RESUMEN
ACL myotoxin (ACLMT) is a K49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus (broad-banded copperhead) that induces necrosis of skeletal muscle. We have previously cloned and sequenced the cDNA coding for ACLMT from a venom gland cDNA library. In order to perform structure and function studies, we have developed an expression system for production of ACLMT as a fusion protein with maltose binding protein (MBP) from the periplasm of bacteria, using the pMAL-p2 expression vector. The cDNA coding for the mature toxin without the signal peptide was amplified by PCR and subcloned into the pMAL-p2 vector. The new plasmid (pMAL-MT) was used to transform BL21(DE3) E. coli cells. Culture of transformed cells induced with IPTG led to the expression of a 60 kDa fusion protein which strongly reacts with anti-native ACLMT antibodies. The fusion protein was purified from the bacterial periplasm by affinity chromatography in an amylose column and by gel filtration. The purified fusion protein (MBP-rACLMT) was able to induce necrosis of skeletal muscle of mice very similar to that caused by the native myotoxin.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Agkistrodon/genética , Proteínas Bacterianas/genética , Venenos de Crotálidos/enzimología , Proteínas de Escherichia coli , Vectores Genéticos , Isopropil Tiogalactósido/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas de Transporte de Monosacáridos , Neurotoxinas/metabolismo , Fosfolipasas A/genética , Fosfolipasas A/toxicidad , Plásmidos/genética , Proteínas Recombinantes de Fusión , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes/genética , Toxinas Biológicas/aislamiento & purificación , Agkistrodon/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Portadoras/química , Cromatografía de Afinidad , Cromatografía en Gel , Venenos de Crotálidos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca de Genes , Inyecciones Intramusculares , Lisina/química , Maltosa/aislamiento & purificación , Proteínas de Unión a Maltosa , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Necrosis , Neurotoxinas/toxicidad , Fosfolipasas A/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Toxinas Biológicas/metabolismo , Toxinas Biológicas/toxicidad , Transformación GenéticaRESUMEN
The alpha2beta1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Here we describe the isolation of a novel metalloproteinase/disintegrin, which is a potent inhibitor of the collagen binding to alpha2beta1 integrin. This 55-kDa protein (alternagin) and its disintegrin domain (alternagin-C) were isolated from Bothrops alternatus snake venom. Amino acid sequencing of alternagin-C revealed the disintegrin structure. Alternagin and alternagin-C inhibit collagen I-mediated adhesion of K562-alpha2beta1-transfected cells. The IC50 was 134 and 100 nM for alternagin and alternagin-C, respectively. Neither protein interfered with the adhesion of cells expressing alphaIIbeta3, alpha1beta1, alpha5beta1, alpha4beta1 alphavbeta3, and alpha9beta1 integrins to other ligands such as fibrinogen, fibronectin, and collagen IV. Alternagin and alternagin-C also mediated the adhesion of the K562-alpha2beta1-transfected cells. Our results show that the disintegrin-like domain of alternagin is responsible for its ability to inhibit collagen binding to alpha2beta1 integrin.