Characterization of Multiple Metalloproteinases with Fibrinogenolytic Activity from the Venom of Taiwan Habu (Trimeresurus mucrosquamatus): Protein Microsequencing Coupled with cDNA Sequence Analysis

Abstract

Three fibrinogenolytic proteases were isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using anion-exchange and gel filtration chromatographies followed by cation-exchange HPLC. Further characterization of these purified fractions with fibrinogenase activity indicated that they are single-chain proteases of approximately 24 kDa, possessing strong cleaving activity mainly on the Aα and less on Bβ and γ chains of fibrinogen subunit chains. Enzyme activities were strongly inhibited by EDTA or 1,10-phenanthroline and not by phenylmethanesulfonyl fluoride, suggesting that these fibrinogenases belong to the family of metalloproteinases and not thrombin-like serine proteases. N-Terminal sequence analysis of these proteases failed to show any free amino-terminal residues, thus hampering the sequence determination by conventional sequencing strategy. Microsequencing on the electroblotted fragments of CNBr-treated proteases separated on SDS-PAGE was then used to determine the partial; sequences. Sequence comparison of the determined partial sequences of these proteins with published sequences of the protein data bank revealed that they showed sequence homology with H2-protease, HR2a and protrigramin, which were all shown to belong to metalloproteinases present in various snake venoms. Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of fresh venom glands of the same snake species to facilitate cloning and sequencing of these proteases. Sequencing several positive clones containing amplified cDNAs revealed the existence of one fibrinogenase in the Taiwan habu, which was contained within one complete cDNA encoding the preproproteinase precursor of hemorrhagic metalloproteinases.