Mechanisms and functions of collagen glycosylations in bone Public Deposited

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  • June 7, 2019
Creator
  • Sricholpech, Marnisa
    • Affiliation: School of Dentistry, Oral and Craniofacial Biomedicine PhD Program
Abstract
  • O-linked glycosylation of hydroxylysine (Hyl) is one of the unique post-translational modifications found in collagens and collagen-like proteins. In type I collagen, some of the helical Hyl residues are galactosylated, forming galactosylhydroxylysine (G-Hyl) which can be further glucosylated into glucosylgalactosylhydroxylysine (GG-Hyl). The critical importance of these glycosylated Hyl residues was implicated since alterations in their levels were associated with several human connective disorders. To date, the multifunctional enzyme, lysyl hydroxylase 3 (LH3), has been shown to be the major glucosyltransferase enzyme, while its galactosyltransferase function is still debatable. For bone type I collagen, little is known about the regulatory mechanisms and the significance of Hyl glycosylation. Therefore, in this study, we have aimed to elucidate the formation mechanism and functions of the glycosylated Hyl in type I collagen by utilizing mouse osteoblast (MC3T3-E1 (MC) cells) culture system. Short hairpin RNA technology was employed to stably suppress the expression of LH3 gene (Plod3) and generate single cell-derived clones (Sh clones). Characterization of type I collagen, synthesized by the Sh clones, showed significant level decrease of GG-Hyl with concomitant increase of G-Hyl while total Hyl remained unchanged, thus indicating the major function of LH3 in G-Hyl glucosylation (Study I). By mass spectrometry, specific molecular loci and forms of glycosylation have been identified at residues α1-87, α1-174 and α2-173. In addition, the effect of lowered LH3-mediated glucosylation was observed in the formation and maturation of intermolecular cross-links, collagen matrix organization and mineralization (Study II). Most recently, novel collagen galactosyltransferase enzymes, glycosyltransferase 25 domain 1 and 2 (GLT25D1 and D2), have been discovered and characterized. We have shown in study I that Glt25d1 is the only isoform expressed in MC cells. By suppressing Glt25d1, the type I collagen synthesized showed significantly lower levels of both G-Hyl and GG-Hyl (Study III). In conclusion, the results from all these studies clearly indicate that for bone type I collagen, Hyl galactosylation is modulated by Glt25d1 and subsequent glucosylation by LH3. Moreover, the glucose units in the GG-Hyl residues appeared to play essential roles in the formation of normal collagen template for the mineralization process.
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  • ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Dentistry (Oral Biology).
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  • Yamauchi, Mitsuo
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