Identification and characterization of a serine protease inhibitor of Paragonimus westermani.
ABSTRACT Paragonimus westermani is a trematode parasite that causes pulmonary and/or extrapulmonary granulomatous disease in humans. In this study, we identified a full-length gene encoding a novel serine protease inhibitor of P. westermani (PwSERPIN) and characterized the biochemical properties of the recombinant protein. PwSERPIN had an open reading frame of 1,164 bp, which encoded 387 amino acid residues. Sequence analysis of the primary structure of PwSERPIN revealed that it had the essential structural motifs which were well conserved among the serine protease inhibitor (serpin) superfamily and had shown 16.5-29.6% sequence identities with previously reported serpins from other helminthic parasites. No signal peptide or N-glycosylation site was found in the sequence. Genomic DNA structure analysis showed that PwSERPIN comprised six exons separated by five introns. The bacterially expressed recombinant PwSERPIN effectively inhibited the activities of trypsin, thrombin, and chymotrypsin in a dose-dependent manner, but showed lower inhibitory capacity on cathepsin G and elastases. Expression of PwSERPIN was detected throughout various developmental stages of the parasite, from metacercariae to adult worms, and the transcription level gradually increased with the maturation of the parasite. PwSERPIN was identified in the soluble extract of the parasite, but not in the excretory and secretory products (ESP) and in the insoluble extract of the parasite. These results collectively suggest that the PwSERPIN is an intracellular serpin of P. westermani and that might play primary roles in regulating the activities of intracellular serine proteases of the parasite.
Article: The biochemical and immunological characterization of two serpins from Clonorchis sinensis.[show abstract] [hide abstract]
ABSTRACT: Serpins (serine proteinase inhibitors) are evidenced to regulate numerous biological processes such as immunoregulation in parasitic helminths. The functions of serpins from Clonorchis sinensis remain unclear to date. In this study, two serpin genes, respectively denominated as CsproSERPIN and CsSERPIN2, had been selected from metacercaria cDNA library of C. sinensis. The biochemical activities of both recombinant proteins (rCsproSERPIN and rCsSERPIN2) were analyzed by assays of inhibition on some serine or cysteine proteases, the results showed that rCsproSERPIN significantly inhibited trypsin, chymotrypsin and thrombin, while rCsSERPIN2 inhibited only chymotrypsin. Moreover, cytokine and antibody measurements indicated that rats subcutaneously immunized with rCsproSERPIN and rCsSERPIN2 respectively developed a strong IFN-γ production and IgG2a levers of sera were higher than IgG1. Besides, immunoblot assays revealed that the rCsproSERPIN and rCsSERPIN2 could be recognized by the sera of rats infected with C. sinensis and the sera of rabbits immunized by excretory/secretory products. Furthermore, immunofluorescence assays illuminated the two were similarly localized in the reproductive organs such as vitelline glands, testis and eggs in adult stage. In short, all the results collectively indicated that CsproSERPIN and CsSERPIN2 might play important role in the parasite development by preventing the parasite from digestion by exogenous serine proteases, as well as CsproSERPIN and CsSERPIN2 probably involved in immunoregulation of host by inducing Th1-biased type cytokines in rats.Molecular Biology Reports 12/2012; · 2.93 Impact Factor