Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from Angiostrongylus cantonensis

Abstract

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, a novel cystatin molecule (AcCystatin) was cloned from a cDNA library of Angiostrongylus cantonensis fourth-stage larvae. The putative 14-kDa protein contained 120 residues with cystatin-conserved motifs known to interact with the active site of cysteine peptidases and showed high identities with cystatins from other nematodes. RT-PCR analysis revealed that the expression pattern of AcCystatin was equal at the time points of third-stage larvae, fourth-stage larvae, and adults of the parasite life cycle. The recombinant AcCystatin (rAcCystatin) expressed and purified from Escherichia coli has been demonstrated to possess an obvious inhibitory activity against cathepsin B and could significantly upregulate nitric oxide production from IFN-gamma activated RAW 264.7 macrophages. Sera from mice (non-permissive host) infected with A. cantonensis detected rAcCystatin by Western blot, while the sera from infected rats (permissive host) could not. The results implied that AcCystatin might be an immunoregulator in A. cantonensis infection.

Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

“…Interestingly, cysteine protease activity was not detected under the experimental conditions tested. This type of protease is the most widely reported class of protease in parasitic nematodes and has been shown to hydrolyze gelatin in addition to other substrates (Yatsuda et al 2006, Kasny et al 2007, Liu et al 2010. Cysteine proteases are associated with several biological processes, such as tissue penetration, feeding and evasion of host immune response (Sajid & McKerrow 2002).…”

AcCystatin, an immunoregulatory molecule from Angiostrongylus cantonensis, ameliorates the asthmatic response in an aluminium hydroxide/ovalbumin-induced rat model of asthma

“…Recombinant AcCystatin was obtained and purified as previously described (Liu et al 2010), and subsequent endotoxin removal was performed using Detoxi-Gel Endotoxin Removing Columns (Thermo Scientific, USA) according to the manufacturer's instructions, resulting in 0.05 endotoxin units per milligram protein as indicated by the Limulus Amebocyte Lysate Chromogenic Endotoxin Quantitation Kit (Thermo Scientific, USA).…”

“…We previously described the in vitro immunoregulatory properties of the purified recombinant cystatin from Angiostrongylus cantonensis (AcCystatin; Liu et al 2010). In this study, we describe a novel mechanism of immunomodulation in which AcCystatin prevents murine allergic airway reactivity, most likely via the following mechanisms: the suppression of the expression of the cytokines IL-4, IL-5, IL-6 and IL-17A; the reduced expression of the chemokines eotaxin-1, eotaxin-3 and MCP-1; and the decreased production of OVA-specific IgE antibodies, concomitant with the enhancement of IL-10 release.…”

Cloning, expression, and characterization of salivary apyrase from Aedes albopictus

“…Numbers of significant proteins have been expressed with the prokaryotic expression system during the past few years (Verma et al 2008;Liu et al 2010;Yang et al 2010). Purification was done by Ni-NTA under denaturing condition.…”