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The highly glycosylated bone sialoprotein (BSP) is an abundant non-collagenous phosphoprotein in bone which enhances osteoblast differentiation and new bone deposition in vitro and in vivo. However, the structural details of its different glycosylation linkages have not been well studied and their functions in bone homeostasis are not clear. Previous studies suggested that the O -glycans, but not the N -glycans on BSP, are highly sialylated. Herein, we employed tandem mass spectrometry (MS/MS) to demonstrate that the N -glycanson the recombinant human integrin binding sialoprotein (rhiBSP) are also enriched in sialic acids (SAs) at their termini. We also identified multiple novel sites of N -glycan modification. Treatment of rhiBSP enhances osteoblast differentiation and mineralization of MC3T3-E1 cells and this effect could be partially reversed by efficient enzymatic removal of its N -glycans. Removal of all terminal SAs has a greater effect in reversing the effect of rhiBSP on osteogenesis, especially on mineralization, suggesting that sialylation at the termini of both N -glycans and O -glycans plays an important role in this regulation. Moreover, BSP-conjugated SAs may affect mineralization via ERK activation of VDR expression. Collectively, our results identified novel N -glycans enriched in SAs on the rhiBSP and demonstrated that SAs at both N - and O -glycans are important for BSP regulation of osteoblast differentiation and mineralization in vitro. [ABSTRACT FROM AUTHOR]

Abstract: Liquid chromatography–mass spectrometry was applied to determine the action pattern of different chondroitin lyases. Two commercial enzymes, chondroitinase ABC (Proteus vulgaris) and chondroitinase ACII (Arthrobacter aurescens), having action patterns previously determined by viscosimetry and gel electrophoresis were first examined. Next, the action patterns of recombinant lyases, chondroitinase ABC from Bacteroides thetaiotaomicron (expressed in Escherichia coli) and chondroitinase AC from Flavobacterium heparinum (expressed in its original host), were examined. Chondroitin sulfate A (CS-A, also known as chondroitin-4-sulfate) was used as the substrate for these four lyases. Aliquots taken at various time points were analyzed. The products of chondroitinase ABC (P. vulgaris) and chondroitinase AC (F. heparinum) contained unsaturated oligosaccharides of sizes ranging from disaccharide to decasaccharide, demonstrating that both are endolytic enzymes. The products afforded by chondroitinase ABC (B. thetaiotaomicron) and chondroitinase ACII (A. aurescens) contained primarily unsaturated disaccharide. These two exolytic enzymes showed different minor products, suggesting some subtle specificity differences between the actions of these two exolytic lyases on chondroitin sulfate A. [Copyright &y& Elsevier]