The Function Study Of DMP1in Minerazation

Dentin matrix protein1(DMP1) was mapped to human chromosome4q21,which encodes an acidic phosphorylated noncollagen protein. DMP1contained anlarge number of acidic domains, taked along negative charge. The acidic nature ofDMP1indicated potentially high calcium ion-binding capacity. In vivo, DMP1promoted the formation of hydroxyapatite and regulated the osteoblast differentiation.In vitro, it had been proved to play an important role in mineralized tissue formation.DMP1existed bone tissues and cells within four chief modality of intact DMP1, a57kD N-terminal, a37kD C-terminal fragment and DMP1-PG. They had differentdistribution and function, which possessed important significance to osteogenesis.Now the main functional fragments of DMP1affected osteoblast differentiationmechanism remains controversial at home and abroad.Therefore, we amplificated37kD C-terminal fragment and the full-length DMP1,analyzed the role they played in the biomineralization process.Objective: To analyze the expression and localization of recombinant proteins ofDMP1different functional fragment in cells, and detect how DMP1gene containingvarious bases at single nucleotide polymorphism (SNP) site impact on cellmineralized function.Methods: Various genotypes of rs10019009were obtained from the DMP1genewhich were reformed by site-directed mutagenesis. The target gene fragment of The37kDa N-terminal DMP1gene and the full-length DMP1gene containing variousbases at SNP site were cloned into eukaryotic expression vector pcDNA3.1by PCRreaction technique and double enzymes site-directed cloning technique. HEK293cells were transiently with the constructed recombinant plasmid pcDNA3.1-DMP1-EGFPin mediation of liposome and observed for expression and subcellular location offusion ptotein DMP1-EGFP by fluorescent microscopy. We used alkaline phosphatase(ALP) activity detection method to detect ALP activity in SAOS-2cells before andafter, and observed for the mineralized nodule formation in SAOS-2cells by alizarinred staining to analyze the function of DMP1in mineralization.Results: DNA sequencing proved that the base sequence of DMP1gene aftersite-directed mutagenes was completely consistent with that designed. PCR and DNAsequencing results showed that the recombinant expression plasmids were constructedsuccessfully. Fusion proteins DMP1-EGFP were expressed in the cytoplasm ofHEK293cells after transfected. ALP activity detection results showed that the virousfunctional fragment of DMP1could improve the ALP activity of the SAOS-2cells,and the recombinant plasmid of the full-length DMP1had higher ALP activity than therecombinant plasmid of the N-terminal DMP1, the recombinant plasmids of DMP1containing mutant T base at rs10019009SNP site had higher ALP activity than therecombinant plasmids of DMP1containing the wild-type A base at rs10019009SNPsite. After ascorbic acid and β-glycerol phosphate mineralization induced, we foundthat the recombinant plasmid of the full-length DMP1better promote the formation ofmineralized nodules than the recombinant plasmid of the N-terminal DMP1, therecombinant plasmids of DMP1containing mutant T base at rs10019009SNP sitecould be enhanced mineralized nodule formation than the recombinant plasmids ofDMP1containing the wild-type A base at rs10019009SNP site by alizarin red stainingexperiments.Conclusion: The recombinant expression plasmids for expression of the37kDaN-terminal fragment DMP1and the full-length DMP1containing various bases atrs10019009-SNP site were constructed successfully and effectively expressed in thecytoplasm of HEK293cells, DMP1containing mutant T base at rs10019009SNP site better promote the biomineralization than DMP1containing the wild-type A base atrs10019009SNP site, as well as the full-length DMP1better promote thebiomineralization than the N-terminal DMP1, which laid the foundation of furtherstudy on the function of DMP1in biomineralization and how the rs10019009SNP siteparticipating into the mechanism of ankylosing spondylitis (AS) pathogenesy, and alsoprovided an experimental basis for its future as a target to treat ankylosing spondylitis.