimus lumborum (LL) and Triceps brachii (TB) at

day 0 postmortem.

Ratio LL TB SEM P-value

CAST1 : calpain-1 0.76a* 0.90a 0.103 0.363

CAST2 : calpain-1 2.35b 3.32a 0.295 0.042

CAST total : calpain-1 3.12b 4.23a 0.344 0.045 *Means with different superscripts within the same row indicate a significant difference (P<0.05).

61

Table 3 Abundance of immunoreactive bands from western blot of calpastatin (CAST), myofibrilar

and sarcoplasmic calpain-1, desmin and troponin-T during aging days of muscles Longissimus

lumborum (LL) and Triceps brachii (TB).

Item

Band

( MW1

kDa)

Day 0 Day 1 Day 7 SEM P-value

LL TB LL TB LL TB Muscle

(M) Day (D) M x D

CAST

115 0.75A* 0.78X 0.19B 0.26Y 0.02C 0.02Z 0.05 0.340 <0.001 0.711 90 1.00B 1.04Y 2.11Aa 1.67Xb 0.74B 0.64Y 0.13 0.039 <0.001 0.123 70 1.11AB 0.97Y 1.96Ab 3.19Xa 0.74B 1.20Y 0.34 0.030 <0.001 0.008 58 0.91 0.83 1.21 1.00 0.96 0.78 0.12 0.241 0.207 0.809

45 0.97a 0.72b 1.01a 0.76b 1.08a 0.80b 0.07 0.023 0.545 0.944 36 0.93 0.93 1.07 0.76 1.30 0.97 0.15 0.140 0.194 0.328

calpain-1

sarcoplasmic

80 0.95A* 0.90X 0.56B 0.59Y 0.02C 0.03Z 0.05 0.972 <0.001 0.515 78 0.05B 0.04Y 0.19Aa 0.12Xb 0.06B 0.09XY 0.02 0.403 <0.001 0.043 76 0.01B 0.01Y 0.06B 0.04Y 0.32Aa 0.24Xb 0.11 0.096 <0.001 <0.001

calpain-1

myofibrilar

80 1.08A 1.05X 0.86A 1.09X 0.09B 0.24Y 0.14 0.392 <0.001 0.277 78 0.21B 0.20Y 1.00A 0.82X 0.42B 0.75X 0.11 0.617 <0.001 <0.001 76 0.09B 0.08Y 0.50B 0.33Y 2.17A 1.82X 0.14 0.085 <0.001 <0.001

Desmin

Intact 1.61A 1.53X 1.44A 1.46X 0.65Bb 1.15Xa 0.13 0.345 <0.001 <0.001

38 0.10C 0.13Y 0.45B 0.28Y 0.83Ab 1.25Xa 0.09 0.093 0.006 <0.001

35 0.10B 0.12Y 0.34B 0.18Y 1.38A 1.11X 0.16 0.304 <0.001 <0.001

Troponin-T

Upper

intact 0.94 0.72 0.95 0.76 0.76 0.73 0.09 0.115 0.280 0.155

Lower

intact 0.86 0.82 1.04 0.86 1.15 0.96 0.12 0.271 0.191 0.485

30 0.13B 0.10Y 0.28B 0.16XY 1.49Aa 0.61Xb 0.14 0.062 <0.001 <0.001

* Means with different capital letters (within the muscle, (A and B was used to compare between

LL; X,Y and Z was used to compare betwwen TB) and small letters (within the day) in the same

row are significantly different.

Values represent the relative density of each band related to the same band in the reference sample.

Reference samples are a mix of same amount of protein from all samples of TB and LL of day 0

and another of day 7. To CAST each band of sample reference from day 0 was considered the

abundance 1 and each band of each sample was compared to the correspondent band. To desmin,

troponin-T, sarcoplasmic and myofibrillar calpain-1, intact bands of reference sample of each

protein form day 0 was considered the abundance 1 and the intact bands of each sample was

compared to. For degraded bands of reference sample of each protein form day 7 was considered

the abundance 1 and the degraded bands of each sample was compared to.

62

Table 4 Proteins identified in the spots of 2D-DIGE analysis of Longissimus lumborum (LL) and Triceps brachii (TB).

Muscle Spot

ID1 Identification

Accession

number Species

(%) Σ

Coverage2

MW3

[kDa]

calc.

pI4

Av.

Ratio5 P-value

LL

1 Tubulin alpha-4A chain P81948 Bos taurus 22.99 49.9 5.06 -7 <0.001

2 ATP synthase subunit beta, mitochondrial P00829 Bos taurus 69.70 56.2 5.27 2.24 <0.001

3 Heat shock cognate 71 kDa protein A2Q0Z1 Equus caballus 38.39 70.9 5.52 3.95 <0.001

4 Succinyl-CoA ligase [ADP-forming] subunit beta,

mitochondrial Q148D5 Bos taurus 33.69 50.1 7.18 -1.28 0.019

5 Prostaglandin reductase 2 Q32L99 Bos taurus 7.69 38.4 5.53 1.77 0.002

6 Desmin O62654 Bos taurus 42.34 53.5 5.27 -3.1 <0.001

7 Heat shock cognate 71 kDa protein A2Q0Z1 Equus caballus 44.58 70.9 5.52 1.33 <0.001

8 Heat shock cognate 71 kDa protein A2Q0Z1 Equus caballus 55.88 70.9 5.52 1.24 0.004

9 Heat shock 70 kDa protein 1B Q27965 Bos taurus 34.63 70.2 5.92 1.45 0.002

10 Heat shock 70 kDa protein 1B Q27965 Bos taurus 41.81 70.2 5.92 1.27 0.019

11 Heat shock 70 kDa protein 1 Q28222 C. aethiops 7.84 69.9 6.11 1.29 0.015

12 Actin, alpha skeletal muscle P68138 Bos taurus 27.32 42.0 5.39 -1.89 <0.001

13 Adenylate kinase isoenzyme P00570 Bos taurus 58.76 21.7 8.32 -1.7 0.003

14 Myosin regulatory light chain 2, skeletal muscle isoform Q0P571 Bos taurus 56.47 19.0 5.01 -1.51 0.024

TB

1 Tubulin alpha-4A chain P81948 Bos taurus 39.96 49.9 5.06 -7.47 <0.001

2 ATP synthase subunit beta, mitochondrial P00829 Bos taurus 71.21 56.2 5.27 2.87 <0.001

3 Heat shock cognate 71 kDa protein A2Q0Z1 Equus caballus 47.52 70.9 5.52 2.14 0.004

4 Succinyl-CoA ligase [ADP-forming] subunit beta,

mitochondrial Q148D5 Bos taurus 47.95 50.1 7.18 -2.57 0.036

5 Prostaglandin reductase 2 Q32L99 Bos taurus 12.25 38.4 5.53 1.35 0.067

6 Desmin O62654 Bos taurus 47.87 53.5 5.27 -3.38 <0.001 1 Identification of spots different expressed among day 0 and 7 of LL and TB represented in figure 6.

2 Percentage of peptides identified of intact protein. 3 Calculated Molecular weight

4 Isoelectrical point

5 Ratio of relative abundance of day 7 to day 0. Positive values represent more relative abundance in day 7 and negative values represent more relative abundance in day 0

63

(Trabalho redigido e normatizado de acordo com as normas da revista Journal of Animal Science)

CAPITULO 3 – Purification and characterization of active peaks of calpastatin from swine

Longíssimus dorsi muscle.

Authors: Leonardo G. Oliveira #, Edward Steadham*, Steven M. Lonergan*, Elisabeth Huff-

Lonergan*

# EVZ, Universidade Federal de Goiás, Goiânia, GO, Brazil.

* Muscle Biology Group, Department of Animal Science, Iowa State University, Ames, IA 50011,

United States

Acknowledgements: Appreciation is extended to the Iowa Muscle Biology group for funding this

research and to CAPES-Brazil to provide the scholarship for the first author.

Abstract: Calapastatin is a specific inhibitor of the calcium dependent proteinases m- and µ-

calpain and is related to various metabolic process in the live animal and during post mortem

tenderization of meat. Composed of four repetitive regions with inhibitory activity (domains1– 4), and

an unique domain L located at N-terminal region. Using an anion exchange chromatography to

separate calpastatin and sequencial chromatography steps to purify each peak was found in each peak.

During the process to purify the calpastatin peak 1 the activity per mg of protein is greatly increased

but lose half activity, for calpastatin peak 2 the purification process, specific activity was increased

139.8 fold and at the end of this process remains 36% of the initial total activity. Bands of

approximately 70 kDa are identified of both peaks and the band from peak 2 was a little higher than

peak 1. The calpastatin was identified in the second dimension gel in a similar molecular weight to

SDS-PAGE gel and western blot and one spots from calpastatin peak 1 and two from calpastatin peak

2 were identified as calpastatin. Sequence of peptides identified in Spot from purified peak 1 as part

of the inhibitory domain III and IV and C terminus and from purified peak 2 a sequence of peptides

identified as part of the inhibitory domain I, II and III. This results lead us to believe that both peaks,

in this case, are products of degradation of the intact molecule and probably the small peptides are

loosed during the process. The results of present study shows that is possible the purification of distinct

forms of active calpastatin, however the intact form of calpastatin was not present in this purification.

Presence of peptides was not conclusive to determine the origin and composition of each active peak.

Keywords: Calpain system, Ion exchange chromatography, Proteomics

64

INTRODUCTION

Specific inhibitor of the ubiquitous calcium dependent proteinases m- and µ-calpain, calpastatin,

is related to various metabolic process in the live animal and during post mortem tenderization of meat

(Geesink et al. 1995; Goll et al., 1998; Geesink 1999, Huff-Lonergan et al., 1995; Huff-Lonergan et

al., 1996). Composed of four repetitive regions (inhibitory domains1– 4), each of this have a possibility

to inhibit calpain activity, and an unique N-terminal region: domain L(Takano et al., 1986; Maki et al.,

1988).

With a widely varying molecular weights have been purified from a number of tissues and

different molecular weights are related. Confusion regarding the molecular weight of calpastatin was

due to factors like a proteolytic degradation, and some of this fragments of calpastatin retain inhibitory

activity (Melgren et al. 1983; Imajoh et al., 1984; Nakamura et al., 1985), because of its unusual amino

acid composition, the molecular weight is overestimated using SDS–PAGE (Maki et al., 1988) and

due to the asymmetry estimation using gel filtration leading to an overestimation of its molecular

weight (2).

Calpastatins from different species, using cDNAs analysis, has shown that the most prominent

form found in all tissues has a predicted molecular weight of 72–77 kDa but anomalously migrates on

SDS–PAGE with an apparent molecular weight of 115–130 kDa (Killifer & Koohmaraie, 1994).

However, chromatography of the muscle extracts to separate calpains and calpastatin led to extensive

fragmentation of calpastatin (Arnold et al. 1995; Geesink et al., 1998) and this fragmentation could

difficult to determine the composition of extracted calpastatin from chromatograph column and purify

the intact molecule. During the extraction of calpastatin using anion exchange column, calpastatin

could be eluted in two distinct peaks (Pontremoli et al., 1992; Cruzem et al., 2013) and the significance

still unclear.

The objective of this work is purify each calpastatin peak to characterize it composition using

two dimensional gel electrophoresis and mass spectrometry.

65

MATHERIALS AND METHODS

A commercial animal was slaughtered following standard humane procedures at the Iowa State

University Meat Laboratory and a sample (approximately 1.5 kg center cut) of the Longissimus dorsi

(LD) from each animal (animal was weighted approximately 100kg at the time of slaughter) was

collected within 10 min post-exsanguination, briefly placed on ice and immediately taken to the

laboratory for extraction procedures, chromatographic separation of calpastatin peaks. All process was

executed at 4 oC in a cold room.

Calpastatin extraction

Was used a methodology proposed by Thompson et al. (2000) to extract calpastatin from muscle

and purify. Using a knife to remove visible fat and connective tissue, 900 grams of muscle Longissimus

dorsi was finely minced and 100 g of muscle per time was immediately homogenized using with a

Polytron PT 3100 (Lucerne, Switzerland) in 6 volumes (w/v) of cold extraction buffer (100 mM Tris–

HCl, 10 mM EDTA, pH 8.3, 4 °C). Before use were added to the buffer 0.1% 2-mercaptoethanol (2-

MCE), 2 μM of E-64, and 100 mg/L trypsin inhibitor. The homogenate was centrifuged at 40,000 ×g

for 30 min at 4 °C, and the supernatant was filtered through cheesecloth and dialyzed in 40 volumes

of TEM (40 mM Tris–HCl, 1 mM EDTA, pH 7.4, 0.1% MCE).

Was added 277 g/L os Ammonium sulfate slowly and stirring during 12 hours. After stirring, the

homogenate was divided in centrifuge tubes, centrifuged at 4 oC at 10000 RPM during 30 minutes.

Was discarded the supernatant after centrifugation and the pellet was ressuspended in 4 volumes of

TEM buffer (40 mM TRIS, 1 mM EDTA, 0,1% 2 mercaptoetanol, pH 7.4). This solution was dialyzed

12 hours with TE (40mM TRIS, 1mM EDTA, pH 7.4) using 20 times the amount of final volume of

ressuspended solution, and repeated 4 times until complete 100 times the volume of ressuspended

solution.

66

After dialysis, were centrifuged at 40,000 ×g for 30 min at 4 °C and the supernatant filtered

through cheesecloth. The filtered was loaded onto a 800 mL Q-Sepharose (GE Healthcare Biosciences,

Pittsburgh, PA) anion exchange column. The column was previously equilibrated with TEM and after

loaded the sample was washed with 1200 mL TEM. Calpastatin was eluted using a flow rate of 2.0

mL/min, fraction volume of 2.5 mL with a linear gradient of 50 to 225 mM KCl in TEM using an

ÄKTA™ prime automated liquid chromatography system (Amersham Pharmacia Biotech Inc.,

Piscataway, NJ).

Calpastatin Activity

To determine calpastatin activity of the fractions were used the caseinolytic method

(Koohmaraie,1990) with some modifications. Was used 50 µL of each fraction, brought to 1 mL with

TE (40 mM Tris–HCl, 1 mM EDTA, pH 7.4) in a glass tube. One milliliter of casein buffer (100 mM

Tris–acetate 7 mg/mL casein, and 1 mM sodium azide, pH 7.5, with 0.2% MCE added just before use)

was added, followed by 100 μl of calcium buffer (200 mM CaCl2). For activity determination, was

added in each sample approximately 0.40 units of m-calpain previously purified from porcine lung.

For positive control, was used 1 mL of TE (without sample), casein buffer and added approximately

0.40 units of m-calpain from porcine lung, made in triplicate. For blank tubes was used 1 mL of TE

(without sample), casein buffer, and made in triplicate. Tubes were briefly vortexed and incubated in

a water bath at 25 °C for 1 h.

To stop the reaction were added 2 mL of 5% trichloroacetic acid in each tube and after vortexed.

Samples were centrifuged at 1500 × g for 20 min at 25 °C and determined the absorbance of the

supernatant at 278 nm. The reading was compared to blank and positive control samples (Koohmaraie

et al., 1995). The first peak of calpastatin activity was eluted between 60 to 90 mM KCl. The second

peak of calpastatin was eluted between 120 to 220 mM KCl. An example of elution peaks is in Figure

1.

67

Fractions that had calpastatin activity for peak 1 was pooled and the same was proceed for peak

2 to follow the purification and determine the total activity of pooled fractions. To determine the total

activity of each pool was used crescent amounts of sample starting with 20 µL until 400µL. To

calculate the total activity of pooled calpastatin I and II, was used the value of 50% inhibition of lung

m-calpain. To discount the amount of protein contained in the pooled fraction of calpastatin fractions,

was used in each tube 1 mL of pooled sample, 100 μl of EDTA buffer (200mM EDTA), one milliliter

of casein buffer and approximately 0.40 units of m-calpain previously purified from porcine lung and

incubated, briefly vortexed and incubated in a water bath at 25 °C for 1 h. One unit of calpastatin

activity was defined as the amount required to inhibit 1 unit of porcine lung m-calpain (Koohmaraie,

1990). Protein concentration of polled Calpastatin peaks was determined using Biuret methodology

(Lowry et al., 1951).

Calpastatin peaks purification

To purify the pooled calpastatin peak 1, was dialyzed against 40 volumes of TE at 4 oC during

12 hours. After dialysis was added of ammonium sulfate (Sigma, St Louis, MO) until reach

concentration 1 mol/L. The solution containing calpastatin peak 1 was loaded onto a Phenyl Sepharose

(GE Healthcare Biosciences, Pittsburgh, PA) anion exchange column. The column was previously

equilibrated with 3 times the volume of column of TEM with 1 Mol of ammonium sulfate (Sigma, St

Louis, MO) and after loaded the sample was washed with the same solution. Calpastatin was eluted

using with a flow rate of 1.5 mL/min, fraction volume of 8.0 mL in a linear gradient of 1.0 to 0 M of

ammonium sulfate in TEM using an ÄKTA™ prime automated liquid chromatography system

(Amersham Pharmacia Biotech Inc., Piscataway, NJ).

Was determined calpastatin activity of fractions from peak 1 eluted of Phenyl Sepharose column

(GE Healthcare Biosciences, Pittsburgh, PA) as described protocol, pooled fractions that had activity

and protein concentration was determinate . The same process was made with peak 2 and protein

concentration of pooled fraction was determined. Calpastatin peak 1 was eluted between 710 to 560

68

mM of ammonium sulfate. Was proceeded the same process to purify the peak 2 and was eluted

between 620 do 460 mM of ammonium sulfate.

Pooled fractions were dialyzed against 40 times the volume of pooled fractions on TEM during

12 hours at 4 oC. Peak 1 was stored at this point and peak 2 follow the purification. Calpastatin peak 2

was loaded onto a Blue Sepharose (GE Healthcare Biosciences, Pittsburgh, PA) column. The column

was previously equilibrated with 3 times the volume of column of TEM and after loaded the sample

was washed with tree times the column volume with the same solution and eluted using a flow rate of

1.5 mL/min, fraction volume of 10.0 mL in a linear gradient 0 to 500 mM of potassium chloride in

TEM using an ÄKTA™ prime automated liquid chromatography system (Amersham Pharmacia

Biotech Inc., Piscataway, NJ). Calpastatin activity was determined in each eluted fraction as described

previously and active fractions were pooled and dialyzed against 40 times the volume of pooled

fractions on TEM during 12 hours at 4 oC. Calpastatin was eluted between 60 to 300 mM of potassium

cloride.

Dialyzed pooled fractions from Calpastatin peak 2 was loaded onto a DEAE CAPTO (GE

Healthcare Biosciences, Pittsburgh, PA) ion exchange column. The column was previously

equilibrated with 3 times the volume of column of TEM and after loaded the sample was washed with

tree times the column volume with the same solution and eluted using a flow rate of 1.5 mL/min,

fraction volume of 10.0 mL in a linear gradient 0 to 250 mM of potassium chloride in TEM using an

ÄKTA™ prime automated liquid chromatography system (Amersham Pharmacia Biotech Inc.,

Piscataway, NJ). Calpastatin activity was determined in each eluted fraction as described previously

and active fractions were pooled and dialyzed against 40 times the volume of pooled fractions on TEM

during 12 hours at 4 oC. Calpastatin was eluted between 100 to 1500 mM of potassium cloride. Protein

concentration were determined after all dialyze steps to determine the fold of purification.

SDS-PAGE and immunoblotting

69

For SDS-PAGE and immunoblotting was used samples taken from end of purification. One mL

of each purified calpastatin peak was mixed with 0.5 mL of SDS sample buffer (30 mM Tris-HC1, 3

mM EDTA, 3% [w/v] SDS, 30% [vol/vol] glycerol, and 30 pg of pyronin Y /mL, pH 8.0) (Wang,

1982), 0.1 mL MCE and stored at -80 oC until analyse.

An 12.5% polyacrylamide separating gel (acrylamide:N,N′-bis-methylene acrylamide = 100:1

[w/w], 0.1% [w/v] SDS, 0.05% [v/v] TEMED, 0.05% [w/v] ammonium persulfate, and 0.5 M Tris–

HCl, pH 8.8). A 5% polyacrylamide gel (acrylamide:N,N′-bis-ethylene acrylamide = 100:1 [w/w],

0.1% [w/v] SDS, 0.125% [v/v] TEMED, 0.075% [w/v] ammonium persulfate, and 0.125 MTris–HCl,

pH 6.8) was used for the stacking gel. Gels (10 cm wide ×8 cm tall ×1.5 cm thick) were loaded with

10 μg protein per lane and run at a constant 20 V in SE 260 Hoefer Mighty Small II (Hoefer, Inc.,

Holliston, MA) electrophoresis units overnight.

Transfer of protein from SDS-PAGE gels to a PVDF membrane was performed as described by

Melody et al. (2004). Membranes were blocked for 1 h at room temperature in PBS with 0.1% Tween-

20 and 5% nonfat dry milk and were then incubated in primary antibody over-night at 4 °C. Primary

antibody dilution was for calpastatin, 1:5000 dilution (MA3-945, Thermo Scientific, Rockford, IL).

Membrane was washed 3 times in PBS–Tween for 10 min each at room temperature. The membrane

was then incubated in a goat anti-mouse horseradish peroxidase (No 2554, Sigma, St. Louis, MO)

secondary antibody at 1:10,000 dilution for 1 h at room temperature. After 3 additional 10 min washes

in PBS–Tween, blots were developed using SuperSignal West Femto Maximum Sensitivity Substrate

(ThermoScientific, Rockford, IL) and imaged using a ChemiImager 5500 (Alpha Innotech, San

Leandro, CA) and Alpha Ease FC software (v 3.03 Alpha Innotech).

Two-Dimensional Difference in Gel Electrophoresis.

To determine a protein profile of calpastatin peaks was used two dimensional DIGE technique

described by Anderson et al. (2012) with some modifications.

70

Preparative gels were loaded with 80μg of protein from either a peak. To first dimension proteins

are separated on the basis of isoelectric point (pI) was carried out on Immobiline DryStrips (13 cm,

pH 4–7, GE Healthcare, Piscataway, NJ) rehydrated with DeStreak Rehydration Solution (GE

Healthcare, Piscataway, NJ) containing 2.5 mM DL-dithiothreitol (DTT). Samples are dispersed in a

tray, which soaked immobilized pH gradient strip, placed on top of rehydration mix and was left to

rehydrate overnight at room temperature in a humidifier chamber. Isoelectric focusing was performed

on an Ettan IPGphor isoelectric focusing system (GE Healthcare) for a total of 14,500 V h.

After isoelectric focusing, strips were equilibrated using two sequential 15 min washes with

equilibration buffer (50 mMTris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS and a trace of

Bromophenol Blue) containing 65 mM DTT first and after in a 135 mM iodoacetamida (Rozanas &

Loyland, 2008).

The equilibrated strips were loaded onto 12.5% SDS-PAGE gels(acrylamide: N,N′-bis-

methylene acrylamide=100:1 [wt/wt], 0.1% SDS [wt/vol], 0.05%N,N,N′N-

tetramethylethylenediamine(TEMED), 0.05%ammonium persulfate [wt/vol], and 0.5 M Tris–HCl, pH

8.8), using agarose as an overlay and run over-night at 80 V at 4 oC on Ettan DALT SIX system (GE

Healthcare).

After second dimension electrophoresis, preparative gels were stained with Colloidal Coomassie

Blue solution (1.7% ammoniumsulfate [wt/vol], 30%methanol [vol/vol], 3% phosphoric acid [vol/vol],

and 0.1% Coomassie G-250 [wt/vol]). To destain was used distilled and deionized water. All buffers

and water used in this process was filtered using Stericup Filter Unit, poresize 0.22 μm (Millipore

Corp., Billireca, MA) to minimize potential contamination.

To identify the proteins showed in each gel the selected spot in the gel was excised and sent to

the Iowa State University Protein Facility for identification. It was performed the in-gel digestion (via

trypsin) using Genomics Solution ProGest (Chelmsford, MA). To dissolve the peptides were used

alpha-cyano-4-hydroxycinnamic acid (5 mg/mL in 50% CH3CN/0.1% Trifluoroacetic acid) and

deposited to a matrix-assisted laser desorption/ionization (MALDI) plate. Matrix-assisted laser

71

desorption/ionization mass spectrometry was performed using a QSTAR XL Quadrupole time-of-

flight mass spectrometer equipped with an orthogonal MALDI ion source (AB/MDS Sciex, Toronto,

Canada). Spectra were processed by MASCOT data-base search version 2.2.07 (MatrixScience,

London, UK).

RESULTS AND DISCUSSION

In the protocol for extraction of calpastatin from muscle using anion exchange chromatography,

calpastatin is eluted in two distinct peaks (Figure 1) and this pattern was reported before in (Pontremoli

et al., 1992; Salamino et al., 1994; Geesink et al., 1998; Averna et al., 2001; Samanta et al., 2010;

Cruzen et al., 2013), even though there are no consensus about the origin of those peaks.

Phosphorylation of calpastatin molecule by protein kinase C was demonstrated in vitro (Pontremoli et

al., 1992; Averna et al. 1999; Averna et al., 2001) that could change the ionic charge of the molecule

and modify the affinity to the column and could be involved in the regulation of . Other possibility

could be an alternative splicing of the gene (Geesink et al 1998; Gaarder et al., 2011). Degradation of

calpastatin molecule is another possibility to this separation in two peaks and the possible reason for

losing activity during the purification process (Geesink et al., 1998). The range of elution this first

calpastatin peak, shows that this peak has less affinity to the column compared to the second peak,

therefore it shows difference in ionic charge between them.

To determine the composition of each peak was proceeded the purification of each calpastatin

peak and the result are presented in Table 1. The process to purify the calpastatin peak 1 goes until

Phenil sepharose anion exchange column and among Q sepharose and Phenil sepharose calpastatin

peak 1 lose half activity but the activity per mg of protein is greatly increased. This fact was attributed

to a possible degradation during the purification process (Geesink et al,. 1998). Without heat treatment,

72

some protease is elute with calpastatin and this could cleave the molecule to small peptides tha could

not bind to column and loose activity (Geesink et al., 1998).

For calpastatin peak 2, the purification process was extended, passing through Bluesepharose

Cibacron Blue 3G Affinity column and DEAE CAPTO anion exchange column (Thompson et al.,

2000). Specific activity was increased 139.8 fold from the separation step and at the end of this process

remains only 36% of the initial total activity of this peak. Result of a peak eluted in the similar range

of KCl was found using similar anion exchange column, even though was used heat treatment before

load into the column and the specific activity was 952 units per mg of tissue (Geesink et al., 1998).

In purification of calpastatin from bovine heart Melgren et al. (1983) obtained a specific activity of

4340 units per mg of protein and 13.9% of recovery from the first column.

Same calpastatin separation in two peaks from rat skeletal muscle was described and was

reported and verified the having different specificities for each of the m and µ-calpain (Pontremoli

et al, 1991). They found that both forms of calpastatin are influenced by postranslation modification.

In another study involving phosphorilation of calpastatin peaks shows an interchangeable efficiency

against both calpains. Calpastatin peak 1 was verified more effective against µ-calpain and peak 2

more effective against m-calpain, after phosphorilation peak 1 turn more effective against m-calpain

and a dephosphorilation of peak 2 turn this peak more effective against µ-calpain (Pontremoli et al.,

1992). This findings and an increase in calpastatin degradation by proteases, suggesting the

existence of a different regulatory mechanism for calpastatin forms (Averna et al., 1999).

The protein profile in SDS-PAGE gel of the pooled fractions of calpastatin of Q sepharose

column and at the end of purification are presented in Figure 2 and shows that only weak bands are

stained. This result agree with specific activity and the fold of each peak purification and the bands

stained by calpastatin antibody (Figure 3). The results shows bands of approximately 70 kDa and the

peak 2 was a little higher than peak. Those molecular weight are similar to a fragment of intact

calpastatin degraded by endogenous proteases (Doumit et al., 1999).

73

The intact calpastatin molecule are present in range of 125 to 145 kDa in SDS PAGE. Estimated

molecular weight based on amino acid sequence of skeletal calpastatin is between 77 to 80 kDa. In

SDS-PAGE calpastatin migrates anomalously, and it is difficult to relate a band migrating at a

particular molecular weight in SDS-PAGE to a known calpastatin isoform, this anomalously slow

migration of calpastatin in SDS-PAGE is a property of the calpastatin polypeptide itself and probably

not due to posttranslational modifications (Maki et al., 1988; Goll et al. 2003).

Intact calpastatin molecule has no found in this study, and a possible reason is because the

calpastatin molecule is labile to degradation by endogenous proteases producing peptides that could

remain inhibitory activity (Emori et al., 1988; Goll et al.; 2003). This peptides coud not bind to the

columns during the process being eluted in early fractions or still bounded to column substrate, and

this is a possible reason to found that single band in this study.

The 70kDa molecular weight is similar to a calpastatin constructed in a non-fusing porcine

skeletal muscle using 1xa promoter and the authors attribute this band to a large proteolitic fragment

product of degradation of intact calpastatin containing the N-terminal epitope intact allowing the anti-

1xa peptide antibody to detect this large peptide fragment (Parr et al., 2004).

The calpastatin was identified in the second dimension gel in a similar molecular weight to

western blot (Figure 4). In the figure 4-A shows all 6 spots from calpastatin peak 1 and figure 4-B five

spots sent to identification and the identified spots are the spot number 2, 7 and 8. The peptide

sequences are presented in Table 2.

Sequence of peptides identified as part of the inhibitory domain III and IV and C terminus and

from purified peak 2 a sequence of peptides identified as part of the inhibitory domain I, II and III

(Figure 5). This results lead us to believe that both peaks, in this case, are products of degradation of

the intact molecule and probably the small peptides are loosed during the process. This result help us

to identify the composition of the peaks during the calpastatin extraction but is not conclusive about

the composition, the origin of peaks and the influence in the tenderness development.

74

CONCLUSIONS

The results of present study shows that is possible the purification of distinct forms of active

calpastatin separated using anion exchange column and purifying by sequential chromatography steps,

however the intact form of calpastatin was not present in this purification. Presence of peptides was

not conclusive to determine the origin and composition of each active peak. More studies are necessary

to improve the characterization and the influence in the tenderization process.

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Tables and Figures

10 Figure 1 Calpastatin activity of eluted fractions (Arbitrary units)

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5Table 1 – Steps of purification of calpastatin peaks.

Column Calpastatin peak 1

Activity/mL Activity/mg protein Total Activity Lose activity (%)

Q sepharose 1.48 0.69 344.84 -

Phenilsepharose 0.99 164.50 171.738 50.2%

Column Calpastatin peak 2

Activity/mL Activity/mg protein Total Activity Lose activity (%)

Q sepharose 3.89 5.29 1981.86 -

Phenilsepharose 8.84 368.33 1343.68 32.2%

Bluesepharose 6.49 721.11 1298 34.5%

DEAE CAPTO* 34 739.13 714 64.0%

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11 Figure 2 – Load check silver stained of initial and final step of calpastatin purification. Lane M)

Broad range molecular marker; 1) Pooled fractions of peak 1 activity of calpastatin from Q sepharose

column; 2) Pooled fractions of peak 2 activity of calpastatin from Q sepharose column; 3) Pooled

fractions of peak 1 activity of calpastatin from Phenil sepharose column; 4) Pooled fractions of peak

2 activity of calpastatin from DEAE CAPTO column.

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12 Figure 3 – Western blott stained by calpastatin antibody of purified calpastatin peak 1 (PK1) and

calpastatin peak 2 (PK2).

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13 Figure 4 – 2D DIGE of calpastatin peaks coomassie stained. A) Calpastatin peak 1; B) calpastatin

peak 2. Numbers of collected spots and sent to identification are presented in boxes.

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6 Table 2 - Identified spots from calpastatin peaks gels.

Spot

ID Protein Specie Gene Acession pI

Mass

kDa Coverage Identified Peptides

2 Calpastatin Sus

scrofa CAST P12675 5.33 77.1 9.68 KPEAAQDPIDALSGDFDR

KLDDALDQLSDSLGQR LDDALDQLSDSLGQR DDTIPPEYR QPDPDENKPIEDK LGEKEETIPPDYR

7 Calpastatin Sus

scrofa CAST P12675 5.33 77.1 8.7 STGEVLK

SLTSSVPAESK SEPELDLSSIK ESQATAPTPVGEAVSR LSVTGVSAASGKPAETK

8 Calpastatin Sus

scrofa CAST P12675 5.33 77.1 8.84 ESQATAPTPVGEAVSR

LSVTGVSAASGKPAETK KSEPELDLSSIK SLTSSVPAESK

STGEVLK

82

MNPTETKAIPVSKQLEGPHSPNKKRHKKQAVKTEPEKKSQSTKPSVVHEKKTQEVKP

KEHPEPKSLPTHSADAGSKRAHKEKAVSRSNEQPTSEKSTKPKAKPQDP TPSDGKLS

VTGVSAASGKPAETKKDDKSLTSSVPAESKSSKPSGKSDMDAALDDLIDTLGGPEE

TEEDNTTYTGPEVLDPMSSTYIEELGKREVTLPPKYRELLDKKEGIPVPPPD TSKPLGP

DDA IDALSLDLTCSSPTADGKKTEKEKSTGEVLKAQSVGVIKSAAAPPHEKKRRVEE

DTMSDQALEALSASLGSRKSEPELDLSSIKEIDEAKAKEEKLKKCGEDDETVPPEYRL

KPAMDKDGKPLLPEAEEKPKPLSESEL IDELSEDFDQSKRKEKQSKPTEKTKESQAT

APTPVGEAVSRTSLCCVQSAPPKPATGMVPDDAVEALAGSLGKKEADPEDGKPVED

KVKEKAKEEDREKLGEKEETIPPDYRLEEVKDKDGKTLPHKDPKEPVLPLSEDFV L

DALSQDFAGPPAASSLFEDAKLSAAVSEVVSQTSAPTTHSAGPPPDTVSDDKKLDDA

LDQLSDSLGQRQPDPDENKPIEDKVKEKAEAEHRDKLGERDDTIPPEYRHLLDKD

EEGKSTKPPTKKPEAPKKPEAAQDPIDALSGDFDRCPSTTETSENTTKDKDKKTASK

SKAPKNGGKAKDSTKAKEETSKQKSDGKSTS

14 Figure 5 – Representative calpastatin molecule aminoacid sequence from Sus scrofa gene: CAST;

713 Aminoacids; Mass (Da):77,124. Inhibitory domains are presented in closed boxes and peptides

identified are in uppercase and in yellow are from peak 1 and from peak 2 as identified by grey.

83

CAPITULO 4 – Análise proteômica do músculo “Longíssimus dorsi” de bovinos da raça nelore

mocho de diferentes grupos de maciez.

Autores: OLIVEIRA, Leonardo G.; FERREIRA, Reginaldo N.; MAGNABOSCO, Claudio U.;

BORGES Clayton L.; PERON, Hugo J.M.C.; MOREIRA André; PÁDUA João T.

RESUMO – Foi avaliado o perfil protéico de animais de extremos valores de força de cisalhamento

com sete dias de maturação da carne, extremo baixo (Macio) e extremo alto (Duro), da raça nelore

mocho de uma população segregante para a maciez da carne. Proteínas relacionadas com a defesa

celular com função anti apoptótica foram encontradas nos dois grupos de animais. Apenas identificada

no grupo D, a proteína citocromo c indica indução do processo apoptótico. Proteínas estruturais foram

identificadas no grupo M, indicando uma possível maior proteólise devido fato destas proteínas quando

intactas não estarem solúveis no tampão de extração utilizado. Maior abundância relativa de proteínas

do processo glicolítico foi evidenciado no grupo M e proteínas do metabolismo oxidativo foram

evidenciadas mais expressas no grupo D, fato que provavelmente está correlacionado à maciez final

da carne. A calpastatina foi somente identificada no grupo D, esta proteína está relacionada com a

maciez final da carne por ser inibidora natural das calpaínas. A maciez da carne está relacionada a

expressão de algumas proteínas ligadas ao estresse, ao tipo de metabolismo energético e a Calpastatina.

Palavras chave: Bos indicus; Força de cisalhamento; LC-MS; Metabolismo; Seleção genética.

ABSTRACT – Proteomic analysis was used to evaluate the protein profile of extreme WBSF values

of 7 days aged meat animals, extreme low values (Tender) and extreme High (Tough), of Nellore owl

breed from a segregating population of meat tenderness. Cell defense proteins related with apoptotic

process were founded in both groups. Only identified in Tough group, the protein citochrome c

indicates induction of apoptotic process. Structural proteins were identified in tender group indicating

possibly more proteolysis due of these intact proteins are not soluble in this used buffer. Higher protein

relative abundance of proteins related to glycolytic metabolic process as evidenced on tender group

and proteins from oxidative process are more expressed in tough group, this fact is probably are

correlated to final meat tenderness. Calpastatin was only founded present in tough group, and are

related to final tenderness due to be a natural inhibitor of calpains. Final tenderness are related to the

expression of some stress proteins, energy metabolism type and to the calpastatin.

Keywords: Bos Indicus; Genetic selection; LC-MS; Metabolism; Shear Force.

INTRODUÇÃO

Dentre os bovinos destinados à produção de carne no Brasil, a grande maioria é composta por

animais da sub espécie Bos indicus, principalmente da raça Nelore, tendo como característica

inportante a rusticidade comparados a animais Bos taurus, porém com carne com menor maciez (1).

A maciez da carne é uma das características organolépticas de grande importância e está

relacionada diretamente à satisfação do consumidor (2,3) e é influenciada por vários fatores ante

mortem e post mortem como a atividade de enzimas proteolíticas presentes no músculo,

disponibilidade de energia pós mortem e velocidade de resfriamento da carcaça (4,5).

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Diferenças consideráveis na maciez da carne podem ser explicadas pela herança genética e

segundo (6) um programa de melhoramento genético com seleção para maciez é uma alternativa

promissora para a produção de carne zebuína naturalmente macia. Trabalhos envolvendo o

melhoramento genético de bovinos vêm sendo desenvolvidos visando selecionar indivíduos por suas

características de interesse (7,8).

A expressão protéica nas células musculares destes animais pode apresentar diferenças devido

às características herdadas dos seus genitores, influenciando todo o metabolismo celular e expressão

das características fenotípicas. Para a investigação destes mecanismos metabólicos a análise

proteômica se apresenta como uma ferramenta útil com grande aplicabilidade (9–11).

Este trabalho tem como propósito a caracterização das diferenças de expressão protéica do

músculo Longissimus dorsi entre animais da raça Nelore Mocho de uma população segregante para

maciez da carne.

MATERIAL E MÉTODOS

Coleta e preparo das amostras

Um grupo de 83 animais contemporâneos, de uma mesma propriedade, oriundos de um programa

de melhoramento genético da EMPRAPA denominado “Macro Programa 2”, fruto de acasalamentos

com o propósito de selecionar animais com característica de carne macia, foram terminados em sistema

de confinamento e abatidos quando atingiram peso médio de 510 kg de peso vivo. Estes animais foram

abatidos em frigorífico inspecionado pelo sistema de inspeção federal (SIF) e sob condições

humanitárias de abate. Uma amostra do músculo Longíssimus dorsi de cada animal foi coletada 30

minutos após a exanguinação, entre a 12ª e 13ª costela, na meia carcaça direita de 1,5cm de espessura

abrangendo toda seção do músculo.

O tecido adiposo e conjuntivo visível de cada amostra foi retirado para após ser cortada em cubos

de aproximadamente 1cm3, imergidas em nitrogênio líquido para congelamento e transporte até o

Laboratório de Fisiologia da Digestão do Instituto de Ciências Biológicas II da Universidade Federal

de Goiás. As amostras foram processadas em triturador com mini container (Waring® SN-04241-11)

durante 3 minutos e armazenadas em freezer a -80ºC para posteriores análises.

Outra amostra com 2,54 cm de espessura foi retirada da meia carcaça direita dos respectivos

animais 24 horas após o abate seguindo o mesmo protocolo. Estas amostras foram embaladas à vácuo

e mantidas sob refrigeração durante 6 dias para a determinação da força de cisalhamento pelo método

Warner-Bratzler Shear Force (WBSF) (12) no dia 7 post mortem.

85

Os animais foram ranqueados de acordo com os resultados da força de cisalhamento (WBSF) e

foram selecionados para as analises os 10 animais dos extremos de alto WBSF (Grupo D) e baixo

WBSF (Grupo M).

Extração proteica

A extração proteica e análise proteômica foram realizadas no laboratório de Biologia Molecular

“José Salum” – Instituto de Ciências Biológicas – Universidade Federal de Goiás. A fração solúvel

das proteínas musculares foi extraída conforme (13). Cada amostra dos grupos baixo WBSF (Macio)

e alto WBSF (Duro) foi solubilizadas em três vezes o volume de solução tampão fria de extração

composto por 50mM de Tris, 1mM de EDTA com o pH ajustado para 8,5 com solução de HCl 6N em

tubo, em equipamento BeadBeater (BioSpec, Bartlesville, USA) em tubos contendo 1/3 do volume da

amostra em esferas de vidro ácido lavadas de 200–500 μm (Sigma Aldrich). Após a solubilização a

amostra foi centrifugada por 20 minutos a uma temperatura de 4ºC a 40.000 x g. As proteínas

solubilizadas na porção sobrenadante foram transferidas para outro tubo e quantificada a concentração

de proteínas pelo método de Bradford et al. (14) em triplicata, utilizando curva padrão previamente

construída com soroalbumina bovina. Três alíquotas foram feitas e armazenadas em freezer -80ºC.

Após a quantificação, foi procedida a eletroforese unidimensional em gel de poliacrilamida para

verificação do perfil de bandas para confirmação da quantificação protéica. Para preparação de cada

amostra, 20 µg de proteína foi colocado em tubo e 15 µL de tampão de amostra (TRIS/HCl pH 6,8

100mM, 4,0% de dodecil sulfato de sódio, 0,2% de azul de bromofenol; 20,0% de glicerol) e aquecido

a 100ºC por 10 minutos. As amostras preparadas foram aplicadas em gel eletroforético de 12,5% de

poliacrilamida com medidas de 10 x 10 cm (N,N’-bis-metileno acrilamida,1% de dodecil sulfato de

sódio, 0,05% de N,N,N’-N tetrametiletiletilenodiamina (TEMED), 0,01% de persulfato de amônia;

0,5M de Tris com pH ajustado para 8.8 com uma solução de HCl) e corridas com voltagem constante

de 120V. Após a corrida eletroforética os géis foram corados pela imersão durante 12 horas em solução

azul brilhante de coomassie (1,7% de sulfato de amônio; 30% de metanol; 3% de ácido fosfórico; 0,1%

de Coomassie G-250). Os géis foram fotografados e estão apresentados no Anexo 1.

Análise proteômica

Uma alíquota de 200µg de proteína de cada animal do grupo D foi utilizada para compor uma

amostra representativa do grupo e feito o mesmo procedimento com as amostras do grupo M para

compor uma amostra representativa do grupo. A quantidade relativa a 200µg de proteínas foi retirada

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de cada amostra representativa de cada grupo, lavadas com quatro volumes de água ultrapura

utilizando filtros Amicon ultra 0,5mL (Merck Milipore, Darmstadt, Alemanha) de 3000 Da, em

seguida com três volumes de solução aquosa de formiato de amônio 20mM. Após as amostras foram

preparadas para a digestão de acordo com protocolo proposto por Borges et al. (11).

Para a digestão das amostras foram adicionados 2µg de Tripsina (Promega, Madison, WI, USA)

diluída em solução de Bicarbonato de amônio (50mM de NH4HCO3) homogeneizada e incubada por

16 horas à 37ºC. Terminada a digestão, foram adicionados 40µL da solução aquosa de Ácido

Trifluoroacético a 5%, homogeneizado e incubado a 37ºC por 90 minutos para a digestão do

RapiGEST e após centrifugadas a 14000RPM a 4ºC por 30 minutos. O sobrenadante foi transferido

para novo tubo e desidratado à temperatura ambiente em centrífuga Speed Vacuum (Eppendorf,

Hamburg, Alemanha) a 3000PRM durante 5 horas. As amostras foram resuspendidas com 120µL de

solução aquosa de bicarbonato de amônio (50mM) e transferidas para frasco ultra limpo (Waters®,

preslit PTFE/silicone caps). Para o ajuste do pH, 5µL da solução aquosa de formiato de amônio e em

seguida 120 fempto Mol de enolase (Waters, Milford, MA, USA) como padrão interno. Em seguida a

separação dos peptídeos digeridos foi procedida utilizando equipamento LC-MS 2D nano ACQUITY

system (Waters, USA) equipado com duas colunas de fase reversa trabalhando em condição ácida e

básica. A primeira coluna foi nanoEase BEH130 C18 (1.7 μm, 100 μm x 100 mm; Waters, USA) e a

segunda coluna NanoAcquity UPLC BEH 130 C18 (1.7 μm, 100 μm × 100 mm; Waters, USA). Para

a espectrometria de massas foi utilizado o Synapt G1 MS (Waters, USA) equipado com uma fonte de

nanoeletrospray e dois analisadores de massas: um quadrupolo e um time-of-flight (TOF) operando

em modo TOF-V. Os dados foram obtidos utilizando o equipamento em modo MSE em alternância de

baixa energia (6 V) e alta energia (20-40 V) com modos de aquisição a cada 0,4 segundos.

Os dados obtidos usando o protocolo em modo MSE foram processados utilizando-se software

ProteinLynx Global Server (PLGS) versão 2.4 (Waters, USA). Os dados foram submetidos a uma

análise para a retirada de interferências, deisotopização e deconvolução de carga. O resultado foi

comparado com banco de dados mundial de sequência de proteínas de Bos taurus UniProt (Universal

Protein Source, http://www.uniprot.org/proteomes/UP000009136) para a identificação proteica.

Modificações como oxidação da metionina e serina e fosforilação da treonina e tirosina foram

consideradas.

A comparação da abundância relativa das proteínas foi feita baseada na intensidade média da

proteína padrão interno (enolase fúngica) e foi usada para converter a intensidade média dos peptídeos

analisados para a quantificação absoluta da amostra injetada no equipamento. O software ExpressionE

informatics v.2.5.2 foi utilizado para a comparação quantitativa. As proteínas identificadas foram

organizadas pela expressão das proteínas do grupo M em relação ao grupo D e selecionadas as

http://www.uniprot.org/proteomes/UP000009136

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proteínas induzidas ou suprimidas no mínimo de 50%. O modelo matemático utilizado para calcular

as relações entre os grupos é parte do software PLGS (Wathers corporation, USA).

Determinação da atividade da enzima superóxido dismutase

Foi determinada a atividade da enzima superoxido dismutease nas amostras dos quatro animais

dos extremos de cada grupo. As proteínas foram extraídas, dosada concentração protéica de cada

amostra extraída e uma alíquota correspondente a 200 fempto Mol foi utilizada para a dosagem da

atividade enzimática utilizado o Kit comercial SOD Assay Kit-WST (Sigma-Aldrich, St. Louis, MO,

USA), seguindo o protocolo descrito pelo fabricante.

Análise estatística

As médias dos valores de WBSF e de atividade da enzima Superóxido dismutase dos animais

selecionados foram submetidas a analise de variância e (P<0,05) com o auxílio do pacote “easyanova”

do software R (R Core Team, 2013).

A comparação da abundância relativa das proteínas foi feita baseada na intensidade média da

proteína padrão interno (enolase) e foi usado para converter a intensidade média dos peptídeos

analisados para a quantificação absoluta da amostra injetada no equipamento. O software ExpressionE

informatics v.2.5.2 foi utilizado para a comparação quantitativa. As proteínas identificadas foram

organizadas pela expressão das proteínas do grupo M em relação ao grupo D e selecionadas as

proteínas induzidas ou suprimidas no mínimo de 50%. O modelo matemático utilizado para calcular

as relações entre os grupos é parte do software PLGS (Wathers corporation, USA).

A força de cisalhamento é a força necessária para cisalhar uma secção transversal de carne, em

kg/cm2, e é uma medida direta na avaliação da maciez da carne (15).

RESULTADOS E DISCUSSÃO

Houve diferença entre as médias de força de cisalhamento entre os grupos de animais (Tabela

1).

7 Tabela 1 – Médias de força de cisalhamento transversal (WBSF) do músculo Longíssimos dorsi

dos animais selecionados para compor o grupo macio (M) e grupo duro (D).

Grupos EPM# P valor*

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Grupo D Grupo M

WBSF* (Kgf/cm2)

5,75 1,24 0,1254 <0,001

#Erro padrão da média

*Valor de probabilidade do teste F da análise de variância.

De acordo com a média de WBSF, a carne dos animais do grupo M pode ser considerada macia

de acordo com Shackelford et al.(16), que definiram valores de WBSF abaixo de 4,5 Kgf.(cm2)-1 como

carne macia. A maciez da carne vem sendo desenvolvido em animais da raça Nelore mocho e visa

selecionar indivíduos que possuam superioridade genética para desempenho e carcaça. Este trabalho

de seleção genética é importante, pois a maciez da carne é um importante atributo organoléptico (2,3)

e de acordo com Castro et al. (8) esta seleção não afeta as características de deposição de gordura

subcutânea, muscularidade e ganho de peso diário médio.

As proteínas são fruto da expressão dos genes do indivíduo e a diferença na produção das

proteínas contribui para a diferença entre os indivíduos. As proteínas são responsáveis por diversas

funções na célula muscular e podem estar solúveis no citoplasma da célula ou das organelas ou

insolúveis, por exemplo, na estrutura celular ou membranas. Proteínas sarcoplasmáticas solúveis

ligadas à defesa celular foram identificadas diferentemente expressas entre os grupos de animais

(Tabela 2).

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8 Tabela 2 – Proteínas relacionadas à defesa celular, diferentemente expressas entre os grupos macio (M) e duro (D).

Descrição Acesso# Score Relação

Macio:Duro* Função

14 3 3 protein epsilon 1433E_BOVIN 4312,4 1,82 Anti apoptose

14 3 3 protein gamma 1433G_BOVIN 3658,7 2,08 Anti apoptose

Mth938 domain containing protein AAMDC_BOVIN 4329,7 0,54 Anti-apoptose

Glutathione S transferase P GSTP1_BOVIN 24269,9 0,502 Detoxificação de radicais de oxigênio

Peroxiredoxin 1 PRDX1_BOVIN 20102,8 0,583 Detoxificação de radicais de oxigênio


Thioredoxin dependent peroxide reductase

mitochondrial PRDX3_BOVIN 1934,2 >100 Detoxificação de radicais de oxigênio


Superoxide dismutase Cu Zn SODC_BOVIN 5107,9 <0,01 Detoxificação de radicais de oxigênio

S formylglutathione hydrolase ESTD_BOVIN 1914,5 0,502 Detoxificação por modificação

Aldo keto reductase family 1 member B1 Q5E962_BOVIN 3095,3 >100 Metabolismo de açucar, glicosídeo,

poliol e carboxilato

Alpha crystallin B chain CRYAB_BOVIN 12623,7 2,203 Receptor transmembrana e sinalizador da

via tirosina quinase

Tripartite motif containing 72 E1BE77_BOVIN 1316,0 >100 Reorganização do citoesqueleto

dependente do ciclo celular

LIM domain binding 3 F1MRX5_BOVIN 1442,6 <0,01 Reorganização do citoesqueleto

dependente do ciclo celular

Protein CutA F1MTI7_BOVIN 2773,3 <0,01 Resposta a estimulos externos

Heat shock 70kDa protein 5 (glucose-regulated

protein, 78kDa) F1N614_BOVIN 1138,0 <0,01 Resposta ao choque térmico

Zeta crystallin QOR_BOVIN 3608,2 0,492 Resposta ao choque térmico

Heat shock protein beta 6 HSPB6_BOVIN 18413,1 2,160 Resposta ao choque térmico

Heat shock protein beta 1 G3X7S2_BOVIN 27819,4 3,065 Resposta ao choque térmico

Heat shock protein HSP 90 beta HS90B_BOVIN 484,5 >100 Resposta ao choque térmico

Heat shock protein 75 kDa mitochondrial TRAP1_BOVIN 436,6 >100 Resposta ao choque térmico

90

Protein DJ 1 PARK7_BOVIN 47840,0 0,482 Resposta ao estresse oxidativo

Aldehyde dehydrogenase 1 family, member L2 E1BDG9_BOVIN 164,7 <0,01 Transferidor de C-1 tetrahidrofolato-

dependente

Aldehyde dehydrogenase E1BMG9_BOVIN 164,7 <0,01 Transferidor de grupos C-1 ativados

Serotransferrin G3X6N3_BOVIN 3555,5 >100 Transporte de íons metálicos (Cu, Fe, etc)

Cytochrome c CYC_BOVIN 3343,2 0,538 Transporte mitocondrial

# Número de acesso no banco de dados UNIPROT.

* Razão entre a abundância relativa da proteína no grupo M pelo grupo D.

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A Heat shock 70kDa protein 5 (HSP-5 70) foi identificada inibida em animais do grupo M e

a Zeta cristalin mais expressa nos animais do grupo D. As Heat shock protein 75 kDa mitochondrial

e Heat shock protein 90 beta foram identificadas somente nos animais do grupo M e as Heat shock

protein beta 1 e beta 6 e αβ-cristalin foram identificadas como mais expressas no grupo dos animais

Macio.

As proteínas de choque térmico (HSPs) fazem parte de uma família de proteínas de uma cadeia

interativa de chaperonas, constitutivamente expressas, tem rápida expressão sob condições de estresse

com uma das principais funções a prevenção contra danos a célula (17). A família das HSP de 70

kDa está ligadas a proteção celular contra a apoptose promovendo a estruturação espacial correta das

proteínas (folding) e prevenindo a translocação de proteínas pró apoptóticas por se ligarem a elas

(17–19). A apoptose foi proposta recentemente como o primeiro estágio do processo de maturação

da carne (20), contribuindo para o processo de amaciamento, favorecendo a ativação das caspases no

processo apoptótico (18).

Outra forma de evitar danos à célula é evitando a formação de espécies reativas de oxigênio

(ERO). Animais do grupo D apresentaram as proteínas Superoxide dismutase Cu Zn (SOD CuZn),

Aldehyde dehydrogenase 1 (ADH1) e Peroxiredoxin 2 (PRX 2) identificada reprimida no grupo M.

Apesar de estar reprimida em animais do grupo M a atividade da SOD CuZn não apresentou diferença

entre os grupos (Figura 1). A SOD é regulada por uma chaperona específica de Cobre para a sua

ativação que introduz íons Cobre e pontes dissulfeto na sua molécula para torná-la ativa (21).

A PRX 2 foi relacionada com a característica maciez com 33% de repressão no dia 7 em relação

ao dia 0 (22). A ADH1 também é um potencial para maciez, atua em processo citoprotetor e

eliminando a conversão de aldeídos potencialmente citotóxicos gerados pela peroxidação lipídica e

atuando também no processo glicolítico convertendo o gliceraldeído a 2-fosfoglicerato (23).

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15 Figura 1 – Atividade da enzima SOD (% inibição da formação do íon super óxido) entre os

grupos baixo WBSF (Macio) e alto WBSF (Duro)

A proteína DJ1 foi identificada reprimida nos animais do grupo M e em conjunto com a proteína

Mth938 domain containing e a PRX2 também está envolvida na defesa celular contra a formação de

ERO e foram relacionadas negativamente com a maciez da carne durante juntamente com a SOD no

processo de maturação (24). Os autores atribuíram os efeitos negativos à prevenção contra a apoptose

e a não ativação das caspases no período post mortem, sendo estas proteases relatadas como

importantes no processo de amaciamento da carne. A proteína DJ1 foi também encontrada menos

expressa em animais com característica de carne mais macia provavelmente envolvida em algum

processo que irá determinar a maciez da carne (25).

A proteína Cytochrome c foi encontrada como reprimida nos animais do grupo M, sendo

possivelmente uma evidencia de maior ativação do processo apoptótico em animais do grupo D. Uma

vez liberada no citoplasma, a proteína cytochrome c induz a formação do apoptossoma ativando o

sistema das caspases, não corroborando com os achados descritos por Picard et al. (24).

Identificada induzida nos animais do grupo M, a proteína Heat shock protein 75 kDa

mitochondrial está relacionada com a regulação negativa da apoptose inibindo a formação de ERO,

a modulação da respiração celular, quando há falta de glicose disponível para evitar a formação de

ERO (26). Esta proteína também foi apontada como possível marcador para maciez em estudo

envolvendo o músculo Semitendinosus corroborando com o achado deste presente estudo (19).

A HSP 90 está envolvida em várias funções celulares e juntamente com a HSP 75 mitochondrial

(TRAP1) desempenha função anti-apoptótica. Quando associada à óxivo nitroso sintase (ONS), a

HSP 90 diminui a atividade proteólitica da µ-calpaína em 80% (27). A formação de uma tríade

protéica na presença do íon Ca+ entre a HSP 90 a ONS e a µ-calpaína, afirmando que este sistema

possivelmente constitui uma forma de regulação da µ-calpaína, possivelmente reduzindo a afinidade

da pelo Ca+ atuando assim como inibidora parcial da sua atividade (28).

0

5

10

15

20

25

Ati

vid

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DBaixo WBSF

Alto WBSF

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Foram detectadas induzidas no grupo M a Thioredoxin dependent peroxide reductase

mitochondrial (PRTDm) e a serotransferina, estas proteínas também estão ligadas à redução da

atividade apoptótica pela redução de ERO (29).

A Proteina tripartite motif containing 72 (TMC72) se mostra identificada reprimida no grupo

M, e pode ser uma possível contribuidora para o processo de amaciamento da carne. Está relacionada

com o reparo da membrana celular e função anti apoptotica, se ligando com a fosfatidilserina e

regulando proteínas de membrana cálcio dependente. Outra função importante é a ação negativa sobre

o hormônio fator de crescimento semelhante à insulina, diminuindo a atividade de calpastatina e não

restringindo a proteólise (30).

As proteínas Heat shock protein beta 1 (HSBb1), Heat shock protein beta 6 (HSPb6) e αβ

cristalin (αβCST) foram evidenciadas induzidas no grupo M. Estas proteínas de menor peso

molecular também fazem parte das chaperonas com a função de proteção da estrutura celular (31). A

atividade destas chaperonas está envolvida na remodelagem da estrutura do citoesqueleto, se ligando

a evitando a agregação e dificultando a ação de proteases, estabilizando e reorganizando as proteínas

estruturais, com grande afinidade pela actina (32). Em estudo envolvendo o músculo Semitendinosus

também foi evidenciado a proteína αβCST como um possível candidato a marcador para maciez (19).

Apesar de estarem altamente relacionadas com o processo de amaciamento da carne (33) o seu

papel de proteção só é efetivo quando estão ligados à miofibrilas, não dissolvidos no sarcoplasma.

Após ocorrer o estresse da fibra muscular, estas chaperonas são rapidamente ativadas e se ligam às

proteínas estruturais, ficando agregadas às miofibras (34), corroborando portanto com os resultados

do presente trabalho. (18) encontraram que a supressão dos genes que codificam estas proteínas estão

ligados com a característica de maciez da carne. Os autores afirmam que a supressão destas

chaperonas leva a uma desorganização das proteínas estruturais presentes na linha Z do músculo

facilitando assim a degradação.

O grupo das proteínas 14-3-3 foi identificado induzido em animais do grupo M e este grupo de

proteínas desempenha as funções de inibição da apoptose e inibição da proteína kinase C. A Proteína

kinase C tem a capacidade de fosforilar a calpastatina (CAST) , alterando as propriedades inibitórias

desta, podendo desta forma atuar no processo de proteólise post mortem, regulando a atividade

inibitória da CAST. A fosforilação da CAST favorece a agregação próximo ao núcleo da célula (35–

37).

Identificada reprimida no grupo M a S formilglutationa hidrolase (SFGH) tem função de

detoxificação pela conversão do formaldeído em glutationa e formato auxiliando na proteção celular

evitando danos à estrutura celular, corroborando com o presente estudo, favorecendo a ativação do

processo apoptótico (38).

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Única proteína estrutural reprimida no grupo M, a myosin light chain 1 3 skeletal muscle

isoform (MCL1-3) (Tabela 3). Proteína ligada ao aparato contrátil do músculo esquelético, ligada a

miosina de cadeia pesada e a actina, somente é encontrada na fração solúvel quando é hidrolisada ou

quando alguma proteína em que ela está ligada é hidrolisada e está associada a proteólise post mortem

inicial, na dissolução do rigor mortis (39). A liberação desta proteína pode estar provavelmente

relacionada com a lesão celular pré abate e várias causas podem estar envolvidas como o estresse

ocasionado no momento do abate. Eleita como provável marcador para maciez da carne, foi descrita

por JIA et al. (25) como diferentemente expressa na fração solúvel em um curto período de tempo

post mortem, resultado do início da proteólise.

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9 Tabela 3 - Proteínas relacionadas a estrutura celular diferentemente expressas entre os grupos macio (M) e duro (D).



Cysteine and glycine rich protein 3 CSRP3_BOVIN 4295,65 <0,01 Citoesqueleto / proteína estrutural

Myomesin (M-protein) 2, 165kDa E1BF23_BOVIN 832,84 >100 Citoesqueleto / proteína estrutural

Myosin light chain 1 3 skeletal muscle isoform MYL1_BOVIN 6546,25 0,55 Citoesqueleto / proteína estrutural

Adenylate kinase isoenzyme 5 KAD5_BOVIN 2139,97 >100 Conversão e regeneração de energia

Vinculin F1N789_BOVIN 705,96 >100 Guia da extensão longitudinal celular

Filamin B, beta E1BKX7_BOVIN 27,05 >100 Microtubulo/ citoesqueleto

Tubulin alpha-1C chain-like F1MNF8_BOVIN 130,93 >100 Microtubulo/ citoesqueleto

Tubulin alpha 4A chain TBA4A_BOVIN 684,28 >100 Microtubulo/ citoesqueleto

Kinectin 1 (kinesin receptor) F1MLU7_BOVIN 50,63 <0,01 Transporte tubulina dependente

Filamin A, alpha F1N169_BOVIN 6,69 >100 Transporte vesicular # Número de acesso no banco de dados UNIPROT.


96

Proteínas estruturais Filanin A alpha (αFILA), myomesin 2 (MYOM2), Tubulin alpha 4A chain

(αTUB4A) e 1C (αTUB1A) da αtubulina e βfilanina B (βFILB) foram identificadas induzidas no

grupo M. As proteínas estruturais encontradas solubilizadas logo após o abate, provavelmente são

produtos da proteólise, pelo fato destas proteínas estarem associadas à fração miofibrilar insolúvel.

Estas proteínas são substratos para a µ-calpaína e para as caspases, e esta proteólise precoce pode

estar associada ao turn-over protéico ou ao processo de apoptose (40–42). A proteólise libera

peptídeos de menor peso molecular do que a molécula intacta e esses peptídeos (40,42,43).

Proteólise precoce da MYOM2 foi relatada por Anderson et al. (39) como uma proteína

candidata a indicadora da maciez final da carne em bovinos corroborando com o presente estudo.

Além desta proteína a abundância da miosina de cadeia leve 1 na fração solúvel foi evidenciada sua

rápida solubilização e teve alta correlação com a taxa de proteólise post mortem também sendo uma

proteína de eleição como marcadora molecular de maciez da carne.

A proteína Kinectin 1 (KNT1) está suprimida no grupo M e está envolvida na estruturação da

integrina e fibronectina no músculo estriado esquelético, proteínas estas presentes na estrutura da

linha Z do sarcômero auxiliando no complexo de adesão e estabilização desta proteína (44).

Foi identificada reprimida no grupo M a enzima Alcohol dehydrogenase class 3 (ADH3)

(Tabela 4). Esta enzima tem a função de converter álcoois a respectivos aldeídos e cetonas. O produto

desta conversão poderá ser convertido à acetil-CoA e ser metabolizado no ciclo do ácido cítrico (45).

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10 Tabela 4 - Proteínas relacionadas ao metabolismo de carboidratos, diferentemente expressas entre os grupos macio (M) e duro (D).



Aconitate hydratase mitochondrial ACON_BOVIN 6093,9 0,48 Ciclo do ácido tricarboxilico

Malate dehydrogenase cytoplasmic MDHC_BOVIN 39534,6 0,50 Ciclo do ácido tricarboxilico

Citrate synthase mitochondrial CISY_BOVIN 3519,9 0,52 Ciclo do ácido tricarboxilico

Malate dehydrogenase mitochondrial MDHM_BOVIN 41957,6 0,53 Ciclo do ácido tricarboxilico

Dihydrolipoyllysine residue succinyltransferase

component of 2 oxoglutarate dehydrogenase complex m ODO2_BOVIN 1026,9 <0,01 Ciclo do ácido tricarboxilico

GLO1 protein (Lactoylglutathione lyase) A4FUZ1_BOVIN 7828,3 0,56 Composto-C e metabolismo de carboidratos

O acetyl ADP ribose deacetylase MACROD1 MACD1_BOVIN 1193,6 <0,01 Composto-C e metabolismo de carboidratos

Alcohol dehydrogenase class 3 ADHX_BOVIN 838,7 <0,01 Composto-C e metabolismo de carboidratos

Myoglobin MYG_BOVIN 92859,8 0,56 Distribuição e troca gasosa

Phosphoglycerate kinase 1 PGK1_BOVIN 145747,7 0,57 Glicólise e gliconeogênese

6 phosphofructokinase liver type K6PL_BOVIN 126,1 2,64 Glicólise e gliconeogênese

6 phosphofructokinase muscle type K6PF_BOVIN 1573,7 3,53 Glicólise e gliconeogênese

6 phosphofructokinase E1BCW3_BOVIN 209,7 >100 Glicólise e gliconeogênese

Creatine kinase B type KCRB_BOVIN 1489,2 47,47 Metabolismo da creatina

Glyceraldehyde 3 phosphate dehydrogenase testis

specific G3PT_BOVIN 3685,5 0,43

Metabolismo de açucar, glicosídeo, poliol e

carboxilato

Mannosidase, alpha, class 2C, member 1 F1MWT0_BOVIN 725,2 >100 Metabolismo de açucar, glicosídeo, poliol e

carboxilato

Glycogen starch synthase muscle GYS1_BOVIN 709,0 >100 Metabolismo de reserva de energia

Phosphorylase kinase gamma 1 Muscle Q29RI2_BOVIN 813,4 >100 Metabolismo de reserva de energia

ATP synthase subunit alpha F1MLB8_BOVIN 1187,4 0,30 Transporte de elétrons e proteína associada a

membrana



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A enzima Dihydrolipoyllysine residue succinyltransferase component of 2 oxoglutarate

dehydrogenase complex m (RDSODO2) , Glyceraldehyde 3 phosphate dehydrogenase testis specific

(G3PT), Aconitate hydratase mitochondrial (ACONm), Citrate synthase mitochondrial (CSIm),

Malate dehydrogenase mitochondrial (MDHm) e Malate dehydrogenase cytoplasmic (MDHc) foram

identificadas suprimidas no grupo M e todas estas enzimas estão envolvidas no ciclo do ácido

tricarboxílico. Os resultados sugerirem que os animais do grupo D apresentam metabolismo

predominante oxidativo e outras proteínas interessantes encontradas suprimidas no grupo M são a

Myoglobin e a ATP synthase subunit α (ATBSα), contribuem na caracterização de músculo dos

animais do grupo D, com característica predominante oxidativa. Contrariamente a este resultado a

ATBSα foi sugerida como uma eleita como possível marcador para a característica de maciez, mas

contrariamente ao presente trabalho os autores utilizaram outro método de extração protéica que

promove a solubilização de proteínas que o presente método utilizado (46). A presença desta proteína

na fração solúvel pode estar ligada possivelmente à proteólise desta proteína.

Apesar de ser um atributo que é influenciado por várias características pré e pós abate, o tipo

do metabolismo predominante na fibra muscular pode estar relacionado à característica de maciez

onde fibras que predominam o metabolismo oxidativo estão relacionadas com carne mais dura

(47,48). Mas são controversos os resultados encontrados na literatura que suportam estas

características (49,50), atribuindo maior maciez a carne apresentando maior atividade oxidativa.

A supressão das proteínas Fatty acid binding protein heart, Prostaglandin F synthase 1,

Prostaglandin reductase 2 e a proteína Phosphoribosylaminoimidazole carboxylase

phosphoribosylaminoimidazole succinocarboxamide synthetase nos animais do grupo M, ajuda a

reforçar a característica oxidativa do músculo de animais do grupo D. A oxidação lipídica também

pode ser uma característica de células que sofreram maior stress (18).

As enzimas envolvidas na glicólise 6 phosphofructokinase (6CPK), 6 phosphofructokinase

muscle type (FKG1m) e 6 phosphofructokinase liver type (6CPKl) foram identificadas induzidas

animais do grupo M. Este resultado reforça a idéia de que a característica de predominância de fibras

glicolíticas está relacionada a maciez da carne. A característica de predominância do metabolismo

glicolítico está relacionada com a presença de menores quantidades de calpastatina, maior fragilidade

na constituição das proteínas que compõe a linha Z e maior pressão osmótica na célula muscular

favorecendo o processo da proteólise post mortem (47).

Foi evidenciada a indução da proteína Creatine kinase B type, outra proteína que corrobora com

a característica de células com predominância do metabolismo glicolítico. Fibras musculares com

predominância do metabolismo glicolítico têm, em média, diâmetro maior quando comparada com

99

fibras com metabolismo oxidativo predominante, e esta característica está relacionada com a

característica de maciez (51).

As proteínas Dihydropteridine reductase (DPR), Calpastatin (CAST), Cytosol aminopeptidase

(APSc) e Peptidyl prolyl cis trans isomerase (PEP), Aspartate aminotransferase mitochondrial

(ASTm) e citoplasmic (ASTc) peptidil prolil cis-trans isomerase (PPCTI) foram identificadas

suprimidas no grupo M (Tabela 5).

100

11 Tabela 5 - Proteínas relacionadas ao metabolismo de proteínas, diferentemente expressas entre os grupos macio (M) e duro (D).



Aspartate aminotransferase cytoplasmic AATC_BOVIN 21150,8 0,57 Biosintese do glutamato

Aspartate aminotransferase mitochondrial AATM_BOVIN 12146,2 0,42 Biosintese do glutamato

KBTBD10 protein A4FV78_BOVIN 2085,9 >100 Degradação proteica citoplasmática e nuclear

Adenosylhomocysteinase SAHH_BOVIN 2039,7 0,56 Degradação da homocisteina

Dihydropteridine reductase DHPR_BOVIN 2521,7 <0,01 Metabolismo da cisteina e grupo aromatico

Transitional endoplasmic reticulum ATPase TERA_BOVIN 1595,0 >100 Modificação por ubiquitinação e deubiquitinação

Calpastatin G3N2N7_BOVIN 1011,0 <0,01 Degradação de proteinas e peptideos

Prolyl endopeptidase PPCE_BOVIN 563,0 <0,01 Degradação de proteinas e peptideos

Cytosol aminopeptidase G3N0I4_BOVIN 637,5 <0,01 Processamento proteolítico

Peptidyl prolyl cis trans isomerase FKBP1A FKB1A_BOVIN 2820,9 0,54 Tradução

nuclear receptor subfamily 5, group A, member 2 F1MCM4_BOVIN 13,7 <0,01 Controle transcripcional

Phosphoribosylaminoimidazole carboxylase

phosphoribosylaminoimidazole succinocarboxamide

synthetase

Q2HJ26_BOVIN 2187,8 0,53 Anabolismo de

nucleotideo/nucleosideo/nucleobase

LIM and cysteine rich domains protein 1 LMCD1_BOVIN 987,8 <0,01 Supressor transcripcional # Número de acesso no banco de dados UNIPROT.


101

Dentre os fatores que influenciam na maciez final da carne um dos principais fatores é a

proteólise post mortem de proteínas estruturais das células musculares (43,52). O sistema proteolítico

calpaína vem sido atribuído como principal no processo de proteólise post mortem (40,43,53–55) e a

calpastatina está correlacionada negativamente com a maciez em bovinos por ser o inibidor natural

das calpaínas (56–59).

A relação entre calpastatina/calpaína tem grande influência na taxa de proteólise post mortem

e a presença de uma concentração maior deste inibidor irá contribuir negativamente para a proteólise

(60).

Identificadas induzidas no grupo D as proteínas LIM domain binding 3 (DLIM3), Cysteine and

glycine rich protein 3 (PRCG3) e LIM and cysteine rich domains protein 1 (PDRC1) fazem parte de

uma família de proteínas regulatórias celulares mediando a interação protéica e o metabolismo de

ácidos nucléico (61). Bernard et al. (18) utilizando a ferramenta transcriptômica, encontraram uma

menor expressão desta proteína em animais com carne macia mas não encontraram associação direta

com os mecanismos de amaciamento da carne.

Foram identificadas suprimidas no grupo M as proteínas nuclear receptor subfamily 5, group

A, member 2 (FTN2), Sodium channel subunit beta 3 (CSβ3) (Tabela 6). possivelmente esta proteína

pode ser negativamente associada a maciez atuando na estabilização destas proteínas estruturais,

dificultando a proteólise post mortem.

102

12 Tabela 6 - Proteínas relacionadas ao metabolismo de lipídeos, ligadas ao transporte de íons, diferentemente expressas entre os grupos macio (M)

e duro (D).

Descrição Acesso* Score Relação

Macio:Duro# Função

ATP binding cassette, subfamily A (ABC1), member 13 E1BM08_BOVIN 13.7 Duro Ligação de ATP

Calsequestrin Q05JF3_BOVIN 1811.5 >100 Ligante de Ca+

Retinal dehydrogenase 1 AL1A1_BOVIN 3922.9 0,51 Não calssificada

Alpha 1B glycoprotein A1BG_BOVIN 809.4 <0,01 Não calssificada

carboxymethylenebutenolidase homolog F1N2I5_BOVIN 1588.2 0,55 Não calssificada

Lumican LUM_BOVIN 958.9 >100 Não calssificada

Sodium channel subunit beta 3 SCN3B_BOVIN 545.3 <0,01 Transporte de cations (Na, K, CA, NH4, etc)

Nuclear transport factor 2 NTF2_BOVIN 3588.3 <0,01 Transporte nuclear

Hemopexin HEMO_BOVIN 1832.0 <0,01 Transporte de íons metálicos (Cu, Fe, etc)

Serotransferrin TRFE_BOVIN 7830.0 <0,01 Transporte de íons metálicos (Cu, Fe, etc)

Fatty acid binding protein heart FABPH_BOVIN 7743.3 0,42 Metabolismo de fosfolipídeos

Prostaglandin F synthase 1 PGFS1_BOVIN 157.7 <0,01 Biosíntese de prostaglandinas

Prostaglandin reductase 2 PTGR2_BOVIN 1846.0 <0,01 Biosíntese de prostaglandinas # Número de acesso no banco de dados UNIPROT.


103

Identificadas somente expressas no grupo M as proteínas Lumican (LUM), Calsequestrina

(CTRN) e Proteína KBTBD10 e mais expressas as proteínas 14-3-3 epsilon e gama. Proteína que se

liga ao Ca+ no retículo sarcoplasmático, a calsequestrina está envolvida na regulação da concentração

deste íon fundamental para a atividade das principais enzimas envolvidas na proteólise post mortem

(62). A proteína serotransferin foi idetificada induzina em animais do grupo M, ela atua na ligação e

transporte do íon Fe++ .

CONCLUSÕES

A expressão aumentada das chaperonas HSP90, αβ cristalina, HSP β1 e β6 são potenciais

marcadores para maciez da carne aos 7 dias de maturação.

A indução das proteínas relacionadas ao metabolismo glicolítico, 6 fosfofrutokinase, creatina

kinase tipo B e fosforilase kinase gamma e a supressão das proteínas ATP sintase subunidade alfa,

gliceraldeído 3 fosfato testículo específico, citrato sintase mitocondrial e malato desidrogenase são

relacionadas a baixos valores de WBSF e são candidatas a marcadores para maciez da carne assim

como a supressão da expressão da calpastatina.

A interação entre as vias metabólicas é complexa e mais estudos no intuito de identificar as

interações e as atividades enzimáticas de cada parte do metabolismo celular para tentar elucidar este

truncado complexo metabólico.

AGRADECIMENTOS

Os autores agradecem a Agropecuária Guaporé e a Empresa Brasileira de Pesquisa

Agropecuária pela oportunidade de fazer parte desta pesquisa.

CAPES por formecer a bolsa de doutorado para o primeiro autor.

Ao Laboratório de Biologia Molecular “José Salum” – ICB – UFG por viabilizar as análises

e ao Laboratório de Fisiologia da Digestão por todo apoio dado à pesquisa.

104

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Anexos

Anexo 1 – Géis de poliacrilamida 12,5% para verificação do perfil de bandas da amostra e

confirmação da quantificação protéica corados por comassie. Os números representam as amostras

carregadas em cada poço, de acordo com o anexo 2.

Anexo 2 – Valores de força de cisalhamento (WBSF-7), grupo

Numero

Amostra

WBSF-

7 valores Grupo

Concentração de

proteína após a

extração (mg/mL)

1 1.38 1.38 M 26,6

2 6.11 6.11 D 22,0

3 1.26 1.26 M 27,4

4 6.71 6.71 D 44,2

5 1.24 1.24 M 39,5

6 5.18 5.18 D 47, 7

7 1.17 1.17 M 21,9

8 5.05 5.05 D 31,7

9 1.23 1.23 M 35,1

10 5.61 5.61 D 27,8

11 1.33 1.33 M 24,5

12 6.47 6.47 D 27,3

13 1.09 1.09 M 33,6

14 5.72 5.72 D 26,9

15 1.32 1.32 M 25,8

16 5.49 5.49 D 32,8

17 1.28 1.28 M 32,8

18 5.91 5.91 D 24,1

19 1.07 1.07 M 29,3

20 5.26 5.26 D 32,3

111

Anexo 3 – Proteínas identificadas nos grupos induzidas ou suprimidas com menos de 50% de diferença.



Trans 1 2 dihydrobenzene 1 2 diol dehydrogenase DHDH_BOVIN 1326.8 0,677 Metabolismo de compostos aromáticos

Phosphatidylethanolamine binding protein 1 PEBP1_BOVIN 51860.6 0,613 Ligante de ATP

Carbonic anhydrase 3 CAH3_BOVIN 88390.0 0,644 Metabolismo de compostos C-1

GPD1L protein A6QQR7_BOVIN 152.0 0,613 Metabolismo de compostos C-3

Glycerol 3 phosphate dehydrogenase NAD cytoplasmic GPDA_BOVIN 24921.8 0,613 Metabolismo de compostos C-3

ATPase, Ca++ transporting, cardiac muscle, slow

twitch 2 F1MPR3_BOVIN 2813.7 0,932 Transporte de cátions (Na, K, CA, NH4, etc)

Sarcoplasmic endoplasmic reticulum calcium ATPase 1 AT2A1_BOVIN 4633.1 1,000 Transporte de cátions (Na, K, CA, NH4, etc)

ATPase, Ca++ transporting, ubiquitous E1BMQ6_BOVIN 1485.9 1,246 Transporte de cátions (Na, K, CA, NH4, etc)

Aldehyde dehydrogenase mitochondrial ALDH2_BOVIN 1896.3 0,613 Composto-C e metabolismo de carboidratos

Protein NDRG2 F1MTZ1_BOVIN 1120.8 0,795 Reorganização do citoesqueleto dependente

do ciclo celular

PDZ and LIM domain protein 3 PDLI3_BOVIN 2833.9 1,020 Reorganização do citoesqueleto dependente

do ciclo celular

Myosin regulatory light chain 2 skeletal muscle

isoform MLRS_BOVIN 3833.0 0,684 citoesqueleto / proteína estrutural

Actin gamma enteric smooth muscle ACTH_BOVIN 3087.3 0,691 citoesqueleto / proteína estrutural

Tropomyosin beta chain TPM2_BOVIN 7428.9 0,741 citoesqueleto / proteína estrutural

Tropomyosin alpha 3 chain TPM3_BOVIN 2226.4 0,980 citoesqueleto / proteína estrutural

Tropomyosin alpha 1 chain Fragment G3X7S7_BOVIN 4846.7 1,150 citoesqueleto / proteína estrutural

Adenylate kinase isoenzyme 1 KAD1_BOVIN 64903.1 0,726 Conversão e regeneração de energia

Triosephosphate isomerase TPIS_BOVIN 117112.1 0,600 Glicólise e gliconeogênese

Phosphoglycerate kinase G3X7N4_BOVIN 106349.3 0,619 Glicólise e gliconeogênese

Fructose 1 6 bisphosphatase isozyme 2 F16P2_BOVIN 6511.3 0,664 Glicólise e gliconeogênese

Glucose 6 phosphate isomerase G6PI_BOVIN 30635.3 0,664 Glicólise e gliconeogênese

Phosphoglycerate mutase 2 F1N2F2_BOVIN 96861.5 0,670 Glicólise e gliconeogênese

Phosphoglycerate mutase 2 PGAM2_BOVIN 96861.5 0,670 Glicólise e gliconeogênese

112

ENO2 protein A6QR19_BOVIN 17506.5 0,691 Glicólise e gliconeogênese

Alpha enolase ENOA_BOVIN 31283.7 0,691 Glicólise e gliconeogênese

Alpha enolase F1MB08_BOVIN 31283.7 0,691 Glicólise e gliconeogênese

Pyruvate kinase Q1JPG7_BOVIN 5700.9 0,705 Glicólise e gliconeogênese

Pyruvate kinase A5D984_BOVIN 87758.4 0,712 Glicólise e gliconeogênese

Beta enolase ENOB_BOVIN 80172.3 0,795 Glicólise e gliconeogênese

Fructose bisphosphate aldolase A6QLL8_BOVIN 123550.6 0,819 Glicólise e gliconeogênese

Fructose bisphosphate aldolase Q3ZBY4_BOVIN 36419.4 0,835 Glicólise e gliconeogênese

heat shock 70kDa protein 6 (HSP70B') F1MWU9_BOVIN 2678.8 0,684 Resposta ao choque térmico

Heat shock related 70 kDa protein 2 HSP72_BOVIN 3006.6 0,684 Resposta ao choque térmico

Heat shock cognate 71 kDa protein HSP7C_BOVIN 7157.0 0,733 Resposta ao choque térmico

Heat shock 70 kDa protein 1B HS71B_BOVIN 12687.0 0,741 Resposta ao choque térmico

Heat shock 70 kDa protein 1A HS71A_BOVIN 8119.3 0,748 Resposta ao choque térmico

Heat shock 70 kDa protein 1 like HS71L_BOVIN 5324.4 0,756 Resposta ao choque térmico

Heat shock protein HSP 90 alpha HS90A_BOVIN 2420.0 0,914 Resposta ao choque térmico

Creatine kinase M type KCRM_BOVIN 81831.2 0,905 Metabolismo da creatina

Creatine kinase S type mitochondrial F1MJT6_BOVIN 1985.4 0,990 Metabolismo da creatina

Creatine kinase S type mitochondrial KCRS_BOVIN 1985.4 1,162 Metabolismo da creatina

Glycogenin 1 F6QLM5_BOVIN 3199.7 0,619 Metabolismo de reserva de energia

Amylo-alpha-1, 6-glucosidase, 4-alpha-

glucanotransferase F1MHT1_BOVIN 4838.6 0,712 Metabolismo de reserva de energia

Glycogen phosphorylase muscle form PHS2_RABIT 45622.7 0,914 Metabolismo de reserva de energia

Glycogen phosphorylase liver form PYGL_BOVIN 8552.9 0,923 Metabolismo de reserva de energia

Glycogen phosphorylase brain form PYGB_BOVIN 15009.1 0,932 Metabolismo de reserva de energia

Phosphorylase F1MJ28_BOVIN 63620.7 0,942 Metabolismo de reserva de energia

Probable C U editing enzyme APOBEC 2 ABEC2_BOVIN 2150.2 0,691 Processamento do mRNA (splicing, 5'- 3'-

final processamento)

Mitochondrial peptide methionine sulfoxide reductase MSRA_BOVIN 13198.5 0,607 Detoxificação de radicais de oxigênio

Glutathione S-transferase mu 3 (brain) F1MUX6_BOVIN 2201.5 0,619 Detoxificação de radicais de oxigênio

Peroxiredoxin 6 PRDX6_BOVIN 21869.7 0,625 Detoxificação de radicais de oxigênio

113

Glutathione S transferase Mu 1 GSTM1_BOVIN 7112.1 0,670 Detoxificação de radicais de oxigênio

GSTM1 protein A4IFG0_BOVIN 2366.8 0,705 Detoxificação de radicais de oxigênio

Ubiquitin 60S ribosomal protein L40 RL40_BOVIN 19072.8 0,691 Anabolismo de proteínas

Prolyl endopeptidase Fragment F6QHN4_BOVIN 582.0 0,803 Degradação de proteína/ Peptídeo

Purine nucleoside phosphorylase PNPH_BOVIN 2291.9 0,638 Metabolismo de


Adenylosuccinate synthetase isozyme 1 PURA1_BOVIN 4594.3 0,607 Anabolismo de


Bifunctional purine biosynthesis protein PURH PUR9_BOVIN 1197.9 0,698 Anabolismo de


UTP glucose 1 phosphate uridylyltransferase UGPA_BOVIN 2264.4 0,726 Metabolismo de pirimidinae


Pyruvate dehydrogenase E1 component subunit alpha

somatic form mitochondrial ODPA_BOVIN 2187.6 0,657 Complexo da piruvato desidrgenase

Pyruvate dehydrogenase E1 component subunit beta

mitochondrial ODPB_BOVIN 2209.1 0,741 Complexo da piruvato desidrgenase

Stress induced phosphoprotein 1 STIP1_BOVIN 1030.5 0,607 Resposta ao estresse

Thioredoxin Fragment G8JKZ8_BOVIN 7535.2 0,664 Resposta ao estresse

Phosphoglucomutase 1 PGM1_BOVIN 79723.9 0,644 Metabolismo de açucar, glicosídeo, poliol e

carboxilato

Glyceraldehyde 3 phosphate dehydrogenase G3P_BOVIN 50248.8 0,741 Metabolismo de açucar, glicosídeo, poliol e

carboxilato

Elongation factor 1 alpha 2 EF1A2_BOVIN 5433.7 0,719 Elogação na translação

Serum albumin ALBU_BOVIN 48995.6 0,589 Transporte de Metais(Cu, Fe, etc)

Isocitrate dehydrogenase NADP mitochondrial IDHP_BOVIN 3654.9 0,595 Ciclo do ácido tricarboxilico

L lactate dehydrogenase B chain LDHB_BOVIN 25425.3 0,619 Ciclo do ácido tricarboxilico

Succinyl CoA ligase ADP GDP forming subunit alpha

mitochondrial F1MZ38_BOVIN 1290.3 0,631 Ciclo do ácido tricarboxilico

L lactate dehydrogenase A chain LDHA_BOVIN 112711.9 0,712 Ciclo do ácido tricarboxilico

Uncharacterized protein G3N3C9_BOVIN 2237.9 0,719 Não classificada

Bridging integrator Q2KJ23_BOVIN 6227.8 0,811 Não classificada

114



115

CAPITULO 5 – Considerações finais

Os fatores que regulam o processo de amaciamento da carne no período post mortem são

complexos e sofrem influência de muitos fatores intrínsecos e extrínsecos, tornando mais desafiador

compreender os mecanismos chave deste processo mas alguns pontos relativos ao animal podem ser

apontados como promissores determinantes para se conseguir produzir carne mais macia.

Um dos pontos é a avaliação da expressão das chaperonas HSP90, αβ cristalina, HSP β1 e β6 e

relacionadas com o proteínas metabolismo glicolítico positivamente relacionadas com a maciez.

Negativamente relacionadas característica maciez da carne as proteínas envolvidas estão ligadas ao

metabolismo oxidativo celular.

O sistema calpaína tem grande destaque na proteólise post mortem e dentro deste sistema, a

calpastatina é um dos principais fatores que irão determinar a taxa e a extensão desta proteólise. Os

fatores que regulam a ação da calpastatina ainda não estão claros necessitando ainda de mais

pesquisas para entender melhor como é regulada .

O campo do metabolismo ainda é pouco conhecido necessitando assim mais trabalhos para de

entender as interações este complexo sistema.

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