Structural and functional characterization of neuwiedase, a nonhemorrhagicfibrin(ogen)olytic metalloprotease from Bothrops neuwiedi snake venom

A fibrino(geno)lytic nonhemorrhagic metalloprotease (neuwiedase) was purifi ed from Bothrops neuwiedi snake venom by a single chromatographic step proc edure on a CM-Sepharose column, Neuwiedase represented 4.5% (w/w) of the cr ude desiccated venom, with an approximate Mr of 20,000 and pI 5.9, As regar ds the amino acid composition, neuwiedase showed similarities with other me talloproteases, with high proportions of Asx, Glx, Leu, and Ser, Atomic abs orption spectroscopy showed that one mole of Zn2+ and one mole of Ca2+ were present per mole olf protein. The cDNA encoding neuwiedase was isolated by RT-PCR from venom gland RNA, using oligonucleotides based on the partially determined amino-acid sequences of this metalloprotease. The fall sequence contained approximately 594 bp, which codified the 198 amino acid residues with an estimated molecular weight of 22,375. Comparison of the nucleotide and amino acid sequences of neuwiedase with those of other snake venom met alloproteases showed a high level of sequential similarity, Neuwiedase has two highly conserved characteristics sequences H(142)E(143)XXH(146)XXG(140) XXH(152) and C164I165M166. The three-dimensional structure of neuwiedase wa s modeled based on the crystal structure of Crotalus adamanteus Adamalysin II. This model revealed that the zinc binding site region showed a I high s tructural similarity with other metalloproteases,, The proteolyitc specific ity, using the B beta-chain of oxidized insulin as substrate, was shown to be directed to the Ala(14)-Leu(15) and Tyr(16)-Leu(17) peptide bonds which were preferentially hydrolyzed. Neuwiedase is a A alpha,B beta fibrinogenas e, Its activity upon the A alpha chain of fibrinogen was detected within 15 min of incubation. The optimal temperature and pH for the degradation of b oth A alpha and B beta chains were 37 degrees C and 7.4-8.0, respectively. This activity was inhibited by EDTA and 1,10-phenantroline, Neuwiedase also showed proteolytic activity upon fibrin and some components of the extrace llular matrix. However, it did not show TAME esterase activity and was not able to inhibit platelet aggregation. (C) 2000 Academic Press.