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ANISERP: a new serpin from the parasite Anisakis simplex.

ABSTRACT: 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).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.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.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.

SUBMITTER: Valdivieso E

PROVIDER: S-EPMC4517634 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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ANISERP: a new serpin from the parasite Anisakis simplex.

Valdivieso Elizabeth E Perteguer Maria J MJ Hurtado Carolina C Campioli Pamela P Rodríguez Esperanza E Saborido Ana A Martínez-Sernández Victoria V Gómez-Puertas Paulino P Ubeira Florencio M FM Gárate Teresa T

Parasites & vectors 20150728

<h4>Background</h4>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).<h4>Methods</h4> ...[more]

PMID: 26215984

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Project description:BackgroundAnisakis simplex sensu stricto and Anisakis pegreffii are sibling species of nematodes parasitic on marine mammals. Zoonotic human infection with third stage infective larvae causes anisakiasis, a debilitating and potentially fatal disease. These 2 species show evidence of hybridisation in geographical areas where they are sympatric. How the species and their hybrids differ is still poorly understood.ResultsThird stage larvae of Anisakis simplex s.s., Anisakis pegreffii and hybrids were sampled from Merluccius merluccius (Teleosti) hosts captured in waters of the FAO 27 geographical area. Specimens of each species and hybrids were distinguished with a diagnostic genetic marker (ITS). RNA was extracted from pools of 10 individuals of each taxon. Transcriptomes were generated using Illumina RNA-Seq, and assembled de novo. A joint assembly (here called merged transcriptome) of all 3 samples was also generated. The inferred transcript sets were functionally annotated and compared globally and also on subsets of secreted proteins and putative allergen families. While intermediary metabolism appeared to be typical for nematodes in the 3 evaluated taxa, their transcriptomes present strong levels of differential expression and enrichment, mainly of transcripts related to metabolic pathways and gene ontologies associated to energy metabolism and other pathways, with significant presence of excreted/secreted proteins, most of them allergens. The allergome of the 2 species and their hybrids has also been thoroughly studied; at least 74 different allergen families were identified in the transcriptomes.ConclusionsA. simplex s.s., A. pegreffi and their hybrids differ in gene expression patterns in the L3 stage. Strong parent-of-origin effects were observed: A. pegreffi alleles dominate in the expression patterns of hybrids albeit the latter, and A. pegreffii also display significant differences indicating that hybrids are intermediate biological entities among their parental species, and thus of outstanding interest in the study of speciation in nematodes. Analyses of differential expression based on genes coding for secreted proteins suggests that co-infections presents different repertoires of released protein to the host environment. Both species and their hybrids, share more allergen genes than previously thought and are likely to induce overlapping disease responses.

Dataset Information

ANISERP: a new serpin from the parasite Anisakis simplex.

Publications

ANISERP: a new serpin from the parasite Anisakis simplex.

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