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

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dc.contributor.authorRodrigues, V. M.
dc.contributor.authorSoares, A. M.
dc.contributor.authorGuerra-Sa, R.
dc.contributor.authorRodrigues, V
dc.contributor.authorFontes, MRM
dc.contributor.authorGiglio, JR
dc.contributor.institutionUniversidade de São Paulo (USP)
dc.contributor.institutionUniv Estadual Minas Gerais
dc.contributor.institutionUniversidade Estadual Paulista (Unesp)
dc.date.accessioned2014-05-20T15:21:20Z
dc.date.available2014-05-20T15:21:20Z
dc.date.issued2000-09-15
dc.description.abstractA fibrino(geno)lytic nonhemorrhagic metalloprotease (neuwiedase) was purified from Bothrops neuwiedi snake venom by a single chromatographic step procedure on a CM-Sepharose column, Neuwiedase represented 4.5% (w/w) of the crude desiccated venom, with an approximate Mr of 20,000 and pI 5.9, As regards the amino acid composition, neuwiedase showed similarities with other metalloproteases, with high proportions of Asx, Glx, Leu, and Ser, Atomic absorption 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 metalloproteases 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 was modeled based on the crystal structure of Crotalus adamanteus Adamalysin II. This model revealed that the zinc binding site region showed a I high structural similarity with other metalloproteases,, the proteolyitc specificity, 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 fibrinogenase, 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 both 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 extracellular matrix. However, it did not show TAME esterase activity and was not able to inhibit platelet aggregation. (C) 2000 Academic Press.en
dc.description.affiliationUniv São Paulo, Fac Med, Dept Bioquim, BR-14049900 Ribeirao Preto, SP, Brazil
dc.description.affiliationUniv Estadual Minas Gerais, Fundação Educ Patos Minas, Ribeirao Preto, SP, Brazil
dc.description.affiliationUniv São Paulo, Fac Med, Dept Microbiol Parasitol & Imunol, BR-14049 Ribeirao Preto, SP, Brazil
dc.description.affiliationUniv Estadual Paulista, Inst Biociencias, Dept Fis & Biofis, Botucatu, SP, Brazil
dc.description.affiliationUnespUniv Estadual Paulista, Inst Biociencias, Dept Fis & Biofis, Botucatu, SP, Brazil
dc.format.extent213-224
dc.identifierhttp://dx.doi.org/10.1006/abbi.2000.1958
dc.identifier.citationArchives of Biochemistry and Biophysics. San Diego: Academic Press Inc., v. 381, n. 2, p. 213-224, 2000.
dc.identifier.doi10.1006/abbi.2000.1958
dc.identifier.issn0003-9861
dc.identifier.urihttp://hdl.handle.net/11449/32500
dc.identifier.wosWOS:000089557300005
dc.language.isoeng
dc.publisherAcademic Press Inc.
dc.relation.ispartofArchives of Biochemistry and Biophysics
dc.relation.ispartofjcr3.118
dc.relation.ispartofsjr1,350
dc.rights.accessRightsAcesso restrito
dc.sourceWeb of Science
dc.subjectmetalloproteasept
dc.subjectcDNA cloningpt
dc.subjectmolecular modelpt
dc.subjectfibrin(ogen)olytic activitypt
dc.subjectBothrops neuwiedipt
dc.subjectsnake venompt
dc.titleStructural and functional characterization of neuwiedase, a nonhemorrhagic fibrin(ogen)olytic metalloprotease from Bothrops neuwiedi snake venomen
dc.typeArtigo
dcterms.licensehttp://www.elsevier.com/about/open-access/open-access-policies/article-posting-policy
dcterms.rightsHolderAcademic Press Inc.
unesp.author.orcid0000-0002-4634-6221[5]
unesp.author.orcid0000-0002-3386-9197[4]
unesp.campusUniversidade Estadual Paulista (Unesp), Instituto de Biociências, Botucatupt

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