cDNA cloning, expression and fibrin(ogen)olytic activity of two low-molecular weight snake venom metalloproteinases.
Two cDNA clones, AplVMP1 and AplVMP2, were isolated from a snake (Agkistrodon piscivorus leucostoma) venom gland cDNA library. The full-length cDNA sequence of AplVMP1 with a calculated molecular mass of 46.61 kDa is 1233 bp in length. AplVMP1 encodes PI class metalloproteinase with an open reading frame of 411 amino acid residues that includes signal peptide, pro-domain and metalloproteinase domains. The full-length cDNA of the AplVMP2 (1371 bp) has a calculated molecular mass of 51.16 kDa and encodes PII class metalloproteinase. The open reading frame of AplVMP2 with a 457 amino acid residues is composed of signal peptide, pro-domain, metalloproteinase and disintegrin domains. AplVMP1 and AplVMP2 showed 85% and 93% amino acid identical to PI class enzyme Agkistrodon contortrix laticinctus ACLPREF and PII class enzyme Agkistrodon piscivorus piscivorus piscivostatin, respectively. When expressed in Escherichia coli, most of recombinant proteins of AplVMP1 and AplVMP2 were in insoluble inclusion bodies, with soluble yields of 0.7 mg/l and 0.4 mg/l bacterial culture, respectively. Both affinity purified recombinant proteins show proteolytic activity on fibrinogen, although having an activity lower than that of crude A. p. leucostoma venom. Proteolytic activities of AplVMP1 and AplVMP2 were completely abolished after incubation with a final concentration of 100 microM of EDTA or 1,10-phenanthroline. Both AplVMP1 and AplVMP2 were active in a fibrin-agarose plate but devoid of hemorrhagic activity when injected (up to 50 microg) subcutaneously into mice, and had no capacity to inhibit platelet aggregation.
Natural Toxins Research Center, College of Arts and Sciences, Texas A&M University, Kingsville, TX 78363, USA.
Ying
Y
Jia
Sara
S
Lucena
Esteban
E
Cantu
Elda E
EE
Sánchez
John C
JC
Pérez
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005342:Fibrinolysis / Q000187:drug effects*; D006470:Hemorrhage / Q000139:chemically induced; D006801:Humans; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010974:Platelet Aggregation / Q000187:drug effects; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012910:Snake Venoms / Q000201:enzymology*
D018076:DNA, Complementary; D012910:Snake Venoms; D045726:Metalloproteases