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dc.contributor.authorValdivieso, Elizabeth
dc.contributor.authorPerteguer, Maria J
dc.contributor.authorHurtado, Carolina
dc.contributor.authorCampioli, Pamela
dc.contributor.authorRodríguez, Esperanza
dc.contributor.authorSaborido, Ana
dc.contributor.authorMartínez-Sernández, Victoria
dc.contributor.authorGómez-Puertas, Paulino
dc.contributor.authorUbeira, Florencio M
dc.contributor.authorGárate, Teresa
dc.date.accessioned2015-09-01T18:13:20Z
dc.date.available2015-09-01T18:13:20Z
dc.date.issued2015-07-28
dc.identifier.citationParasites & Vectors. 2015 Jul 28;8(1):399
dc.identifier.urihttp://dx.doi.org/10.1186/s13071-015-1006-z
dc.identifier.urihttp://hdl.handle.net/10724/31969
dc.description.abstractAbstract Background Serine proteinase inhibitors (serpins) finely regulate serine proteinase activity via a suicide substrate-like inhibitory mechanism. In parasitic nematodes, some serpins interact with host physiological processes; however, little is known about these essential molecules in Anisakis. This article reports the gene sequencing, cloning, expression and preliminary biochemical and bioinformatically-based structural characterization of a new Anisakis serpin (ANISERP). Methods The full AniSerp gene was cloned by specific RACE-PCR after screening an Anisakis simplex (L3) cDNA library. For biochemical assays, the AniSerp gene was subcloned into both prokaryotic and eukaryotic vectors, and the recombinant proteins were purified. The inhibitory properties of the proteins were tested in classical biochemical assays using human serine peptidases and AMC substrates. Immunolocalization of ANISERP, theoretical structural analysis and bioinformatically-based structural modelling of the ANISERP protein were also conducted. Results The AniSerp gene was found to have 1194 nucleotides, coding for a protein of 397 amino acid residues plus a putative N-terminal signal peptide. It showed significant similarity to other nematode, arthropod and mammalian serpins. The recombinant ANISERP expressed in the prokaryotic and eukaryotic systems inhibited the human serine proteases thrombin, trypsin and cathepsin G in a concentration-dependent manner. No inhibitory activity against Factor Xa, Factor XIa, Factor XIIa, elastase, plasmin or chymotrypsin was observed. ANISERP also acted on the cysteine protease cathepsin L. ANISERP was mainly localized in the nematode pseudocoelomic fluid, somatic muscle cell bodies and intestinal cells. The findings of molecular dynamics studies suggest that ANISERP inhibits thrombin via a suicide substrate-like inhibitory mechanism, similar to the mechanism of action of mammalian coagulation inhibitors. In contrast to findings concerning human antithrombin III, heparin had no effect on ANISERP anticoagulant inhibitory activity. Conclusions Our findings suggest that ANISERP is an internal Anisakis regulatory serpin and that the inhibitory activity against thrombin depends on a suicide substrate-like inhibitory mechanism, similar to that described for human antithrombin (AT)-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in the latter of five of the six positively charged residues usually seen at the AT-III-heparin binding site.
dc.titleANISERP: a new serpin from the parasite Anisakis simplex
dc.typeJournal Article
dc.date.updated2015-07-29T19:09:35Z
dc.language.rfc3066en
dc.rights.holderValdivieso et al.


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