ANISERP: a new serpin from the parasite Anisakis simplex

Area of Science:

• Parasitology
• Molecular Biology
• Biochemistry

Background:

• Serine proteinase inhibitors (serpins) regulate serine proteinase activity through a suicide substrate-like mechanism.
• While some nematode serpins interact with host processes, Anisakis serpins remain understudied.
• This study focuses on characterizing a novel Anisakis serpin (ANISERP).

Purpose of the Study:

• To sequence, clone, and express a novel Anisakis serpin (ANISERP).
• To biochemically and structurally characterize ANISERP's inhibitory properties.
• To elucidate ANISERP's mechanism of action and localization within Anisakis.

Main Methods:

• Cloning of the AniSerp gene using RACE-PCR from Anisakis simplex cDNA.
• Expression of recombinant ANISERP in prokaryotic and eukaryotic systems.
• Biochemical assays using human serine peptidases and AMC substrates.
• Immunolocalization, theoretical structural analysis, and bioinformatic modeling of ANISERP.

Main Results:

• The AniSerp gene encodes a 397-amino acid protein with similarity to other serpins.
• Recombinant ANISERP inhibited human thrombin, trypsin, and cathepsin G, but not other tested proteases.
• ANISERP demonstrated a suicide substrate-like inhibition of thrombin and localized to pseudocoelomic fluid, muscle, and intestinal cells.
• Heparin did not affect ANISERP's anticoagulant activity.

Conclusions:

• ANISERP functions as an internal regulatory serpin in Anisakis.
• Its thrombin inhibition mechanism is similar to human antithrombin (AT)-III.
• The lack of heparin modulation is attributed to differences in heparin-binding site residues compared to human AT-III.