The Study On The Target Site Of Porcine Myostatin

Myostatin (also called growth and differentiation factor-8, GDF-8) was recentlyidentified as a member of the TGF-β superfamily that acts as a potent and specificnegative regulator of skeletal muscle mass. Homozygous disruption of the myostatingene in mice results in a significant increase in skeletal muscle mass and a reduction inbody fat. Naturally occurring myostatin mutations in cattle display a similardouble-muscled phenotype, confirming a role for myostatin in muscle development andregulation. Blocking myostatin activity may have the development of novel muscleenhancing agents for both the therapy for the human diseases such as HIV infects andagricultural applications.However, little is known of the mechanism of action on myostatin, such as thestructure of the receptor, the distrubution of the receptor and the signal transductionwhich need a further processed and study.In order to probe the tissues distribution of myostatin and screen the receptor ofmyostatin, we devised four trial: Prokaryotic expression and purification of porcinemyostatin C-terminal polypeptide;the preparation of myostatin C-terminal immuneserum and tissue distribution of porcine myostatin;the analysis of the biological activiesfor the recombinant myostatin;the screening of the myostain receptor.According to sequence of swine myostatin, a pair of primers with two sites ofrestriction endonucleases(BamH I and EcoR I) were synthesized to amplify thefunctional domain coding gene of swine myostatin. The PCR products were clonedinto pGEM-T vector, and then was tranformed into the competent cell of E.coli DH5.The recombinant plasmid pGEM-T-MSTN were screened by α-complementation,identified by PCR and restriction endonucleases digestion. The recombinant plasmidpGEM-T-MSTN and pGEX-4T-1 were digested by restriction endonucleases BamH Iand EcoR I respectively. The Prokaryotic expression plasmid MSTN-pGEX-4T-1 wasconstructed by inserting the coding sequence of myostatin from pGEM-T-MSTN intothe multi cloning sites of Prokaryotic expression vector pGEX-4T-1. The recombinantplasmid MSTN-pGEX-4T-1 was confirmed as mentioned above and transformed intothe competent cell of E.coli BL21 (DE3). The fusion protein was expressed afterinduced by IPTG and a 38kDa special band was detected by SDS-PAGE electrophoresis.Soluble fusion protein was at a level approximately 18% of total protein via theoptimization condition. Lysised supernatants were further purified by GlutathioneSepharose 4B Affinity Chromatography.Purified fusion protein expressed in E.coli was injected in the adultrabbits three times in two mouths. High titer (1:10240) polyclonal antiserumgainst myostatin was isolated from the immunized rabbit by determination ofELISA. At the same time, tissue distribution of porcine myostatin wasinvestigated through immunohistochemistry and Western-blotting. Theresults of western-blot showed: lots of distribution on lung, liver, skeletalmuscle and adipocyte, and the most of distributin is heart;Inferiordistribution on kindey and pancreas. The results of immunohistochemistryshowed: diversity extent of immuno positive stain were on lung, liver,skeletal muscle, spleen, heart muscle, cerebellum, uterus-cells, colon tissue.The distribution is main in the tissues existing the muscle cells.The functional role of the recombinant myostatin in inhibitionmyogenesis in a rart embryonic myoblasts L6 system was determined. Inaddition, myostatin blocked the multinucleus myotube formation in L6 cells.Taken together, the results show that the recombinant myostatin inhibits ratmuscle cells proliferation and differentiation.To gain insight into the mechanism of myostatin regulation, we set out to screen thebinding partners of the membrane proteins for myostatin. To accomplish this, we haveisolated membrane glycoproteins by sucrose density gradient centrifugation and lectinCon A affinity chromatography from the skeletal muscle and heart muscle tissues. Themyostatin-bound membrane proteins were isolated by affinity purification with an GSTagarose conjugated myostatin. These samples were concentrated, run on aone-dimensional SDS-PAGE gel. To identify any additional myostatin binding proteins,the area of the coomassie blue-stained gel corresponding to molecular masses between30 kD and 90 kD was subjected to in-gel trypsin digestion, and the resulting peptideswere identified by LC-MS/MS and database searching. These data were analyzed toprovide two unknown proteins that were found in the myostatin-bonund in heart heartmuscle tissues, respectively. The result indicats that the receptor of myostatin is aunknown protein. The structure of the receptor is further researched.