Localization And Insight Into The Function Of A Human Novel Gene TSBP Of Sperm Binding Protein

The cDNA sequence of novel gene TSBP(Testis Sperm Binding Protein) related to proteins of bovine seminal plasma ( BSP) in human testis was found and cloned when we studied the important roles of BSP in fertilization and the development of zygote, named TSBP and registen in GenBank( AF279147). Its open reading frame( ORF) encoded a protein of 223 amino acids. There were 7 exons 6 introns and 4 fibronectin type II -medules in the amino acid sequence. It is estimated that TSBP is a kind of binding protein whose function and structure are similar to that of BSP. We have proved that the gene is widely expressed in these tissues such as testis kidney intestines stomachy lung muscle liver heart and spleen. Large quantities of exprssed protein have been obtained by pro-karyotic system and we analysed its sequence. BSP protein is the cardinal protein in bovine sperm, which can combine with choline or phosphycholine and then adhere to the surface of sperm to promote the sperm capacitation induced by hepa-rin and high density lipoproteins ( HDL). Moreover, BSP protein can combine with heterogeneous collagen, fibrinogen, heparin, calmoduline IGF-II apoA- I and inhibit the migration of cholesterol on the sperm membrane. All these suggest that BSP proteins play important roles in the lipid metabolism of sperm membrane and in the modulation of sperm capacitation. In former study, we purified an inhibitor of protein kinase C ( PKC ) from bovine seminal plasma, while we found that the sequence of the inhibitor was the same as that of BSP,and BSP inhibited the activity of PKC and TPK( protein tyrosine kinase) from some futher studies of the relationship of BSP and PKC. At present homogeneous proteins of BSP proteins have been found in the* sperm plasma of horse and pig and they have the binding-properties similar to BSP protein. This indicates that BSP pro-tein and its homogeneous protein exist in many kinds of mammals with the same physical feature to some degree. But in the human congenital system, there were no reports about the related protein with the same functions as BSP. It is crucialto study the localization and function of novel human sperm binding protein TSBP related-BSP.Materials and Methods1. Materials and agentsHuman blood was excised from chromosome analysised man in open laboratory of reproductive medicine of Iiaoning Family-planning Institude. We purchased expression vector pGEX-5X-l PcDNA3 and pGEM-T Easy from Pro-mega, RFP vector pDsRedl-Nl from CLONTECH, E. coli JM109 BL-21 competent cells PCR amplification kh\BamH I Xho I and plasmid DNA purification kit from Takara, Dig-Nick Translation Mix and Anti-digoxigenin-fluorescein from Roche, PI G418 products of PKC and TPK activity detecting from Sigma, DNA ligase kit from Promega; Iipofectamine?2000 from Invitrogen; HEK293 cells purchased from Department of Cell Biology in Xiehe Medical University.2. Fluorescent in situ hybridizationUsing the full-length TSBP cDNA cloned into pGEX-5X-l as template, cD-NA fragment was amplified by PCR using TSBPspecial primers. We labled the probe by digoxigenin by means of Nick-translation, and then made it denatured. Human metaphase chromosome were separated and denatured. After hybridizing probe with chromosome , the hybridization singals were detected by FITC-sheep-anti-digoxigenin antibody after PI restaining in fluorescent microscope.3. Subcellular localizationUsing the full-length TSBP cDNA cloned into pGEX-5X-l as template,the PCR reaction introduced the enzyme-incision site of Xho I and BamH I into 5' end and 3'end,respectively, and cloned the PCR products into pGEM-T Easy. The PCR products and RFP expression vector pDsRedl -N1 were incised by Xho I and BamH I. Digested PCR products and vector were separated,purified and collected to complete the ligation reaction,after which E. coli JM109 was transformed. The positive recombinated plasmid pDsRedl-Nl/TSBP was purified and had the sequence analyzed. The stable expression cells line was constructed by HEK293 cells transfected by pDsRedl-N...