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Molecular cloning and expression of catrocollastatin, a snake-venom protein from Crotalus atrox (western diamondback rattlesnake) which inhibits platelet adhesion to collagen.

ABSTRACT: A 50 kDa protein that inhibits platelet adhesion to collagen has been isolated from snake venom of Crotalus atrox (western diamondback rattlesnake) and has been named 'catrocollastatin'. The cDNA cloning of catrocollastatin has been accomplished. A full-length cDNA of 2310 bp with an open reading frame between nucleotides 51 and 1880 was obtained. The deduced amino acid sequence consists of 609 amino acids. The cDNA-predicted amino acid sequence is highly similar to that of haemorrhagic metalloproteinase jararhagin from Bothrops jararaca venom, HR1B from Trimeresurus flavoviridis, Ht-e from C. atrox and trigramin from T. gramineus. Like jararhagin and HR1B, catrocollastatin is a multidomain molecule composed of an N-terminal domain, a metalloproteinase domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. In the disintegrin-like domain, the frequently seen RGD (Arg-Gly-Asp) sequence is replaced by SECD (Ser-Glu-Cys-Asp). This cDNA was expressed in Spodoptera frugiperda (fall armyworm) (Sf9) insect cells using a baculovirus expression system. Like native catrocollastatin, the expressed protein is capable of selectively blocking collagen-induced platelet aggregation. This is the first full-length clone of a high-molecular-mass haemorrhagin to be expressed.

SUBMITTER: Zhou Q

PROVIDER: S-EPMC1136664 | biostudies-other | 1995 Apr

REPOSITORIES: biostudies-other

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Project description:Snake venom metalloproteinases (SVMPs) are a superfamily of zinc-dependent proteases and participate in a number of important biological, physiological and pathophysiological processes. In this work, we simultaneously amplified nine cDNAs encoding different classes of metalloproteinases from glands of four different snake species (Agkistrodon contortrix laticinctus, Crotalus atrox, Crotalus viridis viridis and Agkistrodon piscivorus leucostoma) by RT-PCR with a pair of primers. Among the encoded metalloproteinases, two enzymes (AclVMP-I and AplVMP-I), three enzymes (CaVMP-II, CvvVMP-II and AplVMP-II) and four enzymes (AclVMP-III, CaVMP-III, CvvVMP-III and AplVMP-III) with the characteristic motif (HEXXHXXGXXH) of metalloproteinase belong to type P-I, P-II and P-III enzymes, respectively. Disintegrin domains of CaVMP-II and CvvVMP-II from two Crotatus snakes contain RGD-motif whereas AplVMP-II from Agkistrodon snake has KGD-motif. Instead of R/KGD-motif within disintegrin domain of SVMP-II enzyme, CaVMP-III, CvvVMP-III and AplVMP-III enzymes contain SECD-motif, while AclVMP-III has DDCD-motif in their corresponding position of disintegrin-like domains. There are 12 Cys amino acids in cysterin-rich domains of each P-III enzyme. Moreover, a disintegrin precursor (AplDis) with RGD-motif also simultaneously amplified from the glands of A.p. leucostoma while amplifying AplVMP-II and AplVMP-III, which indicated that different types of SVMPs and related genes are present in a single species of snake and share a consensus sequence at the 3' and 5' untranslated regions. RT-PCR result also showed that P-III is highly expressed in Crotalus snakes than in Agkistrodon snakes. Aligning the deduced amino acid sequence of these enzymes with other SVMPs from GenBank database indicated that this is the first report on the isolation of cDNAs encoding P-II and P-III enzymes from C.v. viridis and A.p. leucostoma snakes. The availability of these SVMP sequences directly facilitated further studies of structure characterization and diversified function analysis.

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Molecular cloning and expression of catrocollastatin, a snake-venom protein from Crotalus atrox (western diamondback rattlesnake) which inhibits platelet adhesion to collagen.

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