Analysis On The Tegument Surface Proteins Of The S. Japonicum At Different Stages And The Preliminary Characterization Of S. Japonicum Phosphodiesterase-5

In China, schistosomiasis, caused by infection with Schistosoma japonicum, affects 365,700 individuals, with more than 67.6 million at risk of infection in 2009, as reported by the China Health Statistics 2010. The entire schistosome surface is covered by a syncytial layer called the tegument. The outer plasma membrane of the tegument is trilaminate in the cercaria, but has a hepalaminate appearance in the adult worm interpreted as a normal plasma membrane overlain by a secreted bilayer termed as membranocalyx. During the migration and growth of the schistosome in the final host, the tegument surface membrane maintains a dynamic turnover and its structure differentiates at different developmental stages of the parasite, along with constant changes in its biological function. However, all of these changes are attributed to the alteration of tegumental surface proteins. In this paper, the study of tegument surface proteins at different stages of Schistosoma japonicum was identified by proteomic techniques, which is an effective method for the discovery of vaccines and drug targets and the speculation of immune evasion.1.Analysis on the tegument surface proteins of the S. japonicum at different stagesParasites of S. japonicum at 8, 11, 14, 17, 20, 23, 26, 29, 32 and 42 days, as well as 42-day-old female and male worms were obtained and then labeled with Sulfo-NHS-SS-Biotin. Tegument surface proteins were firstly isolated by the system of Streptavidin-Biotin, and then separated by SDS-PAGE and reverse phase HPLC (RP- HPLC) followed by tandem MS analysis. Totally, 475, 347, 263, 165, 250, 337, 298, 140, 114, 85, 202 and 331 proteins were identified on the surface of tegument at 8, 11, 14, 17, 20, 23, 26, 29, 32 and 42 days, as well as 42-day-old female and male worms of S. japonicum, including some improtant tegument surface proteins for schistosome, such as 23 KD, 29 KD, TSP, Annexin.β-TGF receptor, 14-3-3, Na + / K + ion channel proteins are signal transduction associated molecules. Glucose transporter protein (SGTP), enolase, phosphodiesterase-5(NPP-5), 3 - phosphoglycerate dehydrogenase(GAPDH) are related to nutritional intake and metabolism. Actin and paramyosin are structural proteins. C3 complement molecules, IgG and IgM heavy chain antibody are homologous with the host. Further analysis and identification of these proteins are in progress.Tegument surface proteins at different stages of S. japonicum were firstly isolated and analyzed, which were conducive to find out some important proteins associated with nutritional intake, signal transduction and immune evasion, speculate the mechanisms of growth and development and discover vaccines and drug targets.2.Cloning, expression and characterization of S. japonicum tegument surface protein phosphodiesterase-5The tegument proteins of schistosome are regarded as potential vaccines candidates and drug targets to control schistosomiasis. Nucleotide pyrophosphatase/phosphodiesterase-5 (NPP-5), which belongs to a multigene family of nucleotide pyrophosphatase/phosphodiesterases (NPPs), is important in the hydrolysis of pyrophosphate or phosphodiester bonds in nucleotides and their derivatives. In the present study, SjNPP-5, identified as one of the tegument surface proteins of S. japonicum in our previous proteomic studies, was cloned on a fragment of 1371 bp and expressed as a recombinant protein of 69 kDa. Real-time RT-PCR analysis showed that SjNPP-5 was up-regulated at 21–42 days, and the expression level in 42-day-old male worms was almost nine times higher than that in females. Western blot analysis revealed that rSjNPP-5 had good antigenicity. Immunofluorescence analysis found that SjNPP-5 was a membrane-associated antigen mainly distributed on the surface of worms of S. japonicum. BALB/c mice vaccinated with rSjNPP-5 three times showed a 29.90% worm reduction (P<0.05) and a 26.21% egg count reduction (P>0.05). Immunization with rSjNPP-5 induced a mixed Th1/Th2 response in which Th1 was dominant. The response was characterized by a reduced IgG1/IgG2a ratio and elevated production of cytokines IFN-γand IL-4. This study suggested that SjNPP-5 may be important in schistosome development, and further investigations are required to fully understand the function of this molecule.In summary, given that tegument surface proteins play an important role in the growth and development of schistosome, those at different stages of S. japonicum were firstly isolated and analyzed, and some important proteins were discovered. SjNPP-5, a tegument surface protein, was firstly cloned and expressed and its efficacy of immune protection was assessed by immunization with mice. This study is conducive to find out some important proteins associated with nutritional intake, signal transduction and immune evasion, speculate the mechanism of growth and development and discover vaccines and drug targets.