Cloning And Expression Of The Catalytic Domain Of Megakaryocyte Protein Tyrosine Phosphatase And Studying On Their Functions

Protein tyrosine phosphorylation is regulated by protein tyrosine kinase and protein tyrosine phosphatase activities.Counteracting interplays between protein kinases and phosphatases control cell growth,proliferation,differentiation and cytoskeletal organization and have major pathological implications.PTPMEG1(also known as PTPMEG or PTPN4) is a cytosolic PTP.It contains a N-terminal FERM domain,a central PDZ domain,and a C-terminal catalytic domain.The protein is also subject to proteolysis by Trypsin and Calpain,which results in activation of the enzyme both in vitro and in vivo.PTPMEG1 interacts with glutamate receptor delta 2 and epsilon subunits is believed to play a role in signalling downstream of the glutamate receptors through tyrosine dephosphorylation.In both Drosophila and murine,there are homologues of PTPMEG1.Some studies have been carried out to demonstrate the expression and some of its potential functions.Drosophila PTPMEG1 and its vertebrate homologs PTPN3 and PTPN4 are expressed in neuronal circuit formation in the Drosophila central brain,regulating both the establishment and the stabilization of axonal projection patterns.PTPMEG1 is highly enriched in the testis in mouse and human and has been termed as a testis-enriched phosphatase'(TEP).It is specifically expressed in spermatocytes and spermatids within seminiferous tubules,suggesting an important role in spermatogenesis.Over-expression of PTPMEG1 in COS-7 cells can inhibit cell proliferation, reduce the saturation density,and block the ability of these cells to grow without adhering to a solid matrix.However,recent study suggested that PTPMEG1 can dephosphorylate the TCRζchain,a component of the TCR complex,but it is dispensable for TCR signal transduction.PTPMEG2(also known as PTPN9) is a widely distributed intracellular phosphatase originally cloned from human MEG-01 megakarocyte and umbilical vein endothelial cell cDNA libraries.The enzyme contains two domains.The catalytic domain located at the C-terminus displays 30%-40%sequence identity to other known PTP.The N-terminal non-catalytic segment has about 250 amino acids and shares 24%-29%sequence identity with cellular retinaldehydebinding protein (CRALBP),a-tocopherol transfer protein,and yeast Sec14p.CRALBP is a watersoluble protein found in the retina and pineal gland.It acts as a carrier protein for 11-cisretinaldehyde or 11-cis-retinol and modulates the interactions of these retinoids with visual cycle enzymes,a-Tocopherol transfer protein(A-TTP) of liver specifically sorts out a-tocopherol from all incoming tocopherols for incorporation into plasma lipoproteins.Sec14p acts as a phosphatidylinositol transfer protein by catalyzing the transfer of phosphatidylinositol and phosphatidylcholine between membrane bilayers.It is required for protein transport through the Golgi complex in Saccharomyces cerevisiae.With significant sequence homology to these lipid-binding proteins,PTPMEG2 may also bind certain lipid molecules that may regulate its activity and/or localization.Despite all previous studies,much is to be understood about PTPMEG1 and PTPMEG2.There is an urgent need for a systematic exploration of their enzymatic properties and production of high sensitive and specific antiboides.The procedures are as follows:1,A cDNA fragment encoding theΔPTPMEG1 protein(amino acid residues 643-926) was amplified by PCR and then cloned into the pT7 vector.Both soluble and insoluble recombinantΔPTPMEG1 proteins were observed after induction by IPTG.The solubleΔPTPMEG1 was purified by using two chromatographic steps,and the purified enzyme was characterized.Q-Sepharose Fast Flow and Sephacryl S-100, were used to isolate and purifyΔPTPMEG1 protein.The purity of target protein was 90%and the Specific activity was 2437U/mg.The results of enzymatic characteristics determination indicated that when p-NPP was added as substrate,optimal temperature was 42℃,the optimal pH was 5.0,and ion concentration was 0.The kinetics analysis of its catalytic reaction showed that the Km was 10.11 mM.ΔPTPMEG1 exhibited typical enzymatic characteristics of classic PTP and classical Michaelis-Menten kinetics.The insoluble fraction ofΔPTPMEG1,which was mainly distributed in the inclusion body of E.coli cells extracts,was purified by preparative electrophoresis gels for preparation of polyclonal antibodies.A rabbit was immunized withΔPTPMEG1 purified by preparative electrophoresis to generate anti-APTPMEG1 antibodies.Anti-serum was collected on 28^th day after initial injection and purified via affinity chromatography.The purified polyconal antibodies displayed a satisfactory titter and sensitivity.In conclusion,this study provides useful materials for the further study of the biological function of PTPMEG1.2,we employed the pT7 vector to express the catalytic domain of PTPMEG2 as a non-fusion protein in E.coli cells.First,a cDNA fragment encoding the catalytic domain of PTPMEG2(amino acid residues 283-593) was amplified by PCR and cloned into the pT7-7 vector.The construct was then used to transform DE3- pLysS E. coli cells(Novegen) to give rise a recombinant DMEG2 protein of 315 amino acids in which the first 4 residues(MARl) were derived from the vector.Expression of the recombinant protein was induced by 50 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG) overnight at 28℃.E.coli cells were broken up by sonication in Buffer A plus protease inhibitors as above.Both soluble and insoluble recombinantΔPTPMEG2 proteins were observed after induction by IPTG.The solubleΔPTPMEG2 was purified by using two chromatographic steps,and the purified enzyme was characterized.Q-Sepharose Fast Flow and SP-Sephadex,were used to isolate and purifyΔPTPMEG2 protein.The purity of target protein was 94.8%and the Specific activity was 54606U/mg.The results of enzymatic characteristics determination indicated that when p-NPP was added as substrate,optimal temperature was 26℃,the optimal pH was 4.5,and ion concentration was 0.The kinetics analysis of its catalytic reaction showed that the Km was 8.965 mM.The insoluble fraction ofΔPTPMEG2,which was mainly distributed in the inclusion body of E.coli cells extracts,was purified by preparative electrophoresis gels for preparation of polyclonal antibodies.A rabbit was immunized withΔPTPMEG2 purified by preparative electrophoresis to generate anti-ΔPTPMEG2 antibodies.3,Ten paraffin-embedded sections of primary rectum tumors and their corresponding lymph node metastases were taken for PTPMEG1 immunohistochemistry analysis.The expression of PTPMEG1 was found to be significantly elevated in the primary sites as well as in the metastases to a less extent. Therefore,the elevated expression of PTPMEG1 is associated with the development and metastasis of rectum cancer,while no significant change of PTPMEG1 expression was observed in colon cancer samples.4,Our osteoporosis rat model was established by oral gavage of tretinoin and this model allowed us to evaluate the relevance of PTPMEG2 expression to osteoporosis through Western Blot analysis.The model was confirmed to have significantly higher ALP and ACP levels as well as higher incidence of developing osteogenesis imperfecta when compared to control group.The PTPMEG2 was found to be expressed in multiple organs of our experimental animals at various levels.However, the findings that PTPMEG2 expression was elevated in kidney but reduced in muscle of our osteoporosis model implicated that PTPMEG2 might play an important role in regulating the functions of kidney and muscle.It was also observed that our rat model had significantly increased expression of PTPMEG2 in the bone marrow cells but not in red blood cells or BMSCs.Since the change of PTPMEG2 expression was not likely to occur in hematopoietic cells or mature red blood cells because the osteoporosis modeling would not cause any alteration in hematopoiesis,and there was no change detected in the undifferentiated BMSCs,it was highly possible that the expression of PTPMEG2 was elevated in maturely differentiated bone cells.Taken together,our findings that osteoporosis resulted in PTPMEG2 overexpression which occurred primarily in maturely differentiated bone cells suggested a critical role of PTPMEG2 in regulating the development of osteoporosis.