ANISERP: a new serpin from the parasite Anisakis simplex

Abstract

BackgroundSerine 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).MethodsThe 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.ResultsThe 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.ConclusionsOur 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.

The genome ofStrongyloidesspp. gives insights into protein families with a putative role in nematode parasitism

“…Screening of potential target mammalian serine proteinases revealed that Bm-SPN-2 inhibits human neutrophil-derived proteinases cathepsin G and elastase (Zang et al 1999). Similarly, the serpin Anisakis simplex serpin (ANISERP) also inhibits cathepsin G, as well as the human blood clotting serine protease thrombin and the digestive serine protease trypsin (Valdivieso et al 2015). In S. ratti, and S. stercoralis there are 12 and 10 genes, respectively, encoding serpins belonging to the trypsin-inhibitor protein family which are significantly upregulated in the parasitic female transcriptome when compared to the free-living female transcriptome.…”

Genetic Diversity of Serine Protease Inhibitors in Myxozoan (Cnidaria, Myxozoa) Fish Parasites

“…Schistosome serpins identified in the blood fluke Schistosoma mansoni are involved in both post-translational regulation of schistosome-derived proteases, as well as in parasite defense mechanisms against the action of host proteases [ 20 ]. In nematodes, serpins interact with endogenous parasite proteinases, counteract the digestion by host proteinases, inhibit the host immune response, or even act as immunomodulators [ 21 , 22 ]. Proteases and their inhibitors have been reported as important factors for invasion and immune evasion of parasites [ 23 , 24 , 25 , 26 ].…”

The highly repetitive genome of Myxobolus sp., a myxozoan parasite of fathead minnows

“…In one such example of gene family expansion, serpins (serine proteases) play an important role in parasitic organisms, like defense against host proteases in Schistosoma mansoni and acting as immunomodulators and inhibit host immune responses in nematodes (Maizels et al, 2001; Valdivieso et al, 2015). Recently, a study analyzed the expression-profiles of M. cerebralis serpins and proposed that they represent good targets for inactivating the parasite at an early stage in the fish host (Eszterbauer et al, 2021).…”