Cloning And Expression Of Human Matrix Extracellular Phosphoglycoprotein And A Primary Research Of Its Biological Effects

Recently, a new bone matrix protein cDNA has been cloned from human, rat, and mouse by independent groups . The human clone, termed MEPE(matrix extracellular phosphoglycoprotein), was isolated from a human oncogenic hypophosphatemic osteomalacia tumor(OHO) cDNA library with polyclonal antibodies that neutralized OHO tumor secreted phosphate uptake-inhibiting factor . MEPE , BSP (Bone sialoprotein), OPN (osteopontin, but sometimes known as SPP1 and Eta-1), DMP I(dentin matrix protein I), DSPP(dentin sialophosphoprotein) are the products of five genes clustered along human chromosome 4q21 between EST markers D4S2785(WI-6336) and D4S2844. The majority of each protein is encoded by the last one or two exons and contains the integrin-binding RGD tripeptide. Another point of similarity of all these genes is that all introns always interrupt between codons (type 0), thus leaving open the possibility of splicing together any two exons without causing frame shifts. We have named this family of proteins the SIBLING family for Small Integrin-Binding LIgand, N-linked Glycoprotein based not on current theories of their functions (which are poorly understood) but based on the simple biochemical and genetic features shared by all members. The human MEPE has 1989 bp cDNA clone and encodes a predicted 525-amino-acid protein rich in Asp, Ser, and Glu residues (26%) containing a 17-amino-acid signal peptide. MEPE contains two N-glycosylation motifs (NNSTandNNSR),a glycosaminoglycan attachment site (SGDG), an RGD cell attachment motif, several predicted phosphorylation motifs,and N-myristoylation sites.In dental tissue, MEPE is expressed in odontoblasts during odontogenesis, Liu has found that Dentonin(a 23-amino-acid peptide derived from MEPE) can promote DPSC proliferation, taking a potential role in pulp repair. That study suggestted that Dentonin affects primarily the innitial cascade of events leading to pulp healing.In this study, we have cloned MEPE cDNA by RT-PCR, expressed MEPE in E.coli and COS-7 cells, purified the recombinant protein, prepared anti-MEPE antibodies. Then we proceeded western blot and immunohistochemistry. Finally, a Steady-State Agarose Gels HA Growth System will be used to study the effects of MEPE to HA.The matrix extracellular phosphoglycoprotein (MEPE) gene is highly expressed in tumors that cause onco-genic hypophosphatemic osteomalacia (OHO). MEPE is also known as one of the bone-tooth matrix proteins and is associated with bone and teeth mineralization. We developed a rabbit polyclonal antibody directed against recombinant human MEPE after cloning its cDNA from the cDNA library of a human brain cDNA library the predicted secreted MEPE protein has a calculated molecular mass of 56 kDa. Like human MEPE, Fourteen of the serine residues in Mepe are potential phosphorylation sites for protein kinase C, casein kinase II, and cAMP-dependent protein kinase. As reported for human MEPE, a specific feature of the MEPE protein is the occurrence in the C-terminus of a serine-rich sequence, DDSSESSDSGSSSES (residues 417–430), that displays homology to repeat motifs found in dentin phosphoprotein, DPP/DSPP (SDSSDSSDSSSSSDSS), DMP1 (SSRRRDDSSESSDSGSSSESDG), osteopontin (DDSHQSDESHHSDESD) and the parent protein, dentin sialophosphoprotein,.. Using this anti-body, we analyzed the distribution of MEPE in dog dental germ tissure by immunohistochemistry. In this specimens, MEPE was predominantly expressed by odontoblasts cell and predentin not by dental pulp cells. Furthermore we use a steady-state agarose gel system, MEPE were incorporated at 5-25μg/ml into steady-state agarose gels and incubated for 5 days, MEPE caused significantly higher levels of calcium phosphate accumulation than control gels didthe results reported here suggest that MEPE can induce HA and we propose that this protein has a potential effect on dental rehabilitation. .