印尼金黃樹蝮蛇毒絲胺酸蛋白酶的純化、基因選殖與基質特擇性

Four serine proteases (designated Tpu1—4) were purified from the venom of Trimeresurus puniceus (golden tree viper) from Indonesia. Upon the amidolytic assays using three chromogenic substrates, Tpu1 preferred to hydrolyze Benzoyl-Pro-Phe-Arg- p-NA, a chromogenic substrate for kallilrein, Tpu2 and Tpu3 preferred to hydrolyze Tosyl-Gly-Pro-Arg-p-NA, a chromogenic substrate for thrombin, but Tpu4 did not hydrolyzed any of these substrates. These purified proteases were able to cleave high molecular weight kininogen (HMWK), fibrinogen, and angiotensinⅠ, but with different efficacies. The estimated molecular weights of Tpu1—4 under reducing condition (with 5% β-mercaptoethanol) were 54 kDa, 30 kDa, 35 kDa, 51 kDa, respectively. After removal of N-glycans by PNGaseF the reduced Tpu1—4 migrated as single bands with an apparent molecular weight of 25~27 kDa in SDS-PAGE. Tpu1 and Tpu4 apparently have higher carbohydrate content than Tpu2 and Tpu3. The N-terminal sequences of these purified proteases are different from each other.
PCR of the venom gland cDNA mixtures using designed specific primers resulted in the amplification of cDNAs encoding these serine proteases. After checking the sequences of about 40 clones, three distinct cDNA sequences were found to match the proteases purified. The predicted molecular weight without post-translation modification (but assuming the formation of six pairs of disulfide bonds) for Tpu1, Tpu2 and Tpu4 are 25624 Da, 25350 Da, and 26109 Da, respectively. The active site residues of serine proteases family, i.e., His57, Asp102 and Ser195 (the chymotrypsin numbering) and location of six pairs of disulfide bonds of the viperid venom proteases are conserved. Tpu1, Tpu2 and Tpu4 contain five, two, and four potentional N-glycosylation sites, respectively.
In addition, three distinct cDNA sequences encoding serine proteases were cloned from the venom gland of T. borneenesis, a species closely related to T. puniceus. The deduced proteases were named Tbo2, Tbo3 and Tbo4, respectively. Interestingly, the N-terminal sequence of Tpu3 is found to be identical to that of Tbo3. Tbo2 and Tpu2 differ by only three residues, confirming a very close relationship between T. puniceus and T. borneenesis. Results of BLASTP search and phylogenetic analyses also revealed that these serine protease sequences of T. puniceus and T. borneenesis are very similar to those of Chinese green tree viper Trimeresurus stejnegeri.