Purification, cloning and sequence analyses for pro-metalloprotease-disintegrin variants from Deinagkistrodon acutus venom and subclassification of the small venom metalloproteases

Acidic and basic hemorrhagic metalloproteases were purified from the venom of Deinagkistrodon acutus (from Fujian Province, China) using gel filtratio n and anion exchange on FPLC and reversed-phase HPLC. Their hemorrhagic act ivities and N-terminal sequences were characterized. Extensive screening of the venom gland cDNA after PCR amplification resulted in the identificatio n and sequencing of a total of seven cDNA clones encoding the multidomain p recursors of six acidic and one alkaline low molecular mass metalloprotease s. Two of the precursors contain a processable disintegrin domain. Disinteg rins of 5 kDa were also purified from the venom. The partial amino-acid seq uences and molecular masses determined by electrospray ionization mass spec trometry of the purified proteins specifically match those deduced from two of the cDNA sequences. Moreover, phylogenetic analyses based on 30 complet e sequences of low molecular mass venom metalloproteases revealed that they may be classified into three functional subtypes: acidic hemorrhagins, bas ic and moderate hemorrhagins, and nonhemorrhagic enzymes. Subtype-specific amino-acid substitutions in the C-terminal regions of the enzymes were high lighted to explore the structure-activity relationships of the enzymes.