RESUMO
Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Through antagonism of integrins alphaIIbbeta3, alpha5beta1, alphavbeta3, and alphavbeta5, contortrostatin inhibits platelet aggregation and disrupts cancer cell adhesion and invasion. We cloned cDNA from a library made from the venom gland cells of Agkistrodon contortrix contortrix using polymerase chain reaction. We found that the contortrostatin gene is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. The precursor cDNA is 2027 bp with a 1449-bp open reading frame. The disintegrin domain is 195 bp encoding 65 amino acids. Like other members of the disintegrin family, each subunit of contortrostatin has an RGD site, and the cysteine alignment is conserved. The disintegrin domain of the cDNA has been expressed in a eukaryotic expression system as a homodimeric fusion protein with an immunoglobulin. The recombinant protein is recognized by an antiserum against native contortrostatin in Western blot. Both the native and recombinant proteins bind to integrins alphavbeta3 and alphavbeta5. Like native contortrostatin, the recombinant fusion protein inhibits platelet aggregation, blocks cancer cell adhesion to fibronectin and vitronectin, and prevents invasion of cancer cells through a Matrigel barrier. The success of functional expression not only validates the cDNA cloning of this disintegrin, but also provides adequate material for functional studies of contortrostatin.
Assuntos
Agkistrodon/genética , Venenos de Crotalídeos/química , Desintegrinas/genética , Desintegrinas/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sequência de Bases , Western Blotting , Adesão Celular/efeitos dos fármacos , Clonagem Molecular , Venenos de Crotalídeos/genética , DNA Complementar/genética , Dimerização , Desintegrinas/química , Desintegrinas/farmacologia , Humanos , Integrinas/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Invasividade Neoplásica/patologia , Agregação Plaquetária/efeitos dos fármacos , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Receptores de Vitronectina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
Contortrostatin is a homodimeric disintegrin from snake venom. We have shown that contortrostatin binds to integrins alphaIIbbeta3, alpha5beta1, and alphavbeta3. We now use several criteria to demonstrate the binding of contortrostatin to alphavbeta5. First, incubation of T24 cells, which express alphavbeta3 and alphavbeta5, with antibody against alphavbeta3 failed to completely inhibit adhesion of cells to vitronectin. However, pretreatment of the cells with contortrostatin or the combination of antibodies against alphavbeta3 and alphavbeta5 completely blocked adhesion to vitronectin. By contrast, either anti-alphavbeta5 alone or contortrostatin blocked adhesion of an alphavbeta3-negative T24 subline. Second, contortrostatin as well as anti-alphavbeta5 inhibits invasion of OVCAR-5, which express only alphavbeta5. Third, contortrostatin binds to purified alphavbeta5 in a saturable manner. Finally, radioligand binding assays yielded a K(d) value of 24 nM for [(125)I]contortrostatin binding to alphavbeta5. This investigation identifies alphavbeta5 as a binding site for contortrostatin. Blockage of alphavbeta5 by contortrostatin inhibits alphavbeta5-mediated adhesion and invasion.
