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dc.contributor.authorValdivieso, Elizabeth
dc.contributor.authorPerteguer-Prieto, Maria Jesus 
dc.contributor.authorHurtado, Carolina 
dc.contributor.authorCampioli, Pamela 
dc.contributor.authorRodríguez, Esperanza 
dc.contributor.authorSaborido, Ana
dc.contributor.authorMartínez-Sernández, Victoria
dc.contributor.authorGomez-Puertas, Paulino 
dc.contributor.authorUbeira, Florencio M.
dc.contributor.authorGarate, Teresa 
dc.date.accessioned2017-09-04T16:35:06Z
dc.date.available2017-09-04T16:35:06Z
dc.date.issued2015-07-28
dc.identifier.citationParasit Vectors. 2015; 8:399
dc.identifier.urihttp://hdl.handle.net/20.500.12105/4854
dc.description.abstractBackground: 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.description.sponsorshipWe thank Biomol-Informatics SL (http://www.biomol-informatics.com/) for bioinformatic consultation. We also thank Juan Francisco Alcaide and Julia Medrano for their invaluable help in the ANISERP patenting process. We thank Dr Raúl Iglesias (Laboratorio de Parasitología, Facultad de Biología, Universidad de Vigo) for his helpful comments on the interpretation of the IHQ studies.
dc.language.isoeng
dc.publisherBioMed Central
dc.relation.isversionofPublisher's version
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/*
dc.subjectSerpin
dc.subjectProteinase
dc.subjectAnisakis
dc.subjectTrypsin
dc.subjectThrombin
dc.subjectCathepsin L
dc.subjectAnticoagulant properties
dc.subjectModelling analysis
dc.subjectHeparin
dc.titleANISERP: a new serpin from the parasite Anisakis simplex
dc.typeArtículo
dc.rights.licenseAtribución 4.0 Internacional*
dc.identifier.pubmedID26215984
dc.format.volume8
dc.format.number1
dc.format.page399
dc.identifier.doi10.1186/s13071-015-1006-z
dc.identifier.e-issn1756-3305
dc.relation.publisherversionhttps://doi.org/10.1186/s13071-015-1006-z
dc.identifier.journalParasites & Vectors
dc.repisalud.centroISCIII::Centro Nacional de Microbiología::Área de Bacteriología, Micología Y Parasitología::Servicio de Parasitología
dc.repisalud.institucionISCIII
dc.rights.accessRightsinfo:eu-repo/semantics/openAccesses_ES


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Atribución 4.0 Internacional
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