Cloning And Expression And Functional Study Of Human Dentin Sialoprotein

Dentin sialoprotein (DSP), comprising 5%-8% of noncollagenous proteins, is found exclusively in the extracts of dentin extracellular matrix. This protein was so named, because of its overall resemblance to bone sialoprotein (BSP). DSP is rich in aspartic acid (Asp) , serine (Ser) , glutamie acid (Glu) and glycine (Gly) . Sedimentation equilibrium analysis revealed that DSP has a molercular weight of about 53,000 and approximately 366 amino acids. The 30% carbohydrate included about 10% sialic acid. The cDNA sequence analysis indicated that DSP has six potential N-glycosylation sites and thirteen potential phosphorylation sites. While the function of DSP is still unknown, DSP transcripts were found to be expressed first in preameloblasts, then in young odontoblasts, and finally substantially in mature odontoblasts actively synthesizing dentin. This transient expression profile may suggest that DSP is an important marker of the odontoblast and related cells and take part in dentinogenesis by serving as a signal molecule between epithelia-mesenchyme.1. Cloning, expression, preparation of potyclonal anti-sera and tissue distribution of hDSPIn the present study, total RNA was extracted from the tooth germs of alegally aborted human embryo by acid guanidinium thiocyanata-phenol-choroform method, the desired DNA product was obtained from the total RNA by RT-PCR with the primers including Oligo(dt) and two gene specific ones. The segment (about 600 bp) was inserted into a pBluescript vector and the interesting plasmid was transformed into E.Coli host strain XL 1-Blue. Reconstructed plasmid of pBS-hDSP was analyzed by restriction endonuclease mapping and DNA sequencing, then hDSP was cloned into expression vector pGEX-3X and expressed in E.coli BL21. The results showed that the restriction endonuclease map and sequence of hDSP encoding mature protein were consistent with those published and hDSP protein could be efficiently expressed in prokaryocytes.Polyclonal antibodies of hDSP were prepared by immunizing New Zealand rabbit with the purified hDSP obtained by prokaryotic expression. The tilter of the prepared antibody of hDSP was 1: 100 000 at least. The expression of hDSP was observed in human tooth germs and other tissues by Western blot. The results showed that hDSP was positive in tooth germs, but negative hi other human tissues including gingiva, liver, kidney and spleen. Its molecular weight was about 60, 000. The size was similar to that of DSP in native dentin.2. Construction and transfection of the expression vector of a full length gene of hDSPForeign protein expression by prokaryocytes possesses the features of high output, low cost and simple methods, but the expressed protein cannot be folded and glycosylated. Thus, such protein often doesn't have the native protein's functions. Since hDSP is a sort of glycoproteins, we selected mammalian cell as expression system. Recombind plasmid of pcDNA3-hDSP was constructed by inserting full length of hDSP into pcDNAS and transfected into COS-7 cell after identified by enzyme analysis and seqencing. Western Blot and immunochemistry showed that hDSP could be expressed in COS-7. This laid a solid foundation forfurther functional study.3. Functional study of h DSPThe expression of hDSP in human tooth germs was examined by immunochemistry. The result showed that hDSP first appeared weakly in enamel epithelial cells of bud and cap stages, then strongly in functional odontoblasts and dentin tubules of the bell stage. With the development of the tooth germs, resting odontoblasts, cells associated with a full thickness of dentin, appeared weakly reactive with anti-hDSP antibodies. Pre-odontoblasts and pre-dentin were always negative. Pre-ameloblasts and ameloblasts were transiently stained positive. This may suggest that hDSP protein is transported via odontoblastic processes through the pre-dentin and deposited directly at the mineralization front during dentin formation and hDSP is involved in cooperation with signaling molecules in pr...