3 research outputs found
ANISERP: a new serpin from the parasite Anisakis simplex
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.We 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
ANISERP: A new serpin from the parasite Anisakis simplex
[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.This work was funded by the Spanish Ministry of Science and Innovation (SAF2002-04057-CO2-01) and the Instituto de Salud Carlos III (ISCIII) as part of project MPY 1404/09, and ISCIII-AESI project MPY 1279/15. Carolina Hurtado and Pamela Campioli were supported by grants from the ISCIII (Fondo de Investigaciones Sanitarias), through VI NP of I + D + I (2008–2011), ISCIII General Sub-Direction of Networks and Centers for Collaborative Research (RETIC-RICET, RD06/0021/0019, RD12/0018/011). Victoria Martínez Sernández holds a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (Programa de Formación del Profesorado Universitario).Peer Reviewe