Abstract
BackgroundProteins belonging to the serine protease inhibitor (serpin) superfamily play essential physiological roles in many organisms. In pathogens, serpins are thought to have evolved specifically to limit host immune responses by interfering with the host immune-stimulatory signals. Serpins are less well characterised in parasitic helminths, although some are thought to be involved in mechanisms associated with host immune modulation. In this study, we cloned and partially characterised a secretory serpin from Schistosoma japonicum termed SjB6, these findings provide the basis for possible functional roles.MethodsSjB6 gene was identified through database mining of our previously published microarray data, cloned and detailed sequence and structural analysis and comparative modelling carried out using various bioinformatics and proteomics tools. Gene transcriptional profiling was determined by real-time PCR and the expression of native protein determined by immunoblotting. An immunological profile of the recombinant protein produced in insect cells was determined by ELISA.ResultsSjB6 contains an open reading frame of 1160 base pairs that encodes a protein of 387 amino acid residues. Detailed sequence analysis, comparative modelling and structural-based alignment revealed that SjB6 contains the essential structural motifs and consensus secondary structures typical of inhibitory serpins. The presence of an N-terminal signal sequence indicated that SjB6 is a secretory protein. Real-time data indicated that SjB6 is expressed exclusively in the intra-mammalian stage of the parasite life cycle with its highest expression levels in the egg stage (p < 0.0001). The native protein is approximately 60 kDa in size and recombinant SjB6 (rSjB6) was recognised strongly by sera from rats experimentally infected with S. japonicum.ConclusionsThe significantly high expression of SjB6 in schistosome eggs, when compared to other life cycle stages, suggests a possible association with disease pathology, while the strong reactivity of sera from experimentally infected rats against rSjB6 suggests that native SjB6 is released into host tissue and induces an immune response. This study presents a comprehensive demonstration of sequence and structural-based analysis of a secretory serpin from a trematode and suggests SjB6 may be associated with important functional roles in S. japonicum, particularly in parasite modulation of the host microenvironment.
Highlights
Proteins belonging to the serine protease inhibitor superfamily play essential physiological roles in many organisms
Identification of S. japonicum serpin Source sequence encoding S. japonicum SjB6 [GenBank: CAX69453.1] was found using BLAST (Basic Local Alignment and Search Tool) [17] with the BLASTP and BLASTN algorithms against S. japonicum Gene Index (SjGI) available at DFCI and a
Analysis of the deduced SjB6 peptide indicated the presence of three conserved motifs, namely a serpin motif, a serpin signature and a reactive centre loop (RCL) (Table 1), located near the C-terminal with the cleavage site of the RCL at position 346C–S347
Summary
Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential physiological roles in many organisms. Serpins participate in many important physiological processes such as signalling cascades [5], blood coagulation [6,7], fibrinolysis [8], inflammation and activation of the complement system [9,10] These inhibitors play key roles in host immune modulation by pathogens, and the suggestion that pathogen serpins may have evolved for the singular purpose of limiting host immune activation by interfering with host immunomodulatory signals [11,12]. In parasitic helminths, these inhibitors appear to perform similar roles [13]. There are limited reports to date presenting the functional characterisation of serpins in the Asiatic schistosomes or their possible role in host-parasite interactions
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