A membrane-associated metalloprotease of Schistosoma japonicum structurally related to the FACE-1/Ste24p protease family.

The CaaX proteases are closely related in the post-translational modification of many membrane-bound or secreted proteins and play a key role in the activation or stabilization of these molecules belonging to the CAAX family. In this study, a full-length cDNA putatively encoding a FACE-1/Ste24p CaaX protease (type I) of the Schistosoma japonicum was isolated. The cDNA, named SjSte24p, composed of 1646 bp and encoded 473 amino acids with predicted Mr/pI as 54.77 kDa/8.04. SjSte24p is a monoexonic gene constantly expressed in the parasite from cercariae to adult stages. It contained the characteristic of CaaX protease topology, including seven trans-membrane domains and a metallo-protease segment with a zinc-binding motif (HEXXH). SjSte24p shared a considerable degree of sequence identity with the type I CaaX proteases. A phylogenetic analysis showed that this protein family is tightly conserved from fungi to vertebrates. The expressed recombinant SjSte24p protein showed a proteolytic activity, which was inhibited by EDTA. Its activity was increased at low doses of the Zn2+ (0.001-0.01 mM); but was reversibly down-regulated at high doses (>0.1 mM). The native SjSte24p appeared to function in insoluble from. The protein was mainly localized in the tegument on the surface of adult worms. These results indicated that the SjSte24p is a practical zinc-dependent metalloprotease, which belongs to the FACE-1/Ste24p protease family.

Isolation and characterization of a novel serine protease inhibitor, SjSPI, from Schistosoma japonicum.

Serine proteinase inhibitor (Serpin, SPI) is a vital superfamily of endogenous inhibitors that monitor proteolytic events active in a number of biological functions. In this study, we isolated a full length gene encoding a novel serine protease inhibitor of Schistosoma japonicum (SjSPI) and characterized its molecular properties. Our result showed that SjSPI contained an open reading frame of 1,218 bp, which encoded 405 amino acid residues. Chromosomal structure analysis showed that SjSPI gene was comprised of six exons separated by five introns. It had essential structural motifs which were well conserved among the Serpin superfamily and showed 17-33% sequence identities with Serpins from other helminthic parasites. Trematode Serpin diverged separately into two different subclades and that the SjSPI clustered Subclade I. Exon-intron structures of trematode Serpins were highly conserved, closely with cestode Serpins. No signal peptide but a strongly transmembrane domain was predicted to exist in SjSPI, suggesting that the protein might be a soluble membrane-associated protein. Homology modeling predicted in silico confirmed that the SjSPI structure also belonged to the Serpin superfamily, containing nine α-helices and a reactive central loop. The bacterially expressed recombinant GST-SjSPI protein effectively inhibited the activities of chymotrypsin, trypsin and thrombin. Expression of SjSPI was detected throughout various developmental stages of the parasite in host and reached its maximal levels at the adult and egg stages, which suggests that SjSPI may be possibly involved in maintaining the physiology of eggs by regulating endogenous serine proteases.

Functionally Expression of Metalloproteinase in Taenia solium Metacestode and Its Evaluation for Serodiagnosis of Cysticercosis.

BACKGROUND: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite's evasion from the host's immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis. METHODS: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis. RESULTS: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity. CONCLUSIONS: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis.

Molecular cloning and characterization of a phospholipid hydroperoxide glutathione peroxidase gene from a blood fluke Schistosoma japonicum.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a major antioxidant enzyme and plays critical roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. A full-length cDNA sequence corresponding to GPx gene from Schistosoma japonicum (designated SjGPx) was isolated and characterized. SjGPx contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in its 3'-untranslated region. Protein encoded by SjGPx demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic domains and absence of the subunit interaction domains. Phylogenetic analysis revealed that the SjGPx was highly related to the other PHGPx-related members, and clustered into the trematode subclade II. Semi-quantitative reverse transcription PCR and western blotting showed that the SjGPx was mainly expressed in the female adults and eggs. RNA interference was employed to investigate the effects of knockdown of SjGPx. SjGPx expression level was significantly reduced on the 5th day post-RNAi. We observed a 53.86% reduction in total GPx activity and the eggs severely deformed. Oxidative stimulation of viable worms with H2O2 or paraquat resulted in 1.6- to 2.1-fold induction of the GPx activity. Our results revealed that the SjGPx protein is selenium-dependent PHGPx, which might actively participate in the detoxification of oxidative damage during egg production.