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UNIVERSIDADE FEDERAL DE PERNAMBUCO
CENTRO DE CIÊNCIAS BIOLÓGICAS
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS
NÍVEL DOUTORADO
Purificação, caracterização estrutural e funcional da lectina do
veneno de Bothrops leucurus: Modulação de eventos
citotóxicos após a irradiação gamma.
ERIKA DOS SANTOS NUNES
RECIFE
2011
ERIKA DOS SANTOS NUNES
Purificação, caracterização estrutural e funcional da lectina do
veneno de Bothrops leucurus: Modulação de eventos
citotóxicos após a irradiação gamma.
Tese apresentada ao Programa de Pós-Graduação em Ciências Biológicas da Universidade Federal de Pernambuco como pré-requisito para a obtenção do título em Doutor em Ciências Biológicas.
Orientadora: Profa. Dra. Maria Tereza dos Santos Correia
Co-orientadora: Profa. Dra. Míriam Camargo Guarnieri
Banca examinadora:
Profa. Dra. Maria Tereza dos Santos Correia (Presidente/UFPE)
Profa. Dra. Luana Cassandra Breitenbach Barroso Coelho (UFPE)
Profa. Dra. Vera Lúcia de Menezes Lima (UFPE)
Profa. Dra. Márcia Vanusa da Silva (UFPE)
Profa. Dra. Jeanne Claíne de Albuquerque Modesto (UFPE/CAV)
“É graça divina começar bem. Graça
maior persistir na caminhada certa. Mas
graça das graças é não desistir nunca.”
Dom Hélder Câmara
AGRADECIMENTOS
A Deus por guiar meus passos todos os dias de minha vida.
A meus pais por me apoiarem em todos os momentos da minha existência.
A meu esposo Roberto, por sua paciência, dedicação, por suas palavras de incentivo e
principalmente por seu amor.
As minhas irmãs e cunhados que me apoiaram e torceram por mim em todas as etapas desse
trabalho.
As profa. Dra Tereza Correia e a profa. Dra. Miriam Guarnieri, pela amizade, atenção e por
acreditar no meu potencial para executar esse trabalho.
A profa. Dra. Teresinha Gonçalves e a Jaciana Aguiar pela amizade e colaboração nesse trabalho.
A profa. Dra. Maria Madalena e André Mariano do Departamento de Veterinária pela amizade e
colaboração na análise de Microscopia de fluorescência.
A profa. Dra. Maria Luiza V. Oliva e a profa. Dra. Rosemeire A. Silva-Lucca pela colaboração
nesse trabalho.
A todos que integram o Laboratório de Glicoproteínas, pela paciência, amizade e por me receberem
de braços abertos....... bem abertos.
A meu “filho” Fernando, a Lidiany, Giselly e Mary, pela paciência, amizade e colaboração......
“vocês moram no meu coração”.
A Thiago e Francis pela ajuda nesse trabalho, amizade e atenção...... “eita dupla arretada de boa”.
Aos meus colegas de Doutorado, em especial a minha amiga Dilênia, por sua amizade e incentivo
sempre presentes.
A todos os meus amigos e parentes que mesmo distantes torceram por mim.
A Universidade do Estado da Bahia (UNEB) pelo afastamento temporário concedido para a
realização desse trabalho.
Muito obrigada!
RESUMO
Lectinas são proteínas ou glicoproteínas de origem não imune cuja ligação reversível e específica a carboidratos resulta em aglutinação celular. Bothrops leucurus, serpente da Família Viperidae, representa um sério problema médico para a região Nordeste do Brasil. Uma lectina ligante de galactosídeo (BlL) foi purificada do veneno da serpente Bothrops leucurus através da combinação de cromatografia de afinidade e gel filtração. BlL aglutinou eritrócitos de coelho e humano, com preferência para eritrócitos de coelho, sendo especificamente inibida por galactose, rafinose e lactose, bem como por glicoproteinas. BlL é uma proteína ácida, com massa molecular de 30 kDa e composta de duas subunidades de 15 kDa, exibiu dependência de cátions divalentes, foi principalmente ativa em pH 4.0 a 7.0 e termoestável até 70°C. O espectro de emissão de fluorescência mostrou resíduos de triptófano completamente encobertos na sua estrutura. Dicroísmo Circular de BlL foi típico de uma proteína toda β estrutural. BlL mostrou ser efetivo contra bactérias gram-positivas (Staphylococcus aureus, Enterococcus faecalis e Bacillus subtilis). A atividade antitumoral de BlL foi avaliada em relação ao seu potencial citotóxico em linhagem tumoral (K562, Hep-2 e NCI-H292) e quanto à sua capacidade hemolítica. BlL apresentou uma significante atividade citotóxica em todas as linhagens tumorais testadas e não exibiu atividade hemolítica na máxima concentração testada (2000 µg/mL). Além disso, foi realizada em células K562, a análise da externalização da fosfatidilserina e potencial de membrana mitocondrial, utilizando microscópio de fluorescência. Tratamento com BlL induziu externalização da fosfatidilserina e despolarização mitocondrial, indicando morte celular por apoptose. Após irradiação gamma, BlL apresentou mudanças estruturais e teve sua atividade hemaglutinante significante alterada. SDS-PAGE indicou que a irradiação causou fragmentação e com posterior agregação da lectina. A análise em cromatografia de fase reversa revelou fragmentação estrutural. A atividade citotóxica de BlL em linhagens tumorais (K562, Hep-2 e NCI-H292) foi abolida após irradiação, indicando que a irradiação de BlL se mostrou uma eficiente estratégia para inativar sua atividade citotóxica. Esses achados demonstram que o veneno de B.leucurus contem uma lectina ligante de galactosídeo com potencial promissor para aplicação terapêutica e biotecnológica.
Palavras-chave: lectina, Bothrops leucurus, apoptose, irradiação gamma, veneno de serpente.
ABSTRACT
Lectins are proteins or glycoproteins of nonimmune origin which specific and reversible binding to carbohydrates resultins in cell clumps. Bothrops leucurus, Family Viperidae snake, represents a serious medical problem for the Northeast region of Brazil. A galactoside-binding lectin (BlL) was purified from the venom of Bothrops leucurus through a combination of affinity chromatography and gel filtration. BlL agglutinate rabbit erythrocytes and human, with a preference for rabbit erythrocytes and is specifically inhibited by galactose, raffinose and lactose, as well as glycoproteins. BlL is an acid protein with a molecular mass of 30 kDa and composed of two subunits of 15 kDa, exhibited dependence on divalent cations, was especially active at pH 4.0 to 7.0 and heat stable up to 70°C. The emission spectra of tryptophan fluorescence showed residue completely buried in its structure. Circular Dichroism BlL was typical of an entire protein β structure. BlL was show to be effective against gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis e Bacillus subtilis). Antitumor activity of BlL was assessed in relation to their cytotoxic potential in type of tumor (K562, Hep-2 e NCI-H292) and the its hemolytic capacity. BlL showed a significant cytotoxic activity in all tumor cell lines tested and showed no hemolytic activity at maximum concentration tested (2000 µg/mL). Furthermore, it was held in K562 cells, analysis of externalization of phosphatidylserine and mitochondrial membrane potential using fluorescence microscope. BlL treatment induced phosphatidylserine externalization and mitochondrial depolarization, indicating cell death by apoptosis. After gamma irradiation, BlL showed structural changes and its hemagglutinating activity was significantly altered. SDS-PAGE indicated that irradiation caused fragmentation and subsequent aggregation of the lectin. The analysis in reverse-phase chromatography revealed structural fragmentation. Cytotoxic activity in tumor cell lines BlL (K562, Hep-2 e NCI-H292) was abolished after irradiation, indicating that the irradiation of BlL proved an efficient strategy for inactivating their cytotoxic activity. These findings demonstrate that the venom contains a B. leucurus galactoside-binding lectin with promising potential for therapeutic and biotechnological application.
Keywords: lectin, Bothrops leucurus, apoptosis, gamma irradiation, snake venom.
LISTA DE FIGURAS DA REVISÃO BIBLIOGRÁFICA
Figura 1: Representação esquemática de lectinas tipo-C (CTLs) e proteínas relacionadas
as lectinas tipo-C (CLRPs) de venenos de serpentes ou snaclecs.
08
Figura 2: Métodos cromatográficos para purificação de proteínas. 10
Figura 3: Características das células tumorais. 13
Figura 4: Vias intrínseca e extrínseca da apoptose. 15
Figura 5: Serpente Bothrops leucurus. 20
LISTA DE FIGURAS DOS ARTIGOS
ARTIGO I
Fig.1. (A) Purification of BlL by affinity chromatography of B. leucurus venom (30 mg
of protein) on a guar gel column. Elution with CTBS buffer (-■-) followed by 200 mM D-
galactose (-▲-; arrow). Specific hemagglutinating activity (SHA, -○-). (B) Purification of
BlL by chromatography in Superdex 75 column coupled to an ÄKTA purifier system. (C)
SDS-PAGE of BlL. (MW) molecular weight markers; BlL under non-reducing (lane 1) or
reducing conditions (lane 2); electrophoresis under native conditions for acidic proteins
(lane 3). (D) Reverse phase HPLC on a C4 column. (E) BlL chain separation after
desalting on a C4 column. The column was equilibrated with 0.1% TFA (solvent A) and
eluted using 90% acetonitrile/10% H2O/0.1% TFA (solvent B) in a non-linear gradient,
where B = 0% at t = 5 min, 45% at t = 10 min, 50% at t = 30 min and 100% at t = 35 min.
(F) Hemagglutinating activity (HA) of EDTA-treated BlL after addition of Ca2+ in
different concentrations.
53
Fig.2. (A) Intrinsic fluorescence emission of BlL excited at 280 nm (---) and 295 nm (—).
(B) CD spectrum of BIL in 50 mM phosphate buffer, pH 7.2, at 25°C. Measurements are
the average of eight scans using a solution containing 0.25 mg of protein/mL. CD
spectrum deconvolution using CDPro software calculated 1% α-helix, 44% β-sheet, 24%
β-turn, 31% unordered structures and an RMS of 2%.
57
ARTIGO II
Fig.1. Effect of BlL in K562 cell population determined by fluorescence microscopy
using Annexin V–FITC Kit, after 48 h incubation. The negative control (NC) was the
vehicle used (DMSO). Etoposide was used as positive control (E). *p < 0.01 in
comparison to control by ANOVA followed by Newman Keulls test. Data are presented
as mean ± S.D. from three independent experiments.
74
Fig.2. Effect of BlL in K562 cell population determined by fluorescence microscopy
using JC-1, after 48 h incubation. The negative control (NC) was the vehicle used
(DMSO). Etoposide was used as positive control (E). *p < 0.01 in comparison to control
by ANOVA followed by Newman Keulls test. Data are presented as mean ± S.D. from
three independent experiments.
74
ARTIGO III
Fig.1. Effect of γ-radiation on lectin activity and molecular weight. (a) The percentage of
remaining specific hemagglutination activity, %SHAREM is represented after irradiation.
Error in the determination of %SHAREM for the different doses was approximately ± 1%,
which is less than the size of the symbols. * Significant difference (p < 0.05) compared to
non-irradiated lectin. (b) SDS-PAGE from irradiated BlL. SDS-PAGE was performed in
a discontinuous system with 15% separating and 5% stacking gels. (MW) Molecular
weight; (C) non-irradiated control; (1) 1 kGy and (2) 2 kGy. (c) Reverse phase
chromatography on an HPLC system: (▬) control and irradiated lectins at (▬) 1 kGy and
(▬) 2 kGy. (d) Light scattering was measured at 90° for the aggregation assays.
90
Fig.2. BlL intrinsic fluorescence. (a) Mass center; Lectin excitation (280 nm) and
emission (295–450 nm). (b) Mass center tryptophan; Lectin excitation (295 nm) and
emission (305-450 nm).
91
Fig.3. BlL bis-ANS fluorescence. Mass center; Lectin excitation (360 nm) and emission
(400–600 nm).
92
LISTA DE TABELAS DOS ARTIGOS
ARTIGO I
Table 1. Summary of B. leucurus lectin (BlL) purification. 52
Table 2. Inhibition of hemagglutinating activity of BlL by carbohydrates and glycoproteins.
55
Table 3. Minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of BlL.
58
ARTIGO II
Table 1. Cytotoxic activity of BlL against tumor cell lines. 70
LISTA DE ABREVIATURAS
AH Atividade Hemaglutinante
APAF-1 Fator Ativador de Apoptose-1
ADP Adenosina difosfato
BlL Lectina de Bothrops Leucurus
Bis-ANS 4.4'-Bis 1-anilinonaphthaleno 8-sulfonato
BJcuL Lectina de Bothrops jararacussu
BmLec Lectina de Bothrops moojeni
CD Dicroísmo Circular
CfLEC-2 Lectina de Chlamys farreri
CFU Unidades Formadoras de Colônia
CI50 Concentração Inibitória Média 50%
CLEC-2 Receptor transmembrana tipo II C-tipo lectina-like
CM Carboximetil
CMB Concentração Mínima Bactericida
CMI Concentração Mínima Inibitória
ConA Concanavalina A
CRD Domínio de Reconhecimento de Carboidratos
CRLPs Proteínas Relacionadas Lectinas tipo-C
CTLs Lectinas tipo-C
CTBS Tampão Tris Base Salina
DEAE Dietilaminoetil
DMEM Meio Dulbecco’s Modificado Eagle’s
DMSO Dimetilsulfóxido
DTT Dithiolthreitol
EDTA Ácido etilenodiaminotetracético
FBS Soro Fetal Bovino
FITC Isotiocianato de Fluoresceína
FPLC Cromatografia Líquida de Rápida Resolução
GPIb Glicoproteína Ib
HPLC-RP Cromatografia Líquida de Alta Resolução em Fase Reversa
IgG2 Imunoglobulina G subclasse 2
MML Lectina de Musca domestica
MMP Permeabilidade da Membrana Mitocondrial
MTT Brometo de (3-(4, 5-dimetiltiazol-2-il)-2, 5-difeniltetrazólio)
PCL Lectina de Polygonatum cyrtonema
PAGE Eletroforese em gel de poliacrilamida
PI Iodeto de propídeo
POL lectina de Polygonatum odoratum
SDS-PAGE Eletroforese em gel de poliacrilamida com dodecilsulfato de sódio
SHA Atividade Hemaglutinante Específica
Snaclecs Lectina Tipo-C de Veneno de Serpente
Th1 Linfócito T auxiliar
ThT Thioflavina
SUMÁRIO
RESUMO
ABSTRACT
Lista de Figuras
Lista de Tabelas
1 INTRODUÇÃO 01
2 OBJETIVOS 03
2.1 Objetivo Geral 03
2.2 Objetivos Específicos 03
3 REVISÃO BIBLIOGRÁFICA 04
3.1 Lectinas 04
3.2 Lectinas de venenos de serpentes 05
3.3 Purificação e Caracterização de lectinas 09
3.4 Câncer 11
3.5 Morte celular (apoptose) 13
3.6 Atividade antitumoral 15
3.7 Atividade antibacteriana 17
3.8 Radiação 18
3.9 Serpente Bothrops leucurus 20
4 REFERÊNCIAS BIBLIOGRÁFICAS 22
5 ARTIGO CIENTÍFICO I
Purification of a lectin with antibacterial activity from Bothrops leucurus snake venom
44
6 ARTIGO CIENTÍFICO II
Cytotoxic effect and apoptosis induction by Bothrops leucurus venom lectin on tumor cell
lines
67
7 ARTIGO CIENTÍFICO III
Gamma irradiation abolish in vitro cytotoxicity of lectin of Bothrops leucurus snake venom
82
8 CONCLUSÕES 98
1 INTRODUÇÃO
O Interesse em lectinas tem se intensificado com a constatação que estas são extremamente
valiosas para a investigação de açúcares na superfície celular, na interação de células com o seu
meio e também em uma variedade de processos patológicos (SHARON, 2007). Lectinas são
proteínas ou glicoproteinas de origem não imunológica, ubiquamente distribuídas na natureza, as
quais reconhecem e ligam-se reversivelmente a carboidratos de maneira não-catalítica (LAM et al.,
2010).
A habilidade de ligar-se seletivamente a glicoconjugados faz dessas proteínas notáveis
ferramentas para pesquisa experimental e clínica. Em microbiologia, as lectinas têm assumido um
importante papel no estudo de glicoconjugados e superfície de células microbianas. Evidências
suportam sua interação com bactérias gram-negativas e gram-positivas, detectando sutis diferenças
na estrutura de carboidratos complexos (LUO et al., 2007), onde lectinas podem atuar interferindo
com o seu crescimento e desempenhando um papel em sistemas de defesa (SUN et al., 2008). Com
o resultado da alta prevalência de bactérias multi-resistentes, a pesquisa por novos protótipos
antimicrobianos são continuamente necessários, uma vez que pode permitir uma otimização no
tratamento de infecções bacterianas relacionadas com cepas resistentes aos antibióticos
convencionais (WORLD HEALTH ORGANIZATION, 2007).
Na Cancerologia Experimental, alterações na glicosilação durante a transformação maligna,
desempenham um importante papel no comportamento metastático das células tumorais e essas
modificações podem ser detectadas por lectinas (DAMODARAN et al., 2008; ARAB et al., 2010).
Interação lectina-carbohidrato causa aglutinação das células tumorais, citotoxicidade, indução da
apoptose e inibição do crescimento tumoral, criando uma variedade de possibilidades para a
produção de medicamentos anticancer (De MEJÍA & PRISECARU, 2005; NAKAHARA et al.,
2005). A identificação de novos compostos citotóxicos que melhorem ou restaurem a capacidade
das células malignas em sofrer apoptose pode ser crucial para terapias mais eficazes contra o câncer
(REYES-ZURITA et al., 2009)
A descoberta também incentiva os pesquisadores a buscarem moléculas similares em
venenos de serpentes, uma vez que esses são uma fonte rica em componentes bioativos, como
enzimas, proteínas e peptídeos com importantes propriedades farmacológicas, que podem levar a
produção de novos agentes com valor terapêutico (KOH et al., 2006).
2
Venenos de serpentes são empregados como imunógenos para produção de soro antiofídico
e vários esforços tem sido realizados para diminuir sua toxicidade, visando minimizar os danos
causados aos animais imunizados, prolongando o seu tempo de vida (GALLACI et al., 2000).
Radiação ionizante vem sendo empregada com sucesso para atenuar venenos de serpentes e suas
toxinas isoladas, diminuindo ou abolindo suas atividades biológicas e tóxicas, sem alterar sua
imunogenicidade, mostrando-se uma potente ferramenta para modificar e destoxicar biomoléculas.
(FERREIRA JUNIOR et al., 2005; BAPTISTA et al., 2009; CAPRONI et al., 2009).
Com base nessas considerações, o estudo das propriedades e funções da lectina de Bothrops
leucurus contribui para o entendimento da interação proteína-carboidrato, como também para
aplicações médicas e biológicas.
3
2 OBJETIVOS
2.1 Objetivo Geral
Purificar, caracterizar estrutural e funcionalmente a lectina do veneno de Bothrops leucurus:
Destoxicação do efeito citotóxico após a irradiação gamma.
2.2 Objetivos Específicos
� Purificar e caracterizar a lectina do veneno da serpente Bothrops Leucurus (BlL);
� Avaliar a atividade antibacteriana da lectina em bactérias patogênicas humanas;
� Determinar a citotoxicidade da lectina em linhagens tumorais humanas (Hep-2, K562 e NCI-
H292) in vitro;
� Examinar a atividade hemolítica da lectina em eritrócitos de camundongos;
� Avaliar o mecanismo de ação envolvida na atividade citotóxica da lectina em células K562
in vitro;
� Determinar o efeito da radiação gamma na estrutura da lectina purificada;
� Avaliar a citotoxicidade da lectina purificada após irradiação gamma em células tumorais
Hep-2, K562 e NCI-H292.
4
3 REVISÃO BIBLIOGRÁFICA
3.1 Lectinas
O termo “lectina” (originado do latim lectus, que significa escolhido, selecionado) foi
primeiro empregado por BOYD & SHAPLEIGH em 1954, para designar um grupo de proteínas que
apresentava a característica comum de seletividade na interação com carboidratos. Em 1980,
GOLDSTEIN et al. (1980) definiram as lectinas como proteínas ou glicoproteínas de origem não
imunológica, que apresentavam dois ou mais sítios de ligação a carboidratos, através dos quais
interagem com carboidratos, aglutinando células vegetais e/ou animais e precipitando
polissacarídeos, glicoproteínas e glicolipídeos de forma irreversível. O termo aglutinina é utilizado
como sinônimo para lectina, porque se refere à habilidade de aglutinar eritrócitos ou outras células
(PEUMANS & VAN DAMME, 1995).
De acordo com a nova definição, são consideradas lectinas proteínas ou glicoproteínas de
origem não imune que possuem pelo menos um sítio-não catalítico, o qual se liga reversívelmente a
mono ou oligossacarídeos específico (PEUMANS & VAN DAMME, 1995, 1998; PEUMANS et
al., 2001). Cada lectina liga-se a um carboidrato específico ou grupo de carboidratos em
oligossacarídeos ou glicoproteínas, através dos seus sítios de ligação que tendem a se localizar na
superfície da molécula protéica e a seletividade da ligação são obtidas através de pontes de
hidrogênio, interações hidrofóbicas e de van der Waals (COMINETTI et al., 2002; SHARON &
LIS, 2002).
As lectinas apresentam ampla variedade estrutural, sendo comum entre elas a presença de,
ao menos, um sítio específico de ligação a carboidrato, denominado “Domínio de Reconhecimento
de Carboidrato” (CRD), o qual se liga a carboidratos ou glicoconjugados em solução ou que estejam
conectadas ao envoltório celular (WEIS & DRICKAMER, 1996).
A presença de lectinas pode ser detectada pelo ensaio de hemaglutinação em eritrócitos
humanos ou de outras espécies animais. Nesse ensaio é realizada uma diluição serial e em seguida,
incubação com os eritrócitos, onde a atividade hemaglutinante é detectada pela formação de uma
malha decorrente da interação entre a lectina e os carboidratos localizados na superfície dos
eritrócitos. A capacidade de aglutinação por lectinas pode ser intensificada quando os eritrócitos são
submetidos a tratamentos com enzimas ou com soluções químicas (COELHO & SILVA, 2000).
5
Para assegurar que o agente aglutinante é uma lectina, são realizados ensaios subseqüentes de
inibição da atividade hemaglutinante (AH) com diferentes carboidratos (KAWAGISHI et al., 2001;
JAYATI et al., 2005).
As lectinas têm ampla distribuição na natureza, sendo encontradas em microorganismos
(SINGH et al., 2010), plantas (YAN et al., 2010; YAO et al., 2010) e animais (BATTISON &
SUMMERFIELD, 2009; CHEN et al., 2010). O reino animal tem demonstrado ser um rico
manancial para obtenção de lectinas, servindo como fonte para o isolamento e caracterização dessas
macromoléculas. A primeira atividade de lectina animal, provavelmente, foi observada no veneno
de serpentes, o qual foi capaz de promover a aglutinação em eritrócitos humanos (KILPATRICK,
2002).
Diferentes funções têm sido atribuídas as lectinas de animais, incluindo reconhecimento de
patógenos (DUTTA et al, 2005; XU et al., 2010), inibição da agregação plaquetária (SARRAY et
al., 2004), atividade antiproliferativa sobre células tumorais (CAO et al., 2010) e indutores da
apoptose (ZHAO et al., 2010).
As lectinas animais podem ser classificadas com base na estrutura molecular em cinco
principais categorias: tipo-C, tipo-S (galectinas), tipo-I (siglecs), tipo-P (receptor de manose-6P) e
tipo-N (SHARON & LIS, 2004). De acordo com a seqüência do CRD, lectinas animais tipo-C
podem ser classificadas dentro de 17 grupos (I ao XVII) (ZELENSKY & GREADY, 2005), estando
as lectinas de venenos de serpentes incluídas no grupo VII (OGAWA et al., 2005).
3.2 Lectinas de venenos de serpentes
Envenenamentos ofídicos representam um problema médico-social de considerável
magnitude, uma vez que cerca de 2.5 milhões de pessoas são picadas por serpentes anualmente,
sendo mais de 100.000 casos fatais. Contudo, venenos de serpentes constituem em uma fonte
biológica natural que contém diversos componentes farmacologicamente ativos, os quais, com o
advento da biotecnologia podem ser isolados e delineados suas propriedades terapêuticas (KOH et
al., 2006; ANGULO & LOMONTE, 2009).
6
Venenos de serpentes possuem uma variedade de proteínas, tais como serinoproteases,
fosfolipases, desintegrinas (LIMA et al., 2005), toxinas three-finger (3FTxs) (PAHARI et al., 2007),
metaloproteases e lectinas tipo-C (DOLEY & KINI, 2009).
Lectinas de serpentes tem sido encontrada em diversas serpentes da família Viperidade,
Elapidae and Crotalidae (GUIMARÃES-GOMES et al., 2004; LIN et al., 2007; SILVA JR. et al.,
2008; WANG, 2008). Essas exibem alta homologia na seqüência primária, com alguns invariantes
resíduos de aminoácidos, incluindo um padrão conservado de ligações dissulfeto. Sua
especificidade de ligação é mais freqüentemente atribuída à galactose, mas também a manose
(SHARON et al., 2003).
De acordo com sua estrutura e funções biológicas, essas proteínas podem ser classificadas
em dois subgrupos: lectinas tipo-C verdadeiras (CTLs), as quais contem um domínio de
reconhecimento de carboidratos (CRD) que se liga a açúcar e aglutina eritrócitos; e proteínas
relacionadas com lectinas tipo-C (CRLPs), com CRDs incompleto e, portanto, apresentando outras
atividades biológicas contra os fatores de coagulação e plaquetas, afetando a hemostasia
(DRICKAMER, 1999; WEI et al., 2002; MORITA et al., 2004). Em recente nomenclatura, esse
grupo foi renomeado para snaclecs (lectinas tipo-C de venenos de serpentes) (CLEMETSON et al.,
2009).
A primeira lectina isolada e parcialmente caracterizada de serpente foi obtida do veneno de
Bothrops atrox. Essa proteína foi denominada de trombolectina e apresentou as seguintes
características bioquímicas e biológicas: proteína homodimérica, ligante de β-galactosídeo, não
glicosilada e dependente de íons cálcio para exercer sua atividade de hemaglutinação (GARTNER
et al., 1980; GARTNER & OGILVIE, 1984).
CTLs consistem em uma família de proteínas estruturalmente homólogas, as quais são
geralmente homodímeros αβ ligados por ponte dissulfeto com dois polipeptídeos homólogos de
aproximadamente 14 kDa, apresentando atividade de aglutinação de eritrócitos e ligação a
carboidratos, principalmente galactose (Figura 1b) na presença de íons Ca2+ (LU et al., 2005;
OGAWA et al., 2005). Esses homodímeros têm sido reportados em várias serpentes, tais como
Bothrops jararaca (OZEKI et al., 1994), Lachesis muta stenophyrs (ARAGON-ORTIZ et al.,
1996), Trimeresurus stejnegeri (XU et al., 1999), Crotalus ruber (HAMAKO et al., 2007) e
Bungarus multicinctus (LIN et al., 2007). Em adição, CTLs podem ser constituídas por
homooligômeros (HIRABAYASHI et al., 1991; WANG, 2008).
7
Várias dessas proteínas têm sido parcialmente ou completamente seqüenciadas (KOMORI et
al., 1999; NIKAI et al., 2000; CARVALHO et al., 2002; HAMAKO et al., 2007) e, em alguns casos
seus genes clonados (GUIMARÃES-GOMES et al., 2004; KASSAB et al., 2004; LIN et al., 2007;
JEBALI et al., 2009). Estudos de cristalografia e dicroísmo circular estão revelando particularidades
na estrutura das lectinas de serpentes. WALKER et al. (2004) isolaram uma lectina de Crotalus
atrox (RSL) e observaram uma organização intrigantemente oligomérica de acordo com os dados
cristalográficos, por exemplo, uma estrutura decamérica formada por cinco dímeros. Espectros de
dicroísmo circular da lectina de Bothrops jararacussu (BJcuL), mostraram 32,2% de estrutura β e
18.8% de estrutura α, estando de acordo com a maioria das lectinas de venenos de serpentes, as
quais pertencem a classe α+β (SILVA JR. et al., 2008). Contudo, a lectina isolada de Lachesis muta
apresentou 78% β de estrutura e 1% α como característica de sua estrutura secundária (ARAGON-
ORTIZ et al., 1989).
Diversos efeitos biológicos das CTLs de venenos de serpentes têm sido relatados, incluindo
atividade mitogênica sobre linfócitos (MASTRO et al., 1986), liberação de cálcio do retículo
sarcoplasmático (OHKURA et al., 1996), inibição da proliferação de várias linhagens tumorais
(PEREIRA-BITTENCOURT et al., 1999), aglutinação de eritrócitos (KASSAB et al., 2001),
citotoxicidade para alguns tumores e células endoteliais (CARVALHO et al., 2001), efeitos renais
(HAVT et al., 2005; BRAGA et al., 2006), aumento a aderência de leucócitos sobre células
endoteliais de vênulas (ELÍFIO-ESPOSITO et al., 2007), inibição viral in vitro (ISLAS et al., 2007)
e ação inibitória sobre patógenos de plantas (RÁDIS-BAPTISTA et al., 2006; BARBOSA et al.,
2010).
Em contraste com CTLs, snaclecs não apresentam o clássico loop de ligação a açúcar/cálcio
e possuem a habilidade de interagir com fatores de coagulação e receptores de membranas nas
plaquetas (XU et al., 2004; LU et al., 2005; CLEMETSON, 2010). Geralmente, apresentam uma
estrutura básica de heterodímeros (αβ) ligadas covalentemente por pontes dissulfeto ou oligômeros
de heterodímeros (Figura 1c, d) (MORITA, 2004, 2005; CHEN et al., 2010).
8
Figura 1: Representação esquemática de lectinas tipo-C (CTLs) e proteínas relacionadas as lectinas
tipo-C (CLRPs) de venenos de serpentes ou snaclecs (Fonte: DOYLE & KINI, 2009).
Snaclecs apresentam uma variedade de atividades biológicas, como anticoagulantes e
agonistas ou antagonistas da agregação plaquetária (CLEMETSON, 2010). Por exemplo,
jerdonuxina purificada de Trimeresurus jerdonii induziu agregação plaquetária, provavelmente,
através da ligação ao receptor GPIb (CHEN et al., 2011). Agretina, isolada do veneno de
Calloselasma rhodostoma, estimula agregação plaquetária através de um novo receptor de
plaquetas, denominado CLEC-2 (SUZUKI-INOUE et al., 2006). Por outro lado, flavocetina-A se
liga a GPIb e fortemente inibe a agregação plaquetária dependente do fator de von Willebrand
(FUKUDA et al., 2000). Muitos membros dessa família interagem com os fatores de coagulação
IX/X ou α-trombina e formam complexos bloqueando as subseqüentes reações da cascata de
coagulação, exibindo atividade anticoagulante (KOO et al., 2002; MONTEIRO & ZINGALI, 2002;
ZANG et al., 2003).
Apesar das diversas atividades descritas para as lectinas de serpentes, o papel destas no
envenenamento não está claramente definido. Entretanto, se supõem que esses componentes tenham
uma função de defesa assim como nos invertebrados, porém de uma maneira mais ofensiva. Tem
sido proposto que CTLs juntamente com as snaclecs, que inibem trombina e fatores de coagulação
(CASTRO et al., 1999), provavelmente, ajudam a causar a desordem hematológica particularmente
evidente nos acidentes com as serpentes do gênero Bothrops. Além disso, a atividade de
hemaglutinação (HAVT et al., 2005), bem como a indução de edema em camundongos e o aumento
da permeabilidade vascular (LOMONTE et al., 1990; PANUNTO et al., 2006) induzidos por essas
proteínas, podem contribuir para os severos efeitos locais observados após o envenenamento pelas
serpentes desse gênero, incluindo Bothrops leucurus.
9
3.3 Purificação e Caracterização de lectinas
A utilização de técnicas cromatográficas purifica as lectinas de acordo com a massa
molecular, carga e afinidade específica de ligação a carboidratos. Na cromatografia de troca iônica a
proteína é separada em função de sua carga ao se ligar a um suporte com carga contrária a sua. A
coluna é lavada com solução tampão e as proteínas com nenhuma ou pouca interação com o
trocador de íons são excluídas. As proteínas adsorvidas na matriz podem ser eluídas pelo aumento
da força iônica ou alteração do valor de pH do meio (DATTA et al., 2001). Como exemplo tem a
dietilaminoetil (DEAE) celulose (LI et al., 2008), um trocador aniônico, e a carboximetil (CM)
celulose um trocador catiônico.
A cromatografia de filtração em gel ou exclusão molecular (ROJO et al., 2003; POHLEVEN
et al., 2009) separa as proteínas de acordo com o tamanho molecular (Figura 2B), onde as proteínas
maiores migram em maior velocidade que as menores devido a sua exclusão dos poros do gel. Este
tipo de cromatografia é usado tanto para obter preparações protéicas homogêneas (FREIRE et al.,
2002) como para definição de massa molecular da proteína (KAWAGISHI et al., 2001). A
cromatografia de afinidade (Figura 2C), técnica mais comumente utilizada, baseia-se na habilidade
das lectinas se ligarem especificamente a suportes polissacarídeos através dos seus sítios
específicos. A proteína desejada pode ser obtida com alto grau de pureza (YE & NG, 2002),
alterando-se as condições de pH (SÁ et al., 2008), força iônica (FREIRE et al., 2002) ou pela
eluição com uma solução contendo um competidor (OLIVEIRA et al., 2002). As matrizes de
afinidade podem ser selecionadas de acordo com a especificidade da lectina a carboidratos. Gel de
guar é uma dessas matrizes composta de um polissacarídeo com cadeias de manose substituídas por
resíduos de galactose α 1-6, sendo uma matriz versátil para isolamentos de lectinas ligantes de D-
galactopiranosil e N-acetil-galactosaminil (COELHO & SILVA, 2000).
10
Figura 2: Métodos cromatográficos para purificação de proteínas. (Fonte: STRYER et al., 2004)
Métodos eletroforéticos são utilizados para caracterizar estruturalmente as lectinas, bem
como para estabelecer o grau de pureza das mesmas. A eletroforese em gel de poliacrilamida
(PAGE) pode ser realizada usando um gel contendo dodecilsulfato de sódio (SDS) ou β-
mercaptoetanol (condições redutoras), que sob condições redutoras revela o grau de pureza, a
composição, a massa molecular de subunidades (REYNOSO-CAMACHO et al., 2003; PAIVA et
al., 2006) e através de coloração específica, a natureza glicoprotéica (COELHO & SILVA, 2000;
FENG et al., 2006).
Cromatografia líquida de rápida resolução (FPLC) e cromatografia líquida de alta resolução
em fase reversa (HPLC-RP) têm sido amplamente utilizadas com um processo final de purificação
mais refinada de lectinas, após a utilização de outros métodos cromatográficos (WONG & NG,
2003; JIANG et al., 2009). FPLC e HPLC podem estabelecer a homogeneidade de lectinas, como
também separar subunidades protéicas, assim determinar se essas biomoléculas são monoméricas
ou não (KAWSAR et al., 2008; SILVA et al., 2009).
Lectinas podem ser caracterizadas através da avaliação da AH em diferentes temperaturas,
bem como a inibição da AH por carboidratos e/ou glicoconjugados e o efeito de íons na atividade
hemaglutinante. Em relação ao pH, a verificação da faixa de estabilidade pode ser realizada
submetendo-se a lectina a tampões em diferentes valores de pH (MANSOUR & ABDUL-SALAM.,
2009). Quanto ao efeito da temperatura algumas lectinas permanecem estáveis até 80°C e a partir de
11
então, com a elevação da temperatura, a AH diminui até ser abolida, como no caso da lectina de
Aplysia kurodai (KAWSAR et al., 2009). Contudo, lectina ativa após aquecimento a 100°C também
foi isolada (SANTOS et al., 2009).
Muitas lectinas necessitam de íons metálicos bivalentes para exibir sua atividade. Ficou
demonstrado que a lectina de Macrotyloma axillare necessita de Ca2+ e Mn2+ para capturar o
carboidrato pelo qual tem afinidade (SANTANA et al., 2008). Por outro lado, a lectina da folha de
Phthirusa pyrifolia não teve sua AH abolida quando tratada com EDTA e, portanto, a AH é
independente de íons metálicos, tais como Ca2+, Mg2+ and Mn2+ (COSTA et al., 2010).
Muitos outros métodos também são ferramentas relevantes para a caracterização das
lectinas, tais como a determinação da seqüência de aminoácidos, cristalização, estudos de
fluorescência e dicroísmo circular (FUJII et al., 2011; DING et al., 2010; VAZ et al., 2010;
VAREJÃO, 2010).
3.4 Câncer
Os primeiros relatos da ocorrência de câncer foram encontrados nos papiros do Egito e data
de aproximadamente 1.600 a.C., os quais são referentes à descrição de oito casos de tumores ou
úlceras de mama que foram tratados por cauterização. A palavra câncer, no grego, significa
caranguejo e está associada a uma analogia entre o crescimento infiltrante do câncer e a forma como
esse crustáceo se prende ao solo usando suas patas (AMERICAN CANCER SOCIETY, 2009).
O câncer constitui-se em um importante problema de saúde pública em países desenvolvidos
e em desenvolvimento, sendo responsável por mais de seis milhões de óbitos a cada ano,
representando cerca de 12% de todas as causas de morte no mundo. Embora as maiores taxas de
incidência de câncer sejam encontradas em países desenvolvidos, dos dez milhões de casos novos
anuais de câncer, cinco milhões e meio são diagnosticados nos países em desenvolvimento
(WORLD HEALTH ORGANIZATION, 2002).
No Brasil, as estimativas para o ano de 2010, válidas para o ano de 2011, apontam para a
ocorrência de 489.270 casos novos de câncer, sendo 236.240 (sexo masculino) e 235.030 para o
sexo feminino. Os tipos mais incidentes, à exceção do câncer de pele do tipo não-melanoma, serão
os cânceres de próstata e de pulmão no sexo masculino e os cânceres de mama e do colo do útero no
12
sexo feminino, acompanhando o mesmo perfil observado na América Latina (INSTITUTO
NACIONAL DO CÂNCER, 2010).
Câncer pode ser causado por dieta incorreta, predisposição genética e fatores ambientais.
Cerca de 35% dos cânceres no mundo são causados por uma dieta incorreta, e no caso do câncer de
cólon, esse fator pode responder por 80% dos casos. Quando adiciona álcool e cigarros na dieta, a
percentagem pode aumentar para 60%. A predisposição genética é responsável por 20% dos casos
de câncer. Então, a grande maioria dos casos de câncer está associada com a carcinogênese
ambiental (REDDY et al., 2003).
O processo de carcinogênese inclui três estágios: iniciação, promoção e progressão tumoral.
No primeiro estágio as células sofrem o efeito de uma agente carcinógeno, levando a formação de
uma célula geneticamente transformada. O segundo estágio da carcinogênese envolve a ação de
substâncias classificadas como oncopromotores, que induzem a expressão de genes envolvidos no
crescimento celular. A célula iniciada é transformada em célula maligna de forma lenta e gradual. O
terceiro e último estágio, o de progressão, caracteriza-se pela multiplicação descontrolada das
células mutadas, onde começam a surgir as primeiras manifestações clínicas da doença (ALMEIDA
et al., 2005).
As mutações envolvem amplificação e/ou supra-expressão de oncogenes aliada à deleção
e/ou silenciamento de genes supressores de tumor (HAHN & WEINBERG 2002). Segundo LUO et
al. (2009), as mutações no câncer promovem a reativação ou modificação de programas celulares
que regulam mecanismos relacionados à embriogênese e a homeostasia, tais como proliferação,
diferenciação, migração e apoptose.
As células tumorais apresentam auto-suficiência para os sinais de crescimento e resistência
aos sinais antiproliferativos, evasão de morte celular programada (apoptose), potencial de
replicação ilimitado, angiogênese, invasão tecidual e metástase (HANAHAN & WEINGERG,
2000). Recentes trabalhos (Figura 3) têm mostrado a adição de outras características, tais como:
escape do sistema imune (KROEMER & POUYSSEGUR, 2008) e fenótipos de estresse, incluindo
metabólico, mitótico, proteotóxico, oxidativo e de dano ao DNA (LUO et al., 2009).
13
Figura 3: Características das células tumorais. (Fonte: LUO et al., 2009)
Atualmente, o tratamento do câncer é considerado como um dos problemas mais
desafiadores da medicina (CHABNER & JR., 2005). A partir do momento que a neoplasia primária
metastatiza o prognóstico se torna ruim, sendo a quimioterapia antineoplásica a principal forma de
tratamento nesse estágio. A vantagem desse tratamento é o de atingir igualmente as metástases
disseminadas pelo corpo. Contudo, esses medicamentos apresentam diversos efeitos colaterais, pois
sua grande maioria possui baixo índice terapêutico, ou seja, dose terapêutica muito próxima da dose
tóxica. Dessa forma, torna-se fundamental estimular as pesquisas, as quais medicamentos
antineoplásicos mais eficazes e seguros sejam descobertos (FUKUMASU et al., 2008).
3.5 Morte celular (Apoptose)
O equilíbrio entre a morte celular e proliferação celular regula e controla o número de
células no organismo. A cascata de eventos, bioquímicos e fisiológicos, que leva a mudança na
síntese de macromoléculas, na homeostase e volume celular, bem como na perda da viabilidade
celular estão relacionadas às alterações morfológicas características de cada tipo de morte celular
(TINARI et al., 2008).
Apoptose é considerado um mecanismo vital em diversos processos, tais como homeostase
dos tecidos, apropriado funcionamento do sistema imune e desenvolvimento embrionário (BRAS et
al., 2005: ELMORE, 2007). Por outro lado, a desregulação da apoptose pode afetar o balanço entre
Invasão tecidual e metástase
Angiogênesesustentada
Inibição de vigilância imune
Estresse metabólico
Estresse Proteotóxico
Estresse mitótico
Estresse oxidativo
Danos no DNA
Potencial replicativoilimitado
Insensível a sinais de anti-crescimento
Auto-suficiente em sinais de crescimento
Evasão da apoptose
Hipóxia Senescência
Aneuploidia
Baixo
pH
Sobrevida e proliferação em novos
ambientes
Invasão tecidual e metástase
Angiogênesesustentada
Inibição de vigilância imune
Estresse metabólico
Estresse Proteotóxico
Estresse mitótico
Estresse oxidativo
Danos no DNA
Potencial replicativoilimitado
Insensível a sinais de anti-crescimento
Auto-suficiente em sinais de crescimento
Evasão da apoptose
Hipóxia Senescência
Aneuploidia
Baixo
pH
Sobrevida e proliferação em novos
ambientes
14
proliferação celular e morte celular, resultando no aparecimento de várias doenças humanas,
incluindo o câncer (ZORNING et al., 2001; DANIAL & KORSMEYER, 2004).
O termo apoptose foi introduzido por KERR et al (1972), para definir um conjunto de
características morfológicas e bioquímicas, como redução de volume nuclear e celular, condensação
da cromatina (OTT et al., 2007), fragmentação do núcleo, formação de prolongamentos da
membrana plasmática (blebbs), fragmentação celular (corpos apoptóticos) (KIECHLE & ZHANG,
2002; KROEMER et al., 2005), exposição da fosfatidilserina (ZIEGLER & GROSCRURTH, 2004)
e mudanças na permeabilidade de membrana mitocondrial com perda do potencial de membrana
(RICCI & ZONG, 2006).
Em condições normais, os fosfolipídios são assimetricamente distribuídos, com o
fosfolipídio fosfatidilserina normalmente confinada na face citoplasmática da membrana plasmática
por um mecanismo de transporte ativo. Essa assimétrica distribuição pode ser perturbada
principalmente durante o processo de apoptose, na qual serve como um sinal primário para remoção
fagocítica de células apoptóticas (BALASUBRAMANIAN & SCHROIT, 2003). A externalização
da fosfatidilserina corresponde a um evento quase universal da apoptose, que ocorre poucas horas
após o estímulo apoptótico, e apresenta um alvo muito abundante (milhões de sítios por célula),
sendo facilmente acessível na face externa da membrana (BOERSMA et al., 2005;
BLANKENBERG, 2008).
Essa alteração da membrana plasmática levou KOOPMAN et al (1994) e outros a delinear
um ensaio de detecção da fosfatidilserina por coloração com isotiocianato fluoresceína (FITC)-
conjugado com Anexina V, uma proteína com forte afinidade natural para fosfatidilserina
(MARTIM et al. 1995; OZGEN et al., 2000; BRUMATI et al., 2008). Por ser capaz de distinguir
entre células apoptóticas e necróticas, as quais têm comprometida a integridade da membrana,
iodeto de propídeo (IP) foi adicionado. Por esse ensaio, células viáveis, apoptóticas e necróticas
podem ser discriminadas por microscopia de fluorescência ou citômetro de fluxo (VERMES et al.,
1995; BOERSMA et al., 2005; GROSSE et al., 2009).
Apoptose pode ser induzida por uma via extrínseca, envolvendo receptores de morte
presentes na superfície celular, ou por via intrínseca induzida por estímulos extracelular que
transmite um sinal para a mitocôndria (Figure 4) (BRENNER & KROEMER, 2000; KUO et al.,
2010). A via extrínseca é caracterizada por interação de um ligante com receptores de morte, que
desencadeia a formação de um complexo multimérico, seguido por recrutamento e ativação da
caspase-8 (LAVRIK et al., 2005).
15
A via intrínseca envolve alteração no potencial de membrana mitocondrial, levando a
permeabilização da membrana mitocondrial (MMP), e seguida por liberação do citocromo C
(CHIPUK et al., 2006; KROEMER et al., 2007). O citocromo C citosólico liga-se a proteína APAF-
1 (“Fator 1 ativador da apoptose”), desencadeando a formação de um complexo protéico chamado
de apoptosomo, o qual permite o recrutamento e ativação da caspase-9 (BAO & SHI, 2007). MMP
é regulada por membros da família Bcl-2 e Bax, que inibe ou promove a permeabilização da
membrana mitocondrial, respectivamente (REED, 2006). Ambas as vias, convergem para a caspase-
3 executora, cuja atividade produz as características morfológicas da apoptose (PORTER &
JANICKE, 1999). Então, MMP e principalmente, a perda do potencial de membrana mitocondrial
marca o ponto de não retorno do processo de morte celular (KROEMER, 2003). Por causa deste
papel central na cascata da apoptose, a avaliação do potencial de membrana mitocondrial fornece
uma importante direcionamento para o mecanismo de morte celular (GOTTLIEB & GRANVILLE,
2002), bem como a caracterização de novos agente indutores da apoptose (BUENZ et al., 2007).
Figura 4. Vias intrínseca e extrínseca da apoptose (Fonte: ANDERSEN et al., 2005)
3.6 Atividade antitumoral
Diversas proteínas produzidas em células de mamíferos ocorrem como glicoproteínas e
recentes avanços na glicobiologia têm revelado que suas cadeias de açúcar desempenham papéis
importantes no reconhecimento celular, sendo essenciais para manutenção da ordem social no
Estímulo apoptótico
(quimioterapia, UV)Intrínseco
Mitocôndria
Morte Celular
Extrínseco
Ligante
Receptor
Estímulo apoptótico
(quimioterapia, UV)Intrínseco
Mitocôndria
Morte Celular
Extrínseco
Ligante
Receptor
16
comportamento das células que constituem organismos multicelulares (KOBATA & AMANO,
2005).
Glicosilação é uma modificação pós-translational comum em proteínas celulares, ocorrendo
durante o desenvolvimento normal das células (LEHLE et al., 2006; CAMPBELL et al., 2007).
Entretanto, a biossíntese das cadeias de oligossacarídeos presentes nas glicoproteínas se encontra
freqüentemente alterada na diferenciação e transformação maligna. Algumas dessas mudanças
podem ser reconhecidas por proteínas ligantes de carboidratos, as lectinas (GAJ et al., 2009).
Nos últimos anos lectinas têm recebido atenção especial devido as suas importantes
atividades biológicas exploráveis, como citoaglutinação, sonda histoquímica, atividade mitogênica,
citotoxicidade, ação antiproliferativa e indutora da apoptose (SOBRAL, 2010; LAM & NG, 2010;
YAN et al., 2010; ZHANG et al., 2010).
O campo da pesquisa terapêutica vem se desenvolvendo no sentido de testar novas formas
de tratamento, bem como novas substâncias potencialmente eficazes contra as neoplasias (SAAD-
HOSNE et al., 2004). Nesse contexto, estudos experimentais utilizando lectinas de serpentes e
células tumorais têm revelado resultados promissores, indicando sua potencial atividade antitumoral
através da prevenção e/ou tratamento do câncer. Lectina de Bothrops jararacussu (PEREIRA-
BITTENCOURT et al, 1999) demonstrou uma potente atividade inibitória para linhagens celulares
de câncer humano (renal e pancreático). Em outro estudo, CARVALHO et al. (2001) sugeriram que
essa lectina pode servir como uma interessante ferramenta para combater a progressão do câncer,
por inibir o crescimento de linhagens celulares humanas de câncer de mama metastático (MDA-
MB-435) e carcinoma de ovário (OVCAR-5).
Recentes trabalhos mostram um efeito inibitório de lectinas de serpentes sobre diversas
funções mediadas por integrinas em células tumorais. Lebectina, uma lectina tipo-C isolada do
veneno de Macrovipera lebetina, apresenta notável atividade anti-integrina, sendo hábil para
prevenir adesão, migração, invasão e proliferação de células tumorais in vitro (SARRAY, et al.,
2004).
Lebecetina, uma segunda lectina tipo-C purificada desse mesmo veneno, também exerce o
mesmo efeito de lebectina sobre células tumorais, e ambas, provavelmente, atuam via interação com
a integrina α5β1 (SARRAY et al., 2001, 2007). De acordo com SARRAY et al. (2009) lebectina foi
capaz de controlar a adesão célula-célula mediada por N-caderina, e em conjunto com seu efeito
17
inibitório sobre a integrina α5β1, pode contribuir para o bloqueio da migração de células tumorais
previamente observado.
3.7. Atividade antibacteriana
A história da humanidade pode ser considerada, do ponto de vista médico, como uma luta
contra microorganismos que causam infecções e doenças. Embora, no início do século XX, doenças
infecciosas se apresentassem como a principal causa de morte no mundo, a introdução dos
antibióticos como a sulfa e penicilina em uso clínico em 1930 e 1940, respectivamente, teve um
notável impacto sobre o tratamento de infecções, diminuindo drasticamente a mortalidade
(COHEN, 2000; BUYNAK, 2004).
No entanto, a euforia no potencial controle das doenças infecciosas teve vida curta, pois
quase tão rapidamente como drogas antibacterianas foram implantadas, bactérias responderam por
manifestar várias formas de resistência (TENOVER et al., 2006; SPELLBERG et al., 2008).
Embora tenham surgido inúmeras classes de antibióticos, observa-se atualmente que existe pelo
menos uma cepa resistente a esses fármacos, seja em comunidade ou em hospitais (FLUIT &
SCHMITZ, 2004; MATLOW & MORRIS, 2009), acarretando um sério problema para a saúde
pública em todo o mundo (LEVI & MARSHALL, 2004; RAGHUNATH, 2008). Permanece,
portanto, a necessidade de novos antimicrobianos ou protótipos antibacterianos, que possam ser
empregados no delineamento de antibióticos contra bactérias multidroga-resistentes.
Peptídeos e proteínas têm sido avaliados como antibióticos para o controle de bactérias
patogênicas (NAIR et al., 2007; WANG et al., 2008). Lectinas tem assumido um importante papel
em interações com patógenos, através do reconhecimento específico de glicoconjugados presentes
na superfície das células bacterianas (TATENO et al., 2002), como peptidioglicanos, ácido teicóico,
lipopolisacarídeos e glicolipídeos (SCHAFFER & MESSNER, 2005; CLOUD-HANSEN et al.,
2006), resultando em atividade antibacteriana (MOURA et al., 2006). Além disso, lectinas podem
ativar proteínas ou enzimas associadas ao processo de eliminação de microorganismos patogênicos
(WANG et al., 2008). Em animais, são ferramentas notáveis para agregar e opsonizar patógenos
(FUJITA, 2002).
Devido a essa habilidade, diversas lectinas de animais vêm sendo avaliadas quanto ao seu
potencial antibacteriano. Lectina do anelídeo Perineresis nuntia foi capaz de inibir o crescimento de
18
Bacillus megeterium e Bacillus subtilis e foi sugerido que a proteína está envolvida na defesa imune
do hospedeiro (KAWSAR et al., 2010). CfLEC-2, uma lectina de Chlamys farreri, apresentou
atividade agregante quando testada frente a bactéria Staphylococcus haemolyticus, atuando como
mecanismo de defesa (ZHENG et al., 2008). A lectina de Holothuria scabra apresentou potente
atividade antimicrobiana contras às bactérias gram-negativas e gram-positivas, indicando seu efeito
antibacteriano de amplo espectro (GOWDA et al., 2008).
Em venenos de serpentes, lectinas tipo-C com ação antibacteriana também tem sido
purificadas em recentes estudos. Crotacetina, isolada de Crotalus durissus terrificus, inibiu
significativamente o crescimento de dois patógenos de plantas: X. axonopodis pv. passiflorae e C.
michiganensis michiganensis, bactérias gram-negativa e gram-positiva, respectivamente (RÁDIS-
BAPTISTA et al., 2006). Uma lectina tipo-C BmLec, isolada de Bothrops moojeni, exibiu potente
efeito antibacteriano contra Xanthomonas axonopodis pv. (bactéria gram-negativa), causando
vacuolização do citoplasma e ruptura da membrana celular (BARBOSA et al., 2010).
A relevância biológica dessa atividade, induzida por proteínas, no envenenamento por
serpentes não está clara, no entanto, alguns autores sugerem que possa contribuir para a baixa
freqüência de infecções bacterianas no local da picada (TALAN et al., 1991; TRABI et al., 2001;
SAMPAIO et al., 2010) ou como mecanismo de defesa contra microorganismos presentes na sua
presa durante a alimentação (SHIVIK, 2006).
3.8. Radiação
Radiação ionizante consiste de ondas eletromagnéticas resultantes de transições nucleares,
que se propagam com alta velocidade, possuindo energia suficiente para quebrar ligações químicas,
bem como a capacidade de promover a ionização e excitação nos meios com elevado poder de
penetração (BREWER, 2004, 2009). Radiação gamma ou raio gamma (γ) é um tipo de radiação
eletromagnética produzida por elementos radioativos, em um processo subatômico como a
aniquilação de um par pósitron-elétron. Possui comprimento de onda de alguns picometros e por
causa das altas energias, constituem um tipo de radiação capaz de penetrar mais profundamente na
matéria. Devido à sua elevada energia irradiação gamma emite elétron e gera radicais a partir da
quebra do isótopo cobalto 60, ao quais afetam a estrutura das proteínas intactas, seguido por um
ataque ao resíduo radio-sensível ou ligações (SEO et al., 2007).
19
Irradiação causa danos ou inativam proteínas através de dois diferentes mecanismos
(KEMPNER, 2001). Primeiro, pode provocar quebra de ligações covalentes em moléculas de
proteínas alvo, como resultado direto de um fóton de energia. Segundo, atua indiretamente via
radiólise da água, produzindo radicais livres e espécies reativas de oxigênio (ROS), responsável
pela maioria dos danos na proteína (ZBIKOWSKA, 2006).
A exposição de proteínas a radiação produz alterações em sua estrutura química e física por
fragmentação, “ cross-linking ”, agregação, desnaturação, formação de novos grupos reativos, e
oxidação por radicais oxigênios que são gerados na radiólise da água, resultando em distorções na
estrutura secundária e terciária, levando a diminuição ou perda da função biológica da proteína
(SHACTER, 2000; MOON & SONG, 2001).
Os radicais hidroxila e anion superóxido que são gerados por radiação podem modificar as
propriedades moleculares das proteínas, que resultam em alterações de proteínas por ligações
cruzadas covalentes em proteínas formadas após a irradiação (SHAWRANG et al., 2008). O efeito
da irradiação sobre a conformação das proteínas depende de vários fatores, como concentração da
proteína, a presença de oxigênio e da sua estrutura quaternária (LEE et al., 2003; GABER, 2005).
Radiação gamma vem sendo empregada como agente atenuante de venenos ofídicos e
toxinas isoladas, resultando em um produto de baixa ou nenhuma toxicidade, preservando, porém,
suas propriedades imunológicas. Crotamina, uma toxina de Crotalus durissus terrificus, irradiada
na dose de 2.0 kGy foi duas vezes menos tóxica para camundongos que a crotamina nativa (BONI-
MITAKE et al., 2001). SOUZA et al. (2002) demonstraram que o veneno irradiado de Bothrops
jararacussu, o qual apresenta miotoxinas em sua composição, não exibiu efeitos miotóxicos nas
preparações musculares in vitro, quando comparado com o veneno nativo. Em outro estudo,
CASARE et al. (2006) observaram que Crotoxina quando submetida à radiação, em diferentes
doses, apresentou diminuição significativa na mortalidade de camundongos. O tratamento de
Bothropstoxina-1 com radiação na dose de 2.0 kGy promoveu modificações estruturais na toxina,
no entanto, manteve muita das propriedades antigênicas da proteína nativa. Esses autores
verificaram também que a toxina irradiada induziu altos títulos de IgG2, sugerindo que células Th1
estão envolvidas na resposta imune (CAPRONI et al., 2009).
Em recente estudo, irradiação gamma foi utilizada como um tratamento alternativo para
abolir alergenicidade de lectinas em alimentos. VAZ et al. (2010) observaram que a irradiação da
lectina isolada da entrecasca de Sebastiana jacobinenses (SejaBL), em altas doses (acima de 1kGy),
20
induziu uma significante perda da atividade hemaglutinante e causou alterações estruturais na
proteína, incluindo fragmentação e agregação após irradiação.
3.9. Serpente Bothrops leucurus
No Brasil, serpentes do gênero Bothrops são responsáveis por 90% de todos os acidentes
ofídicos nos quais a serpente é identificada. Bothrops leucurus (jararaca-do-rabo-branco) é uma
serpente que apresenta uma ampla distribuição na costa brasileira, do estado do Maranhão até o
Espírito Santo (LIRA-DA-SILVA, 2009). Na Bahia, essa espécie é a principal causadora de
envenenamentos por picadas de serpentes em humanos (LIRA-DA-SILVA & NUNES, 1993; MISE
et al., 2007), representando um sério problema médico para a região Nordeste do Brasil (Figura 5).
Figura 5. Serpente Bothrops leucurus (Fonte: LIRA-DA-SILVA et al. 2009)
Em um estudo sobre atividades biológicas de venenos de serpente da América do Sul,
SANCHES et al. (1992) demonstraram que a letalidade, como também as atividades
edematogênica, coagulante, hemorrágica e necrosante de B. leucurus foram semelhantes às de
várias outras espécies botrópicas, incluindo B. jararaca. Em adição, B. leucurus exibiu ação
neuromuscular e miotóxica em preparações de nervo-músculo de aves (PRIANTI JR et al., 2003).
Componentes implicados em uma variedade de efeitos tóxicos locais, assim como em
profundas perturbações no sistema hemostático das vítimas foram isolados do veneno de B.
21
leucurus. BELLO et al. (2006) purificou uma proteinase fibrinolítica, denominada leuc-a, a qual
exibiu efeito inibitório sobre agregação plaquetária induzida por ADP. Uma L-amino oxidase,
purificada desse veneno, inibiu agregação plaquetária estimulada por colágeno, contribuindo para o
sangramento típico de pessoas picadas por essa serpente (SILVA et al., 2007). HIGUCHI et al.
(2007) demonstrou a presença duas fosfolipases no veneno de B. leucurus, e observou que essas
inibiram significantemente a coagulação e foram hábeis em estimular o crescimento tumoral. Outras
toxinas também foram isoladas, incluindo uma enzima trombina-like (MAGALHÃES et al., 2007),
uma P-III metaloproteinase hemorrágica (SANCHEZ et al., 2007), uma metaloproteinase não
hemorrágica, com atividade edematogênica e trombolítica (GREMSKI et al., 2007). Recentemente,
foi purificado uma metaloproteinase fibrinogenolítica não hemorrágica (BleucMP), com habilidade
para diminuir significantemente o nível de fibrinogênio plasmático provocado por incoagulabilidade
sanguínea (Gomes et al., 2010).
22
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5 ARTIGO CIENTÍFICO I
Purification of a lectin with antibacterial activity from Bothrops leucurus snake venom
Artigo submetido ao periódico Comparative Biochemistry and Physiology - Part B:
Biochemistry & Molecular Biology
45
Erika dos Santos Nunesa,*, Mary Angela Aranda de Souzaa, Antônio Fernando de Melo Vaza,
Giselly Maria de Sá Santanaa, Francis Soares Gomesa, Luana Cassandra Breitenbach Barroso
Coelhoa, Patrícia Maria Guedes Paivaa, Rejane Maria Lira da Silvab, Rosemeire Aparecida Silva-
Luccac,e, Maria Luiza Vilela Olivac, Miriam Camargo Guarnierid, Maria Tereza dos Santos Correiaa
aDepartamento de Bioquímica, Universidade Federal de Pernambuco, Avenida Professor Moraes
Rêgo, s/n, Cidade Universitária, 50670-420, Recife, Pernambuco, Brazil
bDepartamento de Zoologia, Universidade Federal da Bahia, Rua Barão de Geremoabo, s/n,
Campus de Ondina, 40170-210, Salvador, Bahia, Brazil.
cDepartamento de Bioquímica, Universidade Federal de São Paulo, Rua Três de Maio, 100, Vila
Clementino, 04044-020, São Paulo, Brazil.
dDepartamento de Zoologia, Universidade Federal de Pernambuco, Avenida Professor Moraes
Rêgo, s/n, Cidade Universitária, 50670-420, Recife, Pernambuco, Brazil.
eCentro de Engenharias e Ciências Exatas, Universidade Estadual do Oeste do Paraná, Rua da
Faculdade, 645, Jardim La Salle, 85903-000, Toledo, Paraná, Brazil.
*Corresponding author. Phone: +558121268540; Fax: +558121268576
E-mail address: [email protected]
ABSTRACT
A novel lectin was isolated from Bothrops leucurus snake venom using a combination of affinity
and gel filtration chromatographies. The lectin (BlL) agglutinated glutaraldehyde-treated rabbit and
human erythrocytes with preference for rabbit erythrocytes. Galactose, raffinose, lactose, fetal
bovine serum and casein inhibited lectin-induced rabbit erythrocyte agglutination. BlL, with a
molecular mass of 30 kDa and composed of two subunits of 15 kDa, showed dependence on
calcium. BlL is an acidic protein with highest activity over the pH range of 4.0-7.0 and stable under
heating to 70 °C. Fluorescence emission spectra showed tryptophan residues partially buried within
the lectin structure. The percentages of secondary structure revealed by circular dichroism were 1%
α-helix, 44% β-sheet, 24% β-turn and 31% unordered. BlL showed effective antibacterial activity
46
against Gram-positive bacteria Staphylococcus aureus, Enterococcus faecalis and Bacillus subtilis
with minimal inhibitory concentrations of 31.25, 62.25 and 125 µg/mL, respectively. In conclusion,
B. leucurus snake venom contains a galactoside-binding lectin with antibacterial activity.
Keywords: antibacterial activity; fluorescence; circular dichroism; Bothrops leucurus; lectin; snake
venom.
1. Introduction
Lectins are proteins or glycoproteins that bind reversibly to carbohydrates and
glycoconjugates (De-Simone et al., 2006). Lectins have been found in a wide range of organisms
from microorganisms to plants and animals (Utarabhand et al., 2007). C-type lectins are a large
family of Ca2+ dependent lectins. Animal C-type lectins can be classified into 17 groups according
to structural and functional characteristics (Zelensky and Gready, 2005). Snake venoms contain C-
type lectins included in group VII, which are true sugar-binding lectins composed by homodimers
or homooligomers and with Ca2+ and generally galactose binding properties (Clemetson, 2010).
Snake venoms also contain C-type lectin-like proteins which are heterodimers or oligomeric
complexes of heterodimers called snaclecs (snake venom C-type lectins); this group is more
abundant and possesses a loop-swapping or higher order multimerization (Ogawa et al., 2005;
Clemetson et al., 2009; Clemetson, 2010). Snake venom lectins are able to inhibit or activate
specific platelet membrane receptors and blood coagulation factors (Morita, 2004, 2005; Ogawa et
al., 2005; Wang, 2008) and can promote a diversity of biological effects, such as lymphocyte
proliferation (Mastro et al., 1986), induction of edema (Lomonte et al., 1990; Panunto et al., 2006),
induction of Ca2+ release from the sarcoplasmic reticulum (Ohkura et al., 1996), inhibition of cancer
cell proliferation (Pereira-Bittencourt et al., 1999), erythrocyte agglutination in vitro (Kassab et al.,
2001), cytotoxicity to tumors and endothelial cell lines (Carvalho et al., 2001), renal effects (Havt et
al., 2005) and induction of rolling of leukocytes (Elífio-Esposito et al., 2007).
Glycoconjugates present on bacterial cell surfaces, such as peptidoglycans,
lipopolysaccharides and teichoic acids, constitute potential lectin targets (Lee et al., 1998; Santi-
Gadelha et al., 2006). Recently, it was reported that snake venom lectins are able to inhibit growth
of phytopathogenic bacteria (Rádis-Baptista et al., 2006; Barbosa et al., 2010); however, the
interactions between snake venom lectins and human pathogenic bacteria have not been studied.
Bothrops leucurus (white-tailed-jararaca) is an important venomous snake that inhabits
northeastern Brazil. B. leucurus was responsible for all cases of envenoming after snakebite
47
recorded in the metropolitan region of Salvador (State of Bahia, northeastern Brazil) from January
to June 1990 (Lira-da-Silva and Nunes, 1993) and an epidemiological study in Bahia in 2001
revealed that this species was responsible for all confirmed cases of envenoming by Bothrops
species in this state (Mise et al., 2007). Recently, active components from B. leucurus venom were
isolated, including a fibrinolytic proteinase (Bello et al., 2006), a thrombin-like enzyme (Magalhães
et al., 2007), phospholipase A2 (Higuchi et al., 2007), a P-III metalloproteinase (Sanchez et al.,
2007), L-amino acid oxidases (Silva et al., 2007; Torres et al., 2010) as well as the
metalloproteinases leucurolysin-a (Gremski et al., 2007; Ferreira et al., 2009) and BleucMP (Gomes
et al., 2011).
This paper reports the purification, characterization and antibacterial activity of a novel
galactoside-binding lectin isolated from snake venom of B. leucurus, a species with great medical
importance in northeastern Brazil.
2. Material and Methods
2.1. Chemicals
Reference samples of 4.4'-Bis 1-anilinonaphthalene 8-sulfonate (bis-ANS) were purchased
from Molecular Probes Inc., USA. Broad-range protein molecular mass markers, sugars and
glycoproteins were purchased from Sigma-Aldrich (USA). All the solvents and other chemicals
used were of analytical grade from Sigma-Aldrich (USA) or Merck (Germany). All solutions were
prepared with water purified by the Milli-Q® system (Millipore).
2.2. B. leucurus venom
B. leucurus venom was kindly supplied by the Núcleo Regional de Ofiologia e Animais
Peçonhentos da Bahia, Universidade Federal da Bahia, Salvador, Bahia, Brazil.
48
2.3. Protein content and neutral carbohydrate analysis
Protein concentration was determined according to Bradford (1976) using bovine serum
albumin as a standard. Neutral carbohydrate content was determined by the phenol-sulphuric acid
method (Dubois et al., 1956) using a mannose as a standard.
2.4. Hemagglutinating activity and carbohydrate specificity
Hemagglutinating activity (HA) was assessed in microtiter plates according to Correia and
Coelho (1995) using rabbit and human A, B, AB and O-type erythrocyte suspensions (2.5% v/v; 50
µL) treated with glutaraldehyde (Bing et al., 1967). HA was defined as the lowest lectin
concentration able to promote erythrocyte agglutination. Specific hemagglutinating activity (SHA)
corresponded to the ratio between HA and protein concentration (mg). Carbohydrate binding
specificity was evaluated by determining HA in the presence of sugars (D-galactose, D-glucose, D-
fructose, D-lactose, D-mannose, methyl-α-D-glucopyranoside, D-arabinose, L-rhamnose methyl-α-
D-mannopyranoside, N-acetyl-D-glucosamine, D-xylose and L-raffinose) and glycoproteins
(asialofetuin, casein, fetuin and fetal bovine serum).
2.5. Purification of B. leucurus venom lectin
Lyophilized B. leucurus venom (30 mg) was dissolved in 1 mL of calcium-Tris-buffered
saline buffer (CTBS; 20 mM Tris-HCl, 150 mM NaCl and 5 mM CaCl2, pH 7.5) and centrifuged
(2000 g, 5 min, 25°C) to remove insoluble material. The resulting supernatant was applied to a
column (10 x 1.0 cm) of guar gel previously equilibrated with CTBS at a flow rate of 10 mL/h.
Protein elution was monitored by absorbance at 280 nm. After washing to remove unbound
proteins, the adsorbed proteins were eluted from the column with 200 mM galactose in CTBS.
Adsorbed fractions with HA were pooled, dialyzed, lyophilized and applied to a Superdex 75 HR
10/300 GL column coupled to an ÄKTATM purifier system (GE Pharmacia). The column was
equilibrated and eluted with 50 mM Tris–HCl buffer (pH 8.0) containing 150 mM NaCl at a flow
rate of 0.5 mL/min; fractions of 1 mL were collected and protein elution was monitored by
absorbance at 280 nm. Subsequently, active peak from Superdex 75 chromatography (B. leucurus
lectin; BlL) was submitted to reverse-phase chromatography in a C-4 column (Vydac-Protein
Peptide Ultrasphere) performed on an HPLC system (Shimadzu LC-10AD-Tokyo, Japan), with
elution monitored at 280 nm. The column was equilibrated with 0.1% TFA (solvent A) and eluted
49
using 90% acetonitrile/10% H2O/0.1% TFA (solvent B) in a non-linear gradient, where B = 0% at t
= 5 min, 45% at t = 10 min, 50% at t = 30 min and 100% at t = 35 min.
2.6. Effects of divalent ions, pH and temperature on HA
To evaluate the effect of divalent cations on BlL-induced HA, the lectin was previously
dialyzed against 5 mM EDTA (16 h at 4°C) followed by 150 mM NaCl (6 h at 4°C) to eliminate
EDTA. Subsequently, the HA of dialyzed BlL was evaluated in the presence of 50, 100 and 200
mM Ca2+, Mn2+ or Mg2+ in 150 mM NaCl. The effects of pH and temperature on HA were evaluated
by incubating (45 min at 25°C) of BlL in selected buffers (10 mM citrate phosphate, pH 4.0-6.0; 10
mM sodium phosphate, pH 7.0; 10 mM Tris-HCl, pH 8.0-9.0) or after heating (30 min) at 30, 40,
50, 60, 70, 80, 90 and 100°C.
2.7. Polyacrylamide gel electrophoresis (PAGE)
BlL was evaluated by native PAGE for basic [15% (w/v) gel] or acidic [15% (w/v) gel]
proteins according to Reisfeld et al. (1962) and Davis (1964), respectively. Electrophoresis in the
presence of SDS and β-mercaptoethanol was performed on 15% (w/v) gel according to Laemmli
(1970). Polypeptide bands were stained with Coomassie Brilliant Blue in 10% acetic acid (0.02%,
v/v). Glycoprotein staining was performed using the periodic acid-Schiff method (Zacharius et al.,
1969).
2.8. Analysis of polypeptide chains
Polypeptide chain analyses were performed after reduction of disulfide bridges and
alkylation. Lyophilized samples were reduced by the Friedman reaction (Friedman et al., 1970)
with some modifications: BlL (0.5 mg) was dissolved in 250 µL of a solution containing 50 mM
Tris–HCl, pH 8.6, 6 M urea, 10 mM EDTA and 179 mM DTT; the mixture was incubated for 3 h at
37°C in the dark before N2 purging. Free sulphydryl groups were then exposed to 100 µL of
iodoacetate and the reaction continued for another 2 h under the same initial conditions. Iodoacetate
derivative chains were desalted and separated by HPLC on a reverse-phase C4 column with the
elution profile monitored at 280 nm.
50
2.9. Fluorescence spectroscopy
Intrinsic fluorescence emission of BlL in solution (0.07 mg/mL in 10 mM phosphate buffer
pH 7.0) was measured at 25°C using a spectrofluorimeter (JASCO FP-6300, Tokyo, Japan) and a
cuvette (1-cm pathlength rectangular quartz). The excitation wavelengths were 280 and 295 nm;
emission spectra were recorded at a range of 305 to 450 nm with band passes of 5 nm.
2.10. Circular dichroism (CD) measurements
CD measurements were carried out on a J-810 JASCO spectropolarimeter. The instrument
was calibrated with D-10-camphorsulfonic acid. The measurement was carried out at 25°C with a
protein concentration of a 0.250 mg/mL (8 µM) in a 1 mm pathlength cuvette. C spectrum was
recorded at the 191-250 nm range as an average of eight scans. The results were expressed as the
mean residue ellipticity, [θ], defined as [θ]= θobs/(10.C.l.n.), where θobs is the CD in millidegrees, C
is the protein concentration (M), l is the pathlength of the cuvette (cm) and n is the number of amino
acid residues assuming a mean number of 272 residues. The CDPro software was used to estimate
the fractions of secondary structures (Sreerama and Woody, 2000) and the Cluster program was
used to determine tertiary structure class of BlL (Sreerama et. al., 2001).
2.11. Antibacterial activity
Gram-positive (Bacillus subtilis ATCC-6633, Staphylococcus aureus ATCC-6538 and
Enterococcus faecalis ATCC-6057) and Gram-negative (Escherichia coli ATCC-25922 and
Klebsiella pneumoniae ATCC-29665) bacterial strains were provided by the Departamento de
Antibióticos, Universidade Federal de Pernambuco, Brazil. Stationary cultures were maintained in
nutrient agar and stored at 4°C.
Bacteria were cultured in nutrient broth and incubated under continuous shaking at 37°C
overnight. The culture concentrations were turbidimetrically adjusted at 600 nm to 105–106 colony
forming units (CFU)/mL. Purified lectin (BlL) was diluted (1:2048) in a microtiter plate containing
nutrient broth (50 µL per well). Subsequently, 20 µL of bacterial suspension was applied in each
well and the plate was incubated at 37°C for 24 h. After incubation, the optical density at 490 nm
(OD490) was measured using a spectrophotometer for microplates. The assays were performed in
triplicate. The minimal inhibitory concentration (MIC) corresponded to the lowest lectin
51
concentration able to inhibit the growth of 50% or more of microorganisms relative to the negative
control (Amsterdam, 1996). Thereafter, aliquots (20 µL) of each well in which inhibitory activity
was observed were transferred to petri plates containing nutrient agar. The plates were incubated at
37°C for 24 h. The minimal bactericide concentration (MBC) corresponded to the lowest
concentration of lectin able to reduce the number of CFU to 0.1% relative to the negative control.
Antibacterial activity of BlL was also determined in presence of 200 mM galactose.
3. Results and Discussion
Crude extract of B. leucurus venom showed high lectin activity (SHA 136.5 units/mg)
towards rabbit erythrocytes. The inhibition of HA by galactose suggested the presence of a
galactoside-binding lectin; this result encouraged us to evaluate the use of affinity chromatography
on guar gel matrix to purify lectin.
Lectin activity from crude venom adsorbed on guar gel column and only one active (SHA of
29,257) peak was detected after elution with 200 mM galactose (Figure 1A). The use of guar gel
was an inexpensive and innovative protocol for isolation of a snake venom lectin. Guar gum
consists of straight chains of mannose substituted with α(1-6) galactose residues and is a versatile
and viable matrix for the isolation of D-galactopyranosyl- and N-acetyl-galactosaminyl-binding
lectins (Lonngren and Goldstein, 1976; Gupta et al., 1979). Guar gum has been used as an efficient
and inexpensive affinity support for the purification of lectins from Bauhinia monandra leaves
(Coelho and Silva, 2000) and Moringa oleifera seeds (Santos et al., 2009) as well as from the alga
Vidalia obtusiloba (Melo et al., 2004).
The adsorbed fractions from guar gel affinity chromatography were loaded onto gel
filtration column (Figure 1B); three peaks can be seen in the chromatographic profile. The lectin (B.
leucurus lectin; BlL) was eluted at 18 mL, corresponding to an apparent molecular weight around 8
kDa; however, this major peak (SHA 10,240) showed a molecular mass of 30 kDa in SDS-PAGE.
In the presence of reducing agent β-mercaptoethanol, BlL was revealed to be a dimeric protein
composed of two subunits with a molecular mass of 15 kDa (Figure 1C). Because lectins may
interact in undesirable ways with Superdex beads, a possible interaction of BlL with the stationary
phase may have affected the retention time and apparent molecular weight of BlL in gel filtration
chromatography as well as may be responsible for the reduction of SHA observed (Table 1).
52
Evaluation of BlL by native electrophoresis showed a single polypeptide band in PAGE for
acidic proteins (Figure 1C). No polypeptide band was detected in native PAGE for basic proteins.
As showed by Lomonte et al. (1990) using isoelectric focusing, other snake venom lectins are
characterized as acidic proteins. BlL was eluted from C-4 column with about 50% of acetonitrile
(Figure 1D). The reduction and alkylation reactions of BlL were performed using DTT. After
desalting on the C-4 column, only one peak was obtained (Figure 1E), suggesting the presence of
homodimeric chains covalently linked by disulfide bridges; this result agrees with that observed in
electrophoresis. Several lectins from snake venoms are constituted by disulfide-linked homodimers,
such as the lectins from Agkistrodon piscivorus piscivorus and Crotalus ruber (Komori et al., 1999;
Hamako et al., 2007). However, the crystal structure of a galactoside-binding lectin from Crotalus
atrox revealed a decameric structure composed of two 5-fold symmetric pentamers (Walker et al.,
2004).
Table 1 summarizes the BlL purification. The amount of protein recovered after gel
filtration chromatography was less than 1%. The content of BlL in snake venom (< 1%) was similar
to those found for other lectins isolated from snake venoms (Ogilvie et al., 1986; Lomonte et al.,
1990; Carvalho et al., 1998; Nikai et al., 2000; Guimarães-Gomes et al., 2004).
Table 1
Summary of B. leucurus lectin (BlL) purification
Sample
Protein (mg)
Total HAa
SHA (HA/mg)
Purification (fold)b
Crude venom
30
4,096
136.5
1
Affinity chromatography 0.21 6,144 29,257 214.3
Gel filtration 0.1 1,024 10,240 75 aHemagglutinating activity (HA) with rabbit erythrocytes. SHA: specific HA (ratio between HA and
protein content). bPurification fold corresponds to the ratio between SHA of BlL and SHA of crude
venom. The data corresponds to one purification process.
The HA of BlL was abolished after treatment with the chelating agent EDTA (5 mM); Mn2+
and Mg2+ did not restore BlL HA but the activity was gradually increase by addition of Ca2+ and
completely restored when this ion was added at 200 mM (Figure 1F); this result indicates that Ca2+
is essential for the carbohydrate-recognizing property of BlL. The homodimeric structure and
53
calcium dependence indicate that BlL may be included in the group of true galactoside-binding
lectins from snake venoms and did not belong to the group of snaclecs described by Clemetson et
al. (2009). Several other lectins isolated from snake venoms are dependent on calcium (Ozeki et al.,
1994; Kassab et al., 2001; Guimarães-Gomes et al., 2004; Clemetson, 2010). However, this
statement can only be confirmed after N-terminal sequencing and homology studies.
Fig. 1. (A) Purification of BlL by affinity chromatography of B. leucurus venom (30 mg of protein)
on a guar gel column. Elution with CTBS buffer (-■-) followed by 200 mM D-galactose (-▲-;
arrow). Specific hemagglutinating activity (SHA, -○-). (B) Purification of BlL by chromatography
in Superdex 75 column coupled to an ÄKTA purifier system. (C) SDS-PAGE of BlL. (MW)
molecular weight markers; BlL under non-reducing (lane 1) or reducing conditions (lane 2);
54
electrophoresis under native conditions for acidic proteins (lane 3). (D) Reverse phase HPLC on a
C4 column. (E) BlL chain separation after desalting on a C4 column. The column was equilibrated
with 0.1% TFA (solvent A) and eluted using 90% acetonitrile/10% H2O/0.1% TFA (solvent B) in a
non-linear gradient, where B = 0% at t = 5 min, 45% at t = 10 min, 50% at t = 30 min and 100% at t
= 35 min. (F) Hemagglutinating activity (HA) of EDTA-treated BlL after addition of Ca2+ in
different concentrations.
BlL was not detected by glycoprotein staining using periodic acid-Schiff´s reagent and no
carbohydrate was detected using the phenol-sulphuric acid method. The absence of carbohydrate
moiety was also reported for lectins from the snakes Bothrops atrox, Lachesis muta, Dendroaspis
jamesonii, and Bothrops jararacussu (Gartner et al., 1980; Gartner and Ogilvie, 1984; Ogilvie et al.
1986; Carvalho et al., 2002). BlL recognized the structure of saccharides comprising the surface of
erythrocyte membranes since it agglutinated glutaraldehyde-treated erythrocytes from rabbits (SHA
10,240) and human types A, B and O (SHA of 320, 320 and 160, respectively). The difference in
erythrocyte agglutination, depending on cell type (A, B, AB and O) and species of origin, may be
due to the presence of different glycoproteins on the erythrocyte surface. Similar to other lectins
from snakes, HA of BlL was abolished in the presence of galactose, lactose and raffinose (Table 2),
indicating that that BlL is a galactoside-binding protein. Inhibition assays revealed that BlL was
partially inhibited by fetal bovine serum and casein, but not by fetuin and asialofetuin (Table 2).
Casein is a protein with a small carbohydrate fraction and contains phosphorus in its structure
(Roman and Sgarbieri, 2005). Two N-glycosidic carbohydrate complex-type rich in galactose are
present in the structure of α-fetoprotein, a protein found in fetal bovine serum (Krusius and
Ruoslahti, 1982); the presence of galactose may be responsible for inhibition of BlL in presence of
fetal bovine serum. The results indicate that BlL recognizes complex glycoproteins.
55
Table 2
Inhibition of hemagglutinating activity of BlL by carbohydrates and glycoproteins
Inhibitor Minimal inhibitory concentrationa
D-galactose 0.78
D-lactose 1.56
L-raffinose 1.56
D-glucose 12.5
N-acetyl-D-glucosamine NI
D-arabinose NI
D-mannose NI
D-fructose NI
D-xylose NI
L-rhamnose NI
Methyl-α-D-mannopyranoside NI
Methyl-α-D-glucopyranoside NI
Asialofetuin NI
Fetuin NI
Casein 0.25
Fetal bovine serum 0.25
Assays were performed with rabbit erythrocytes and in triplicate. aMinimal inhibitory concentration
corresponds to lowest carbohydrate and glycoproteins concentrations able to inhibit HA of BlL.
Highest carbohydrates and glycoproteins concentrations used were 200 mM and 0.5 mg/mL,
respectively. NI indicates that no inhibition was detected. SHA of BlL in absence of sugars or
glycoproteins: 10,240.
56
BlL HA was heat-stable up to 70°C, with total loss of activity after heating to 80°C
indicating that HA depends on BlL native conformation. The HA of BlL was not affected at a pH
range of 4.0 to 7.0, unlike Bothrops jararacussu lectin, which was more active in neutral pH (Elífio-
Esposito et al., 2007). The intrinsic protein fluorescence spectra of BlL (Figure 2A) revealed a
single major peak at 344 nm. Tryptophan residues exposed to water show a maximal fluorescence
emission at wavelengths around 340-350 nm whereas completely buried residues fluoresce at about
330 nm. The displacement of the mass center of the aromatic residues indicates partially buried
hydrophobic domains within the lectin structure. C-type lectins are usually a dimer of two identical
polypeptides, each containing two tryptophan residues and one tyrosine residue (Morita, 2005). The
individual subunits are able to bind carbohydrates but for the lectin-like function they need at least
bivalency, which is achieved through a simple interchain disulfide linkage. Although dimerization
is essential, the two tryptophan residues and the tyrosin are essential to stabilize intra-subunit
contacts and for biological activities in C-type lectins (Doyle and Kini, 2009).
The CD spectrum of BIL (Figure 2B) indicated that BlL possessed a large amount of β-sheet
structure, characterized by a maximum at approximately 195 nm and a minimum at the range 216-
220 nm (Venyaminov and Yang, 1996). Analysis of the secondary structure content using CDPro
software yielded following results: 1.0% α-helix, 44% β-sheet, 24% β-turn, 31% unordered
structures and an RMS (root-mean-square) of 2.0%. Cluster analysis classified BlL as a β-class
protein (proteins containing mainly β structure), corroborated by results of CDPro analysis (68% β
structures). The secondary structure content of BlL was similar to that determined for Lachesis
muta snake venom lectin (Aragón-Ortíz et al., 1989); however, the majority of lectins from snake
venoms belong to the α+β class, such as the C-type lectin from B. jararacussu venom which
possesses 18.8% α-helix and 32.2% β-sheet (Silva Jr. et al., 2008).
57
Fig.2. (A) Intrinsic fluorescence emission of BlL excited at 280 nm (---) and 295 nm (—). (B) CD
spectrum of BIL in 50 mM phosphate buffer, pH 7.2, at 25°C. Measurements are the average of
eight scans using a solution containing 0.25 mg of protein/mL. CD spectrum deconvolution using
CDPro software calculated 1% α-helix, 44% β-sheet, 24% β-turn, 31% unordered structures and an
RMS of 2%.
In the present study, antibacterial assays demonstrated that BlL exhibited antibacterial
effects against the human pathogenic Gram-positive bacteria S. aureus, E. faecalis and B. subtilis.
Minimal inhibitory (MIC) and minimum bactericidal (MBC) concentrations were determined for
BlL (Table 3). The lectin was not effective against Gram-negative bacteria E. coli and K.
pneumoniae. A possible reason for the difference in susceptibility is the difficulty that BlL
encounters in crossing the outer cell wall of Gram-negative bacteria to reach the periplasmic space.
BlL may interact with the peptidoglycan present in Gram-positive bacteria cell wall while the lectin
may not be able to bind peptidoglycans of Gram-negative bacteria whether it does not enter in the
periplasmic space. BlL showed absence of antimicrobial activity in presence of 200 mM galactose
assuring that the antibacterial effect involves the carbohydrate-binding property of lectin. MIC
values of BlL against S. aureus and E. faecalis show the clinical relevance of lectin since these
concentrations are below the range of 64-100 µg/mL (Gibbons, 2004). The MBC of BlL against B.
subtilis (250 µg/mL) was lower than that (500 µg/mL) described for Phthirusa pyrifolia leaf lectin
(Costa et al., 2010).
58
The MBC for bactericidal drugs is generally the same or not more than four-fold higher than
the MIC. In contrast, the MBC of bacteriostatic drugs are many-fold higher than their MIC
(Levison, 2004). The term tolerant is applied to bacterial strains which growth stops in the presence
an antimicrobial concentration but do not rapidly die leading to high values of MBC (Charpentier
and Tuomanen, 2000). On the basis of MBC/MIC ratio, S. aureus showed to be tolerant to BlL
since the MBC was 15.8-fold greater than MIC (Ishida et al., 1982). On the other hand, Canillac and
Mourey (2001) reported that if the MBC/MIC ratio was found to be less than or equal to 4, the
bacteria were considered to be susceptible. Therefore, B. subtilis was susceptible to BlL. Recently,
Torres et al. (2010) showed that Bothrops leucurus total venom (BleuTV) inhibited the growth of S.
aureus. Therefore, according to our results we can suggest that BlL is involved in the antibacterial
activity of the venom.
Table 3
Minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of BlL
Bacteria MICa MBCa
Staphylococcus aureus (+) 31.5 500
Enterococcus faecalis (+) 62.5 330
Bacillus subtilis (+) 125 250
Escherichia coli (-) ND ND
Klebsiella pneumoniae (-) ND ND
aMIC and MBC expressed as µg/mL of lectin. ND: antibacterial activity not detected at 1000
µg/mL of BlL. Gram-positive (+) and Gram-negative (-) bacteria.
In conclusion, a new galactoside-binding lectin was isolated from B. leucurus venom. BlL
showed antibacterial activity against human pathogenic Gram-positive bacteria. Further studies are
required to determine the mechanisms involved in this bactericidal activity.
59
Acknowledgements
The authors express their gratitude to the Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq) and to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) for research grants. The authors thank Maria Barbosa Reis da Silva and João Antônio
Virgínio for technical assistance, and Scott V. Heald for reviewing the English of the manuscript.
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67
6 ARTIGO CIENTÍFICO II
Cytotoxic effect and apoptosis induction by Bothrops leucurus venom lectin on
tumor cell lines
Artigo a ser submetido ao periódico Toxicology in vitro
68
Erika S. Nunesa,*; Mary A.A. Souzaa; Antônio F.M. Vaza; Teresinha G. Silvab; Jaciana S. Aguiarb;
André M. Batistac; Maria M.P. Guerrac; Miriam C. Guarnierid; Luana C.B.B. Coelhoa; Maria T.S.
Correiaa
aDepartamento de Bioquímica, CCB, Universidade Federal de Pernambuco, Avenida Professor
Moraes Rêgo, s/n, Cidade Universitária, 50670-420, Recife, Pernambuco, Brazil
bDepartamento de Antibióticos, Universidade Federal de Pernambuco, Avenida Prof. Arthur de Sá,
s/n, Cidade Universitária, 50670-901, Recife, Pernambuco, Brazil.
cDepartamento de Medicina Veterinária, Rua Dom Manoel Medeiros, s/n, Dois Irmãos, 52171-900,
Universidade Federal Rural de Pernambuco, Recife, Pernambuco, Brazil.
dDepartamento de Zoologia, CCB, Universidade Federal de Pernambuco, Avenida Professor
Moraes Rêgo, s/n, Cidade Universitária, 50670-420, Recife, Pernambuco, Brazil.
*Corresponding author. Phone: +558121268540; Fax: +558121268576.
E-mail address: [email protected]
ABSTRACT
Neoplastic transformation results from cell changes that escaped of control mechanisms leading to
an increased growth potential as well as alterations in cell surface and other attributes that give to
the tumor cells the ability to invade and metastasize. These transformations are often related to
changes in cell surface glycoconjugates which can be detected by lectins. In this study we evaluated
the anti-tumor potential of BlL, a galactoside-binding lectin isolated from Bothrops leucurus
venom. BlL was evaluated for its cytotoxicity using MTT assay against tumor cell lines (K562,
NCI-292 and Hep-2) and hemolysis assay on mice erythrocytes. The annexin-V and JC-1 assays
were used to determine the phosphatidylserine externalization and mitochondrial membrane
potential in K562 cells, respectively. BlL exhibited cytotoxic activity against all tumor cell lines
tested with IC50 values of 6.63, 11.75 and 15.42 µg/mL for Hep-2, NCI-H292 and K562,
respectively, but was not able to induce hemolysis in mice erythrocytes. BlL treatment induced
phosphatidylserine externalization and mitochondrial depolarization, indicating cell death by
69
apoptosis.Our results suggest that BlL has a promising potential for application in cancer therapy
and/or diagnosis.
Keywords: antitumor activity; apoptosis; Bothrops leucurus; lectin; cytotoxicity; mitochondria.
1. Introduction
Cancer is the second cause of mortality worldwide (Hemalswarya and Doble, 2006) and in
Brazil the estimate for the year 2010 (also valid for 2011) is the occurrence of 489,270 new cases of
cancer (Instituto Nacional do Câncer, 2009). Etiologic factors associated with cancer include
improper diet, genetic predisposition and environment conditions; the majority of human cancers
result from exposure to environmental carcinogens (Reddy et al., 2003).
Glycosylation is the most frequent form of post-translational modifications of proteins
(Chen et al., 2007; Rek et al., 2009) and alterations in the pattern of cell surface glycoconjugates
are remarkable characteristic of malignant cells associated with induction of tissue invasion and
metastasis (Hakomori, 2002; Kobata and Amano, 2005; Reis et al., 2010). Due to their peripheral
location, oligosaccaride epitopes of glycoproteins and glycolipids are recognized by membrane-
anchored carbohydrate-recognition domains of different molecules, including lectins (Jiménez-
Castells et al., 2008).
Lectins comprise proteins or glycoproteins which bind specifically to mono or
oligosaccharides and glyconconjugates (Wu et al., 2009). Carbohydrate-specificity of lectins has
been shown to be a versatile and useful molecular tool for study of glycoconjugates on cell surface,
in particular the changes that cells suffer in malignancy (Sharon and Lis, 2004). Thus, lectins are
excellent candidates to be explored in cancer research as therapeutics agents.
Lectins from snake venoms exhibit several biological activities like ability to inhibit
integrin-dependent proliferation, migration and invasion of tumor cells (Sarray et al., 2004; Sarray
et al., 2007) as well as to reduce the growth of tumor and endothelial cells (Carvalho et al., 2001).
However, the induction of tumor cell apoptosis by snake venom lectins has not been studied.
The BlL is a lectin isolated from the venom of Bothrops leucurus (white-tailed-jararaca).
BlL is a Ca2+-dependent and galactoside-binding protein of 30 kDa composed by dissulfide-linked
dimers of 15 kDa and exhibits antibacterial activity against human pathogenic Gram-positive
bacteria (Nunes et al., 2011).
70
Apoptosis (programmed cell death) is an essential cellular homeostasis mechanism that
ensures the correct development and function of multi-cellular organisms. However, cancer cells
show a reduced sensitivity towards apoptosis and tumors are dependent on the mechanisms of this
resistance to continue alive. Therefore, it is of enormous therapeutic interest the discovery of drugs
that selectively affect the balance of tumor cellular functions towards apoptosis. According
Taraphdar et al. (2001), induction of apoptosis is an important strategy for cancer therapy and
prevention.
The aims of this study were to evaluate the in vitro cytotoxicity of BlL on different human
tumor cell lines (K562, NCI-292 and Hep-2), its lytic property on mouse erythrocytes and its ability
to induce apoptosis in human tumor cells.
2. Material and Methods
2.1. Chemicals
Phosphate buffered saline (PBS), penicillin, streptomycin and DMEM (Dulbecco’s Modified
Eagle’s Medium) were purchased from GibcoTM. MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide) was purchased from InvitrogenTM. Eposide (Etoposide) was purchased
from Blausiegel. Fetal bovine serum, glutamine, Triton X-100 and JC-1 (5,5′,6,6′-Tetrachloro-
1,1′,3,3′ tetraethylbenzimidazolocarbocyanine iodide) were purchased from Sigma-Aldrich®.
Annexin V FITC Apoptosis Kit was purchased from Calbiochem. DMSO (Dimethil sulfoxide) was
purchased from Vetec.
2.2. BlL purification
BlL was purified according to the protocol previously described by Nunes et al. (2011).
Lyophilized crude venom of B. leucurus (30 mg) was dissolved in 1 mL of CTBS buffer (20 mM
Tris-HCl, 150 mM NaCl and 5 mM CaCl2, pH 7.5) and centrifuged (2000 g, 5 min, 25 °C) to
remove insoluble material. The resulting supernatant was applied to a column (10 x 1.0 cm) of guar
gel previously equilibrated with CTBS at a flow rate of 10 mL/h. BlL was eluted from the column
with 200 mM galactose in CTBS.
71
2.3. Cell lines and cell culture
The cell lines used in cytotoxicity assays were K562 (chronic myelocytic leukemia), NCI-
H292 (human lung mucoepidermoid carcinoma cells) and Hep-2 (human larynx epidermoid
carcinoma cells) obtained from Instituto Adolfo Lutz (São Paulo, Brazil). The cells were maintained
in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin and
100 µg/mL streptomycin and maintained at 37 °C with 5 % CO2.
2.4. MTT assay
Cytotoxicity of BlL was tested on K562, NCI-H292 and Hep-2 tumor cell lines. The cells
(105 cells/mL for adherent cells or 0.3x106 cells/mL for suspended cells) were plated in 96-well
microtiter plates and after 24 h, BlL (0.07–50 µg/mL) dissolved in DMSO was added to each well
and incubated for 72 h at 37 °C. Then, MTT (5.0 mg/mL) was added to the plate and growth of
tumor cells was estimated by the ability of living cells to reduce the yellow tetrazolium to a blue
formazan product (Mosmann, 1983; Alley et al., 1988). Negative control groups received only
DMSO; etoposide (1.25–20 µg/mL) was used as positive control. After 3 h (for suspend cells) or 2
h (for adherent cells), the formazan product was dissolved in DMSO and absorbance was measured
using a multi-plate reader (Multiplate Reader Thermoplate). The BlL effect was quantified by
measuring the absorbance at 450 nm resulting from MTT reduction. The results were compared
with negative control absorbance and the values of 50% inhibition of cell proliferation (IC50) were
calculated.
2.5. Hemolytic assay
Assay was performed in 96-well microtiter plates according to the method described by
Costa-Lotufo et al. (2002). Each well received 100 µL of 0.85% NaCl solution containing 10 mM
CaCl2. The first well was the negative control and contained only the vehicle (10 % DMSO). To the
second well, 100 µL of BlL (10–2000 µg/mL) were added and serial dilution was performed until
1:1024. Positive control used 20 µL of 0.1% Triton X-100 (in 0.85% NaCl) to obtain 100%
hemolysis. Then, each well received 100 µL of a 2 % suspension of mice (Mus musculus)
erythrocytes in 0.85 % NaCl containing 10 mM CaCl2. After incubation at 28 ºC for 30 min and
centrifugation, the supernatant was removed and the haemoglobin released by hemolysis was
determined by measurement of absorbance at 450 nm.
72
2.6. Annexin/PI cell death assay
The K562 suspension (0.3 x 106 cells/mL) was seeded in 96-well microtiter plates and
incubated at 37 °C at 5 % CO2 for 24 h; after this period, BlL at IC50 was added. After 48 h the cells
were stained with annexin V and propidium iodide using Annexin V–FITC Kit (Calbiochem®)
following the protocol provided by the manufacturer and analysed by epifluorescence microscope
(Carl Zeiss, Gottingen, Germany) with increase of 1000x under oil immersion with filters for LP
515 nm emission and BP 450-490 nm for excitement. A minimum of 200 cells was counted in
every sample.
2.7. Measurement of mitochondrial membrane potential
Mitochondria depolarization was evaluated by incorporation of JC-1 (5,5´,6,6´-tetrachloro-
1,1´,3,3´-tetraethilbenzimidazolcarbocyanine iodide), a fluorescent lipophilic cationic probe (Kang
et al., 2002; Guthrie and Welch, 2006). The probe JC-1 is freely permeable to cells and undergoes
reversible transformation from a monomer to an aggregate form (Jagg). K562 suspension (0.3 x 106
cells/mL) was seeded into 96-well microtiter plates and incubated at 37 °C and 5 % CO2; after 24 h,
BlL at IC50 was added and plates incubated for 48 h. Then, 50 µL of treated cell suspension were
collected and incubated with JC-1 (10 µL/mL) for 30 min in the dark followed by washing two
times with PBS. The cells were fixed with paraformaldehyde 4% (10 µL), mounted on glass slides
and observed using an epifluorescence microscope (Carl Zeiss, Gottingen, Germany), with increase
of 1000x under oil immersion with filter for LP 515 nm emission and BP 450-490 nm excitement.
A minimum of 200 cells was counted in every sample. Cells with high potential of mitochondrial
membrane were stained in red while cells with low membrane potential were stained in green.
2.8. Statistical analysis
All data are presented as mean ± S.D. The IC50 and EC50 values were obtained by nonlinear
regression with 95% confidence interval using the SigmaPlot software (Systal Software Inc., San
Jose, USA). The differences between experimental groups were determined using one-way analysis
of variance (ANOVA) followed by Newman-Keuls test at significance level of 1%.
73
3. Results
Cytotoxicity of BlL on tumor cell lines was evaluated after 72 h using MTT assay and the
results are presented in Table 1. BlL exhibited cytotoxic activity against all cell lines with IC50
values of 6.63, 11.75 and 15.42 µg/mL for Hep-2, NCI-H292 and K562, respectively.
Table 1
Cytotoxic activity of BlL against tumor cell lines.
IC50 (µg/mL) Sample
Hep-2 NCI-H292 K562
BlL 11.75±0.035 6.63±0.052 15.42±0.060
Etoposide 6.10±0.19 2.75±0.10 4.48±0.23
The IC50 values at 95% confidence interval were obtained by non linear regression. Etoposide was
used as positive control.
In order to verify whether the cytotoxicity was related to injury of cell plasma membrane,
BlL was tested for lytic activity on mice erythrocytes. The results showed that it does not cause
membrane damage even at the upper concentration (2000 µg/mL).
The involvement of apoptosis induction on K562 death was verified by evaluation of
phosphatidylserine externalization using the Annexin V-FITC kit and fluorescence microscope. We
observed that after treatment with BlL (15.42 µg/mL), the number of cells in early apoptosis (Ann
Vpos/PIneg) corresponded to 70.5% (Figure 1). Treatment with BlL exhibited values less than 1% of
late apoptotic cells (AnnVpos/PIpos) and values less than 2% of cell necrosis (AnnVneg/PIpos).
74
Fig. 1. Effect of BlL in K562 cell population determined by fluorescence microscopy using
Annexin V–FITC Kit, after 48 h incubation. The negative control (NC) was the vehicle used
(DMSO). Etoposide was used as positive control (E). *p < 0.01 in comparison to control by
ANOVA followed by Newman Keulls test. Data are presented as mean ± S.D. from three
independent experiments.
Figure 2 shows that the treatment of K562 cells with BlL caused mitochondrial membrane
potential loss, as fluorescence microscopy analysis determined that BlL treatment induced
significant increase in cells with depolarized mitochondria (63.8%) as compared to control cells, as
measured by JC-1 incorporation.
Fig. 2. Effect of BlL in K562 cell population determined by fluorescence microscopy using JC-1,
after 48 h incubation. The negative control (NC) was the vehicle used (DMSO). Etoposide was used
as positive control (E). *p < 0.01 in comparison to control by ANOVA followed by Newman
Keulls test. Data are presented as mean ± S.D. from three independent experiments.
0
10
20
30
40
50
60
70
NC E BlL
Mit
och
on
dri
al
De
po
lari
zati
on
(%
)
**
0
10
20
30
40
50
60
70
NC E BlL
Mit
och
on
dri
al
De
po
lari
zati
on
(%
)
**
0
10
20
30
40
50
60
70
80
90
NC E BlL
Ce
ll n
um
be
r (%
)
Viable Apoptos is Necros i s
*
*
*
*
0
10
20
30
40
50
60
70
80
90
NC E BlL
Ce
ll n
um
be
r (%
)
Viable Apoptos is Necros i s
*
*
*
*
75
4. Discussion
Uncontrolled proliferation and decreased apoptotic signals are attributes of oncogenic
transformation (Hill et al., 2003), and activation of apoptosis constitutes a fundamental mechanism
by which drugs may kill tumor cells (Debatin, 2004). Therefore, compounds with the ability to
induce apoptosis in tumor have potential for anticancer agents (Reed, 2003).
MTT assay demonstrated that BlL showed significant cytotoxic effect against human tumor
cell lines Hep-2, NCI-H292 and K562 indicating that the activity of this lectin was not specific to a
particular tumor cell type. However, different IC50 values were determined for the lectin revealing a
different cytotoxic activity on the different cell lines. Glycoconjugates or saccharides present on the
surface of tumor cells are binding sites for lectins (Luo et al., 2007) and differences in sugar pattern
between different tumor cells may be a reason for the differential effect of BlL. Differences in the
effect snake venom lectins towards human tumor cell lines have been reported (Pereira-Bittencourt
et al., 1999; Carvalho et al., 2001). In addition, cells that not express specific carbohydrates may be
insensitive to cytotoxic lectins (Gorelik et al., 2001).
The presence of 10% fetal bovine serum in culture medium did not exert any effect in
cytotoxicity induced by BlL on tumor cell lines. Pereira Bittencourt et al. (1999), studying the
action of C-type lectin from Bothrops jararacussu (BJcuL) on the proliferation of human cancer
cell lines, observed marked toxicity in presence of 5% fetal bovine serum, however incubation with
10% fetal bovine serum decreased the inhibitory activity of BJcuL lectin by approximately 50%.
The lectin BJcul was highly cytotoxic to OVCAR-5 cells in the presence of 5% fetal bovine serum
and has no inhibitory effect on cell growth when 10% fetal bovine serum was used (Carvalho et al.,
2001). One possible explanation for this is that fetal bovine serum contains specific sugars that
inhibit the cytotoxic action of BJcuL. Our results suggest that fetal bovine serum contains a low
amount of glycoligands specific for BlL in comparison with BJcuL.
Despite their cytotoxic action, BlL was inactive against erythrocytes of mice, suggesting that
the cytotoxic mechanism is not related to lytic property or induction of membrane instability by
BlL.
Morphological and biochemical characteristics of apoptosis are nuclear chromatin
condensation, DNA fragmentation, membrane blebbing (Okada and Mak, 2004; Vermeulen et al.,
2005), externalization of phosphatidylserine (Hengartner, 2000) and depolarization of the
membrane potential (Ly et al. 2003). In this study, apoptosis induction in BlL-treated K562- cells
76
was assessed by fluorescence microscopy analysis of phosphatidylserine externalization on cell
surface and mitochondrial membrane potential.
The loss of plasma membrane asymmetry represents and early event of apoptosis resulting
in translocation of phosphatidylserine from the inner to the outer surface while membrane integrity
remains unchanged (Van Engeland et al., 1998, Fadok et al., 2000; Kagan et al., 2000); this
externalization provides the recognition and removal of apoptotic cells by phagocytes
(Zimmermann et al., 2001; Taylor et al., 2008). The phospholipid-binding protein annexin V has a
high affinity for phosphatidylserine and binds to cells fluorescently labeled with FITC (Reyes-
Zurita et al., 2009). However, translocation de phosphatidylserine also occurs during necrosis, so
propidium iodide is often used to bind to nucleic acids (Gong et al., 2007). We observed by staining
with annexin V-FITC simultaneously with dye propidium iodide that BlL was able to increase
significantly the number of apoptotic cells. The results suggest that the cytotoxic effect is due to
induction of apoptosis in K562 cells.
The mitochondrial apoptotic pathway is one of the major routes to initiate apoptosis (Kuo et
al., 2010). Different stimuli cause changes in the inner mitochondrial membrane leading to the
opening of the mitochondrial permeability transition pore, loss of the mitochondrial transmembrane
potential (Ly et al., 2003; Saelens et al., 2004) and pro-apoptotic proteins release from the
intermembrane space into the cytosol (Mayer and Oberbauer, 2003; Borutaite, 2010). One of these
proteins is the cytochrome C which triggers the formation of the apoptosome complex culminating
with the activation of caspases (Gogvadze et al., 2009).
The loss of mitochondrial membrane potential is the early change in the mitochondria-
mediated apoptosis (Zhao et al., 2010). Our studies demonstrated that treatment with BlL increased
mitochondrial membrane potential loss, which may indicate cell death by apoptosis in K562 cells.
Some lectins such as Con A, POL, PCL and MLL may cause disruption of the mitochondrial
membrane potential as an event associated with apoptosis (Liu et al., 2009a; Liu et al., 2009b; Liu
et al., 2009c; Zhao et al., 2010).
In conclusion, the galactoside-binding lectin from B. leucurus snake venom (BlL) exhibited
cytotoxic activity on tumor cells and induced apoptosis in K562 cells, as verified by
phosphatidylserine externalization analysis and mitochondrial membrane potential determination.
These results suggest that BlL has a promising potential for application in therapy and / or diagnosis
of cancer. Future studies are needed to elucidate the details of BlL induced-apoptosis mechanism in
several tumor cell lines.
77
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors express their gratitude to the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) for research grants and fellowship (LCBBC and MTSC) and to the
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for research grants.
Authors are deeply grateful to Maria Barbosa Reis da Silva, Maria D. Rodrigues and João Antônio
Virgínio for the technical assistance.
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7 ARTIGO CIENTÍFICO III
Gamma irradiation abolish in vitro cytotoxicity of lectin of Bothrops leucurus
snake venom
Artigo a ser submetido ao periódico Toxicon
83
Erika dos Santos Nunesa*, Mary Angela Aranda de Souzaa, Antônio Fernando Melo Vaza, Maria
Luiza Vilela Olivab, Jaciana Santos Aguiarc, Teresinha Gonçalves da Silvac, Miriam Camargo
Guarnierid, Luana Cassandra Breitenbach Barroso Coelhoa, Ana Maria M. de Albuquerque Meloe,
Maria Tereza dos Santos Correia.a
aDepartamento de Bioquímica, Universidade Federal de Pernambuco, Avenida Professor Moraes
Rêgo, s/n, Cidade Universitária, 50670-420, Recife, Pernambuco, Brazil
bDepartamento de Bioquímica, Universidade Federal de São Paulo, Rua Três de Maio, 100, Vila
Clementina, 04044-020, São Paulo, Brazil.
cDepartamento de Antibióticos, Universidade Federal de Pernambuco, Avenida Arthur de Sá, s/n,
Cidade Universitária, 50670-901, Recife, Pernambuco, Brazil.
dDepartamento de Zoologia, Universidade Federal de Pernambuco, Avenida Professor Moraes
Rêgo, s/n, Cidade Universitária, 50670-420, Recife, Pernambuco, Brazil.
eDepartamento de Biofisica e Radiobiologia, Universidade Federal de Pernambuco, Avenida
Professor Moraes Rêgo, s/n, Cidade Universitária, 50670-420, Recife, Pernambuco, Brazil.
*Corresponding author. Phone: +558121268540; Fax: +558121268576
E-mail address: [email protected]
ABSTRACT
Gamma radiation alters the molecular structure of biomolecules and has been able to mitigate snake
venoms and its isolated toxins. The aims of this work was to evaluate the effects of ionizing
radiation in Bothrops lecurus venom lectin (BlL) by fluorescence spectroscopy and in vitro
cytotoxicity. BlL has been structurally altered and hemagglutinating assay shows significant change
after gamma irradiation. SDS-PAGE analyses indicated that irradiation caused fragmentation and
aggregation of lectin. The reverse phase chromatography analysis revealed a loss of the peak area
with structural fragmentation. The effect of γ-radiation on the folding of the lectin was measured by
intrinsic and bis-ANS fluorescence. Cytotoxic activity of BlL on Hep-2, K562 and NCI-H292
tumor cell lines was abolished after irradiation. To improve antivenom and extend the useful life of
84
immunized horses effort has been devoted to decrease chronic venom toxicity, the irradiation may
acts as a detoxification strategy in snake venoms avoiding its cytotoxicity.
Keywords: Bothrops leucurus; lectin; fluorescence; Bis-ANS; gamma rays.
1. Introduction
Snake venoms are complex mixtures of bioactive proteins and polypeptides (Koh et al.,
2006). These toxins are enzymatic and non enzymatic proteins and synergistic interactions between
venom proteins enhanced the lethal potency of the snake venom. Complexes of proteins families,
such as metalloproteases, serine proteases, C-type lectins (CTLs), C-type lectin-related proteins
(CLRPs) and three-finger toxins (3FTxs), have also been reported in venoms (Doley and Kini,
2009). CTLs are true sugar-binding lectin composed by homodimers or homooligomers and with
Ca2+ and generally galactose binding properties (Clemetson, 2010). C-type lectin-like proteins are
heterodimers or oligomeric complexes of heterodimers called Snaclecs (Snake venom C-type
lectins) (Ogawa et al., 2005; Clemetson et al., 2009; Clemetson, 2010). Lectins are proteins or
glycoproteins that are ubiquitous in nature and bind reversibly to carbohydrates (Sharon and Lis,
2004).
Ionizing radiation causes changes in the function and integrity of biomolecules, including
proteins, by two different effects. First, interacting directly on target proteins (Kempner, 2001); and
second, the formation of major products from radyolisis water and its subsequent interaction with
proteins are described as indirect mechanism, which are responsible for most of the radiation effects
on proteins (Riley, 1994; Wang and Wang, 2007). The exposure of proteins at low dose radiation
produces chemical and physical damage that may result in changes in its primary structure,
secondary or tertiary, keeping intact their immunological properties (Nascimento et al., 1996).
The intimate relationship existing structure-activity proteins may be affected by the use of
ionizing radiation, which functions to an important tool in the study of attenuation of snake venoms.
This aspect has received attention from researchers, on many occasions show the effects of gamma
radiation the molecular level, with the involvement of biological activity de snake venom, which
result in a decrease or loss of enzymatic and toxic actions (Gallaci et al., 2000; Souza et al., 2002;
Casare et al., 2006). This phenomenom has led scientists to the formulation of concepts and ideas
extremely relevant, increasing interest in experiments such as the detoxification from snake venoms
by radiation, without affecting its immunogenic properties in order to optimize the production of
antiserum (Moreira et al., 1997; Netto et al., 2002; Ferreira Junior et al., 2005; Ferreira Junior et al.,
85
2006; Ferreira et al., 2009). Traditional methods to reduce unwanted or intolerant immunological
effects of lectins have been ineffective (Sathe et al., 2005). However, an alternative treatment was
recently reviewed to abolish allergenicity of lectins in food using gamma irradiation (Vaz et al.,
2011).
A valuable feature of intrinsic protein fluorescence is the high sensitivity of tryptophan to its
local environment. Changes in the emission spectra of tryptophan often occur in response to
conformational transitions, subunit association, substrate binding, or denaturation (Lakowicz,
2006). Bis-ANS have proved to be sensitive probes for partially folded intermediates in protein-
folding pathways. The basis of these applications is the strong fluorescence enhancement exhibited
by these amphiphilic dyes when their exposure to water is lowered (Semisotnov et al., 1991). The
majority of misfolding states are involving the self-aggregation of specific proteins (or protein
fragments) into filamentous deposits known as amyloid fibrils, which adopt a characteristic cross-β-
sheet structure. Thioflavin T (ThT) dye fluorescence is used regularly to quantify the formation of
amyloid fibrils in the presence of stress physics or oxidative (Hawe et al., 2008).
Bothrops leucurus venom lectin (BlL) has been purified by affinity chromatography of
venoms snake. The galactoside-binding, a protein of 30 kDa composed of 15 kDa subunits, presents
inhibitory activity against antibacterial, citotoxicity on human tumor cell lines as well as induction
of apoptosis (Nunes et al., 2011). Here, the interest lies in studying the effects of ionizing radiation
in the lectin isolated from venom of Bothrops leucurus and evaluated if gamma radiation attenuates
its citotoxicity in tumor cell.
2. Material and Methods
2.1. Chemicals
Reference samples of 4.4'-Bis 1-anilinonaphthalene 8-sulfonate (bis-ANS) were purchased
from Molecular Probes Inc., USA. The broad-range standard marker proteins were purchased from
Sigma Chemical Co. USA. Phosphate buffered saline (PBS), penicillin – streptomycin liquid and
DMEM (Dulbecco’s Modified Eagle’s Medium) were purchased from GibcoTM. MTT (3-(4,5-
Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) were purchased from InvitrogenTM.
Eposide (Etoposide) was purchased from Blausiegel. Fetal bovine serum (FBS) and glutamine were
86
purchased from Sigma-Aldrich®. All the solvents and other chemicals used were of analytical grade
from Merck, Germany. All solutions were made with water purified by the Milli-Q system.
2.2. Cell line and Cell culture
The cell lines used in vitro cytotoxicity were K562 (chronic myelocytic leukemia), NCI-
H292 (human lung mucoepidermoid carcinoma cells) and Hep-2 (human larynx epidermoid
carcinoma cells) all obtained from Adolph Lutz Institute (São Paulo, Brazil). The cells were
maintained in DMEM supplemented with 10% fetal bovine serum, 2mM glutamine, 100 U/mL
penicillin, 100 µg/mL streptomycin at 37 °C with 5% CO2.
2.3. Purification of Bothrops leucurus lectin (BlL)
B. leucurus venom was kindly supplied by the Núcleo Regional de Ofiologia e Animais
Peçonhentos da Bahia, Universidade Federal da Bahia, Salvador, Bahia, Brazil. BlL was purified
according to the protocol previously described by Nunes et al. (2011). Lyophilized crude venom of
B. leucurus (30 mg) was dissolved in 1 mL of CTBS buffer (20 mM Tris-HCl, 150 mM NaCl and 5
mM CaCl2, pH 7.5) and centrifuged (2000 g, 5 min, 25 °C) to remove insoluble material. The
resulting supernatant was applied to a column (10 x 1.0 cm) of guar gel previously equilibrated with
CTBS at a flow rate of 10 mL/h. BlL was eluted from the column with 200 mM galactose in CTBS.
2.4. Lectin irradiation
The lectin aliquots (0.07 mg/mL) in phosphate buffer (pH 7.0) in borosilicate glass vials
(16-125 mm) were frozen and irradiated under atmospheric O2 using a Gammacell 220 Excel 60Co
gamma ray irradiator (Ontario, Canada) with doses of 1 and 2 kGy at a rate of 7.2 kGy/h. Each
sample was analyzed after irradiation by the following methods.
2.5. Hemagglutination activity and protein concentration
Hemagglutination activity (HA), which was defined as the lowest sample dilution that
showed hemagglutination, was evaluated as described by Correia and Coelho (1995). Specific HA
(SHA) corresponded to the relationship between the HA and protein concentration measured
according to Bradford (1976) using bovine serum albumin (BSA) as a standard. The percentage of
87
the remaining SHA (%SHAREM) was calculated according to the equation: %SHAREM = (SHA)GM /
(SHA)GO x 100, where GM is the lectin SHA of each radiation dose (1 and 2 kGy) and G0 is the of
non-irradiated lectin (control).
2.6. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE was performed according to Laemmli (1970). Protein samples were mixed with
loading buffer (60 mM Tris-HCl, pH 6.8, with 2% SDS, 25% glycerol, and 0.1% bromophenol
blue), resolved on a 15% separating gel and stained using Coomassie Brilliant Blue (Sigma). The
following standard molecular weight markers were used: rabbit muscle myosin (205 kDa), E. coli
β-galactosidase (116 kDa), rabbit muscle phosphorylase b (97.4 kDa), rabbit muscle fructose-6-
phosphate kinase (84 kDa), bovine serum albumin (66 kDa), bovine liver glutamic dehydrogenase
(55 kDa), egg albumin (45 kDa), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (36
kDa), bovine erythrocyte carbonic anhydrase (29 kDa), bovine pancreas trypsinogen (24 kDa),
soybean trypsin inhibitor (20 kDa), bovine milk α-lactalbumin (14.2 kDa) and bovine lung aprotinin
(6.5 kDa).
2.7. Reverse phase chromatography analysis
BlL (0.07 mg/mL) was irradiated (1 and 2 kGy) and submitted to reverse phase
chromatography on a C-4 column (Vydac-protein peptide ultrasphere), performed on a HPLC
system (Shimadzu LC-10AD, Kyto, Japan) and monitored at 280 nm. The column was equilibrated
with solvent A (0.1% TFA in H2O) at a flow rate of 0.7 mL/min, and a non-linear gradient elution
was used with solvent B (90% acetonitrile, 10% H2O, 0.1% TFA) in A, with 0% B at t = 5 min;
45% B at t = 10 min; 50% B at t = 30 min; and 100% B at t = 35 min.
2.8. Fluorescence spectroscopy
The fluorescence emission intensity of the irradiated 0.07 mg/mL BlL solution in phosphate
buffer at pH 7.0 was measured at 25°C using a spectrofluorometer (JASCO FP-6300, Tokyo, Japan)
in a rectangular quartz cuvette with a 1-cm path length. The excitation wavelengths were 295 and
280 nm; emission spectra were recorded in the range of 305 to 450 nm, and band passes were 5 nm.
Light scattering was measured at 90° for the aggregation assays; light scattering values at 320 nm
88
were monitored (300 to 340 nm). The spectra displayed in the figures are the average of three scans
that were corrected for the solution signal by subtracting the solution spectrum.
2.9. Hydrophobic surface analysis
The lectin hydrophobic surface was measured using the same conditions employed for the
intrinsic fluorescence experiment. Samples were transferred to a quartz cuvette and then mixed with
5 µM bis-ANS; fluorescence was measured in the JASCO spectrofluorometer. The fluorescence
emission was obtained at 400 to 600 nm with an excitation at 360 nm (Bhattacharyya et al., 2000).
2.10. Thioflavin T (ThT) fluorescence assay
The amyloid fibrils detection was measured using the same conditions employed for the
intrinsic fluorescence experiment. Samples were transferred to a quartz cuvette and then mixed with
13 µM ThT; fluorescence intensity of each sample was recorded every 5 min in the JASCO
spectrofluorometer. The fluorescence emission was obtained at 465 to 550 nm with an excitation at
450 nm (LeVine, 1999).
2.11. MTT assay
The cytotoxicity of the BlL irradiated in dose of 1 and 2 kGy was tested against K562, NCI-
H292 and Hep-2 tumor cell lines. The cells (105 cells/mL for adherent cells or 0.3x106 cells/mL for
suspended cells) were plated in 96-well microtiter plates and after 24 h, irradiated BlL (0.07
mg/mL) dissolved in DMSO was added to each well and incubated for 72 h at 37 °C. Then, MTT
(5.0 mg/mL) was added to the plate and growth of tumor cells was estimated by the ability of living
cells to reduce the yellow tetrazolium to a blue formazan product (Mosmann, 1983; Alley et al.,
1988). Negative control groups received only DMSO; etoposide (1.25–20 µg/mL) was used as
positive control. After 3 h (for suspend cells) or 2 h (for adherent cells), the formazan product was
dissolved in DMSO and absorbance was measured using a multi-plate reader (Multiplate Reader
Thermoplate). The BlL effect was quantified by measuring the absorbance at 450 nm resulting from
MTT reduction. The results were compared with negative control absorbance and the values of 50%
inhibition of cell proliferation (IC50) were calculated.
89
2.12. Statistical analysis
Data are presented as mean ± S.D. The IC50 values and their 95% confidence intervals were
obtained by nonlinear regression using the SigmaPlot graphing software Inc. San Jose, USA. The
differences between experimental groups were compared by one-way of variance (ANOVA)
followed by Newman-Keuls test and the significance level was also set at 1%.
3. Results and Discussion
Ionizing radiation has been widely employed to attenuate venoms and isolated toxins,
preserving and even enhancing their immunogenic properties. However, little is know about the
molecular changes that irradiated proteins undergo. Thus, we compared native and irradiated
Bothrops leucurus snake venom lectin (BlL) aiming to characterize the structural modifications that
radiation induces. BlL SHA was determined after irradiation. In dose of 1 kGy, no significant
change was observed. However, in dose of 2 kGy shows significantly loss of SHA (Figure 1a). C-
type lectins exhibit biological activities like adhesion, via CRDs (carbohydrate recognition domain),
for recognition of oligosaccharides in cell surface. The CRD can function independently of the rest
of the protein and thus may be employed to assess whether agglutination or clumping is maintained
or disrupted after irradiation (Taylor and Drickamer, 1991). Thus, the loss of HA after irradiation
suggests modification of the dimmers interact which can result in dissociation pentamer and loss of
interaction sites on surface cells (Walker et al., 2004).
The precipitate was run on SDS-PAGE after centrifugation to detect any insoluble
aggregates that formed, and the supernatant was separated by RP-HPLC. In dose of 1 kGy, initial
degradation of the BlL (MW 30.0 kDa) was observed. Degradation of the main band was detected
in dose of 2 kGy, indicating that the aggregate formed was composed of fragmented polypeptides
(Figure 1b). The reverse phase chromatography analysis showed the loss of the peak area with the
structural fragmentation (Figure 1c) and aggregation can be evaluated by light scattering (Figure
1d). According Puchala and Schuessler (1995), in fragile bonds in the polypeptide chain break
points occurring protein caused by radiation. Because of the generation of inter-protein cross-
linking reactions, formation of disulfide bonds, as well as hydrophobic and electrostatic
interactions, proteins can be converted to higher molecular weight aggregates (Moon and Song,
2001; Xu and Chance, 2005). The analysis of molecular weight pattern and RP-HPLC shows
conditions that induce denaturation, with subsequent precipitation into insoluble amorphous
aggregates.
90
Fig. 1. Effect of γ-radiation on lectin activity and molecular weight. (a) The percentage of
remaining specific hemagglutination activity, %SHAREM is represented after irradiation. Error in the
determination of %SHAREM for the different doses was approximately ± 1%, which is less than the
size of the symbols. * Significant difference (p < 0.05) compared to non-irradiated lectin. (b) SDS-
PAGE from irradiated BlL. SDS-PAGE was performed in a discontinuous system with 15%
separating and 5% stacking gels. (MW) Molecular weight; (C) non-irradiated control; (1) 1 kGy and
(2) 2 kGy. (c) Reverse phase chromatography on an HPLC system: (▬) control and irradiated
lectins at (▬) 1 kGy and (▬) 2 kGy. (d) Light scattering was measured at 90° for the aggregation
assays.
Nascimento et al. (1996) observed that crotoxin, the main neurotoxin isolated from snake
venom Crotalus durissus terrificus, when irradiated with 2 kGy dose of gamma rays produced
protein aggregation and generation of lower molecular weight breakdown products. Such clusters
applaud less myotoxic, devoid of phospholipase activity and are virtually non-toxic in mice when
compared with native crotoxin. The bothropstoxin-1, the main myotoxic component of Bothrops
jararacussu snake venom, after irradiation com 60Co gamma rays, promoted structural
modifications in the toxin characterized aggregates and oligomers (Caproni et al., 2009).
0
20
40
60
80
100
Control 1 2
Dose (kGy)
SH
A R
em (%
)
205116
6655
4536
2420
14.2
6.5
29
84
97.4
MW 1 2C
*
ab
0
50
100
150
200
250
300
300 310 320 330 340
Wavelength (nm)
Flu
ore
sc
en
ce
inte
ns
ity
(A
.U.)
c
-50
150
350
550
750
950
0 10 20 30 40
Time (min)
Ab
so
rban
ce a
t 280 n
m (
mA
U)
d
non-irradiated
1 kGy
2 kGy
0
20
40
60
80
100
Control 1 2
Dose (kGy)
SH
A R
em (%
)
205116
6655
4536
2420
14.2
6.5
29
84
97.4
MW 1 2C
*
ab
0
50
100
150
200
250
300
300 310 320 330 340
Wavelength (nm)
Flu
ore
sc
en
ce
inte
ns
ity
(A
.U.)
c
-50
150
350
550
750
950
0 10 20 30 40
Time (min)
Ab
so
rban
ce a
t 280 n
m (
mA
U)
d
non-irradiated
1 kGy
2 kGy
91
CTLs display anticoagulant and hemagglutinating activity. Its activity depends on the extent
of association to dimmers. The intrinsic fluorescence of BlL was used to study its association into
dimmers. CTLs is usually a dimer of two identical polypeptides, each containing two tryptophan
residue and one tyrosine residues (Morita, 2005). The emission spectrum of the BlL dimer displays
emission from both tyrosine and tryptophan when excited at 280 nm. Only tryptophan emission is
seen for 295 nm excitation. The intrinsic fluorescence emission decreased (1 and 2 kGy) with
changing the λmax at approximately 347 and 345 nm for hydrophobic and thryptophan residues,
respectively, after irradiation (Figure 2 a-b). To tryptophan residues that are exposed to water have
a maximal fluorescence at wavelengths around 340-350 nm, whereas completely buried residues
fluoresce at about 330 nm. In CTLs individual subunits are able to bind to carbohydrates, but for the
lectin-like function they need at least bivalency, which is achieved through a simple interchain
disulfide linkage. Although dimerization is essential, two Trp residues (one at each end) and a Tyr
residue in the middle of the interaction interface are essential to stabilize intra-subunit contacts and
for the expression of their biological activities (Doyle and Kini, 2009). The displacement of the
mass center of the aromatic residues indicates a possible protein denaturation and exposure of
hydrophobic domains to the solvent. Thus, the subtle differences in the positioning of interactive
segment may lead to distinct quaternary structures of these proteins after irradiation.
Fig. 2. BlL intrinsic fluorescence. (a) Mass center; Lectin excitation (280 nm) and emission (295–
450 nm). (b) Mass center tryptophan; Lectin excitation (295 nm) and emission (305-450 nm).
The aromatic amino acid residues of proteins are major targets of reactive oxygen species by
oxidation (ROS) (Stadtman and Levine, 2003), which are produced after water radiolysis. Partial
aromatic amino acid substituition caused by hydrogen abstraction promote a decrease in the
intensity fluorescence emission being the magnitude of the intensity very informative in itself.
338
339
340
341
342
343
344
345
346
control 1 kGy 2 kGy
Dose (kGy)
Cen
ter
of m
ass
(nm
)
0
100
200
300
400
500
300 320 340 360 380
Wavelength (nm)
Flu
ores
cenc
e in
tens
ity
(A.U
.)
342
342.5
343
343.5
344
344.5
345
345.5
346
346.5
347
347.5
Control 1 kGy 2 kGy
Dose (kGy)
Cen
ter
of m
ass
(nm
)
0
150
300
450
600
750
900
300 320 340 360 380 400
Wavelength (nm)
Flu
ores
cenc
e in
ten
sity
(A.U
.)
a b
338
339
340
341
342
343
344
345
346
control 1 kGy 2 kGy
Dose (kGy)
Cen
ter
of m
ass
(nm
)
0
100
200
300
400
500
300 320 340 360 380
Wavelength (nm)
Flu
ores
cenc
e in
tens
ity
(A.U
.)
342
342.5
343
343.5
344
344.5
345
345.5
346
346.5
347
347.5
Control 1 kGy 2 kGy
Dose (kGy)
Cen
ter
of m
ass
(nm
)
0
150
300
450
600
750
900
300 320 340 360 380 400
Wavelength (nm)
Flu
ores
cenc
e in
ten
sity
(A.U
.)
a b
92
Oxidative damage distorts the intra-subunit contacts and the hydrophobic core of proteins, allowing
dissociation of chains and denaturation.
Compared with the buffer, bis-ANS was weakly detected in the non-irradiated control BlL.
Bis-ANS fluorescence, after irradiation, increased in two doses used in this study, with a maximum
to 500 nm to above 1 kGy (Figure 3). Hydroxyl radicals may attack hydrophobic surfaces in
proteins, causing changes in the protein hydrophobicity, which are considered the determining
factor for structural collapse. Molten globule intermediates and insoluble amorphous aggregates are
characterized by particularly high bis-ANS fluorescence intensities due to the exposure of
hydrophobic core regions that are inaccessible to the dye in the native structure (Semisotnov et al.,
1991). Despite of misfold states involve the self-aggregation of specific proteins (or protein
fragments) into filamentous deposits know as amyloi fibrils, was not observed fluorescence
Thioflavin T after irradiation. For a lectin isolated from Sebastiana jacobinensis, a variety of
conditions was observed that induce denaturation, with subsequent precipitation into insoluble
amorphous aggregates or structured intermediates after irradiation (Vaz et al., 2011).
Fig. 3. BlL bis-ANS fluorescence. Mass center; Lectin excitation (360 nm) and emission (400–600
nm).
In previous reports, was observed by MTT assay showed that BlL significant cytotoxic
effect against human tumor cell lines Hep-2, NCI-H292 and K562 (Nunes et al., 2011). In this
work, we found that in contrast to native BlL, BlL irradiated (1 and 2 kGy) did not show cytotoxic
activity on tumor cell lines used in the study cited above. Baptista et al. (2010) showed that the
465
470
475
480
485
490
495
500
505
510
Control 1 kGy 2 kGy
Dose (kGy)
Cen
ter
of m
ass
(nm
)
0
50
100
150
200
250
400 435 470 505 540 575
Wavelength (nm)
Flu
ores
cenc
e in
ten
sity
(A
.U.)
465
470
475
480
485
490
495
500
505
510
Control 1 kGy 2 kGy
Dose (kGy)
Cen
ter
of m
ass
(nm
)
0
50
100
150
200
250
400 435 470 505 540 575
Wavelength (nm)
Flu
ores
cenc
e in
ten
sity
(A
.U.)
93
irradiated bothropstoxin-I was 5 folds less toxic than its native counterpart, but still immunogenic.
Souza-Filho et al. (1992), in a study on detoxification of the crotoxin complex by gamma radiation
found structural changes that occurred in this neurotoxin venom of the rattlesnake, were due to the
formation of intermolecular covalent bonds and to the rupture of disulfide bonds. Gallaci et al.
(1998), suggest that the breaking of the disulfide bonds of crotoxin by gamma radiation may be an
important role in the loss of its neuromuscular blocking action. The homodimer BJcuL, a lectin
isolated from Bothrops jararacussu snake proved to be active only as a dimeric configuration
(Carvalho et al., 2002). According to Kassab et al (2004), the presence of the monomers without the
formation of a correct inter-chain disulfide bond, as well as structural instability of dimer
configuration avoiding may be favorable activity.
Based on these considerations, we suggest that the loss in cytotoxicity ascribed to
irreversible may be structural changes in BlL, induced by gamma radiation, which may have
affected the interaction of lectin with glicoconjugates exposed on the surface of tumor cells. In
conclusion, our results indicates that gamma irradiation of BlL can induce significant modifications
in their structure, as well as promote its effective detoxification.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors express their gratitude to the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) and to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) for research grants. We are grateful to the Departamento de Energia Nuclear from the
universidade Federal de Pernambuco (UFPE). The authors thank Maria D. Rodrigues and João
Antônio Virgínio for technical assistance.
94
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8 CONCLUSÕES
- Num protocolo eficiente, cromatografias de gel de Guar e Superdex, purificaram uma lectina tipo-
C (BlL) do veneno da serpente Bothrops leucurus.
- BlL é dependente de cálcio e inibida por açúcares contendo galactosídeos. Estruturalmente BlL é
um dímero composto de subunidades de 15 kDa unidas por ligações dissulfeto;
- O Dicroísmo Circular revelou que BlL pode ser classificada como uma proteína toda β (contendo
principalmente estrutura β);
- BlL possui atividade antibacteriana contra bactérias gram-positivas (Staphylococcus aureus,
Enterococcus faecalis e Bacillus subtilis);
- BlL apresentou significante atividade citotóxica em células tumorais (K562, Hep-2 e NCI-H292) e
induziu morte celular por apoptose em células tumorais K562;
- A radiação gamma de 60Co causou alterações funcionais e estruturais em BlL, além de abolir a sua
atividade citotóxica em linhagens tumorais.
