The Effect Of Glycosylation Of Bone Sialoprotein On Osteogenesis

Objective:To investigate the effect of glycosylation of bone sialoproein (BSP) on osteogenesis and its possible mechanisms.Methods:1. Establishment of a cell culture model of osteoblast differentiation using MC3T3-E1Subclone14pre-osteoblast cell line. Cellular morphology was observed under an inverted microscope, the characteristic biological markers of osteoblastic cells were examined at the mRNA level and at the protein level by RT-PCR and Western blot, respectively. Alkaline phosphatase (ALP) and von Kossa stains were performed to detect osteoblast differentiation and bone matrix mineralization.2. Cell O-Linked oligosaccharide was detected with fluorescein isothiocyanate labeled structure-specific lectin VVL after adding rhBSP and rhBSP digested by O-glycosidase to the cultured osteoblasts, and then confirmed by flow cytometry. Meanwhile, mRNA over-expression profiles of ppGalNAcT1to ppGalNAcT7in the process of osteogenesis induced in vitro were detected by RT-PCR. ppGalNAcTs siRNA vectors were amplified and screened using RNA interference technology and transfected into cultured MC3T3-E1Subclone14osteoblastic cells to observe the effect of BSP O-glycosylation on osteogenesis.3. Cell surface a2,3sialic acid was detected with fluorescein isothiocyanate labeled structure-specific lectin MAL-Ⅱ after adding different concentrations of α2,3neuraminidase to the cultured osteoblasts, and then confirmed by flow cytometry. The expression level of BSP was detected by Western blot. The effect of BSP a2,3sialic acid on osteogenesis was examined by measuring the expression levels of osteoblast markers at the mRNA and protein levels.4. Cell N-Linked oligosaccharide was detected with fluorescein isothiocyanate labeled structure-specific lectin LEA after adding rhBSP and rhBSP digested by N-glycosidase F and different concentrations of Tunicamycin (TM) that inhibits the N-linked glycosylation of the newly synthesized proteins to the cultured osteoblasts, and then confirmed by flow cytometry. The effect of BSP N-linked oligosaccharide on osteogenesis was examined by ALP and von Kossa staining, RT-PCR, and Western blot.5. Protein expression levels of BSP in cultured MC3T3-E1Subclone14cells was detected by Western blot after adding PNGase-F, Sialidase,β-Galactosidase, Glucosaminidase, and O-Glycosidase to the cultures. We also explored the binding ability of lectin to sugar-chains and the methods of analyzing BSP sugar-chain structure by lectin-labeling, flow cytometric, and mass spectrometric analyses.Results:1. A cell culture model of osteoblast differentiation using MC3T3-E1Subclone14cells was successfully established. The data demonstrated positive ALP staining in MC3T3-E1Subclone14cell cultures at10day, which indicated the formation of osteoblastic cells; while positive von Kossa staining in MC3T3-E1Subclone14cells at14day indicated mineralization of newly synthesized bone matrix.2. After adding rhBSP and rhBSP digested by O-glycosidase to the cultured osteoblasts, the results of ALP and von Kossa stains showed that BSP O-glycosidase effective the formation of osteoblastic cells and bone matrix mineralization. Over-expressions of ppGalNAcT1-1545and ppGalNAcT4-784were observed during the osteoblastic differentiation of MC3T3-E1Subclone14cells. ppGalNAcT1-1545siRNA and ppGalNAcT4-784siRNA vectors were constructed successfully and transfected into cultured MC3T3-E1Subclone14cells. Knockdown of the specific glycosyltransferases (ppGalNAcT1and ppGalNAcT4) effectively inhibited osteoblast differentiation and matrix mineralization, suggesting that ppGalNAcT1and ppGalNAcT4are responsible for the osteogenic function of BSP O-glycosylation.3. BSP terminal sialylation (primarily a2,3sialic acid) mainly affected bone mineralization, but had no significant effects on osteogenic differentiation of MC3T3-E1Subclone14cells. It is possible that BSP terminal sialylation affects bone mineralization though its regulatory role in expression of vitamin D receptor (VDR), which regulates bone mineralization.4. After adding rhBSP, rhBSP digested with/by N-glycosidase F and different concentrations of Tunicamycin to the cultured osteoblasts, the results of ALP and von Kossa stains etc showed that BSP N-glycosidase and inhibition of the synthesis of N-Linked oligosaccharide have no significant changes in osteoblast function in the cultures.5. N-linked polysaccharides of glycoprotein were released by PNGase F. MALDI analysis demonstrated that the terminal sialic acid of BSP N-linked Oligosaccharide was released and1142(H5N4F1),1366(H6N5F1),1504(H7N6) were presented in the mass spectrogram after treatment with sialidase. This was consistent with the identification of BSP by Western blot. Flow cytometry demonstreted that biotin-labeled lectins, such as MAL-II, VVL,VVA, and LEA, could effectively bind to α2,3Sialidase, O-Glycosidase, PNGase F, p-Galactosidase, and Glucosaminidase etc., which further confirm that BSP is a glycosylated protein consisting of N-Linked oligosaccharide, O-Linked oligosaccharide and terminal sialic acid.In conclusion, we have successfully established a cell culture model of osteoblast differentiation using MC3T3-E1Subclone14pre-osteoblast cells. Our results demonstrated that the two glycosyltransferases, ppGalNAcT1and ppGalNAcT4, are essential for the process of osteoblast differentiation and bone matrix mineralization. BSP terminal sialylation (mainly a2,3sialic acid) positively affects bone mineralization., but relative N-Linked oligosaccharide of BSP did not show significant effects on osteoblast. We have also analyzed BSP oligosaccharide chains by mass spectrometry and BSP protein expression by Western blot, which further confirm that BSP is a glycosylated protein consisting of N-Linked oligosaccharide, O-Linked oligosaccharide and terminal sialic acid. O-glycosylation of BSP plays an essential role in BSP-mediated osteogenesis.