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Background: Type I collagen is the most abundant organic component in bone, providing form and stability. Results: Lysyl hydroxylase 3-mediated glucosylation occurs at specific sites in collagen, including cross-linking sites, and suppression of this modification results in defective collagen and mineralization. Conclusion:The data indicate the critical importance of this modification in bone physiology. Significance: Alterations of this collagen modification may cause bone defects.

Lysyl hydroxylase 3 (LH3), encoded by Plod3, is the multifunctional collagen-modifying enzyme possessing LH, hydroxylysine galactosyltransferase (GT), and galactosylhydroxylysine-glucosyltransferase (GGT) activities. Although an alteration in type I collagen glycosylation has been implicated in several osteogenic disorders, the role of LH3 in bone physiology has never been investigated. To elucidate the function of LH3 in bone type I collagen modifications, we used a short hairpin RNA technology in a mouse osteoblastic cell line, MC3T3-E1; generated single cell-derived clones stably suppressing LH3 (short hairpin (Sh) clones); and characterized the phenotype. Plod3 expression and the LH3 protein levels in the Sh clones were significantly suppressed when compared with the controls, MC3T3-E1, and the clone transfected with an empty vector. In comparison with controls, type I collagen synthesized by Sh clones (Sh collagen) showed a significant decrease in the extent of glucosylgalactosylhydroxylysine with a concomitant increase of galactosylhydroxylysine, whereas the total number of hydroxylysine residues was essentially unchanged. In an in vitro fibrillogenesis assay, Sh collagen showed accelerated fibrillogenesis compared with the controls. In addition, when recombinant LH3-V5/His protein was generated in 293 cells and subjected to GGT/GT activity assay, it showed GGT but not GT activity against denatured type I collagen. The results from this study clearly indicate that the major function of LH3 in osteoblasts is to glucosylate galactosylhydroxylysine residues in type I collagen and that an impairment of this LH3 function significantly affects type I collagen fibrillogenesis.Collagens are a large family of extracellular matrix proteins comprising at least 29 different genetic types (1, 2). Among those types, fibrillar type I collagen is the most abundant protein, and it is the major structural component in most connective tissues, including bone. One of the critical steps in collagen biosynthesis, which contributes to the functional integrity of the tissues, is the post-translational modifications, including the hydroxylation of specific proline (Pro) and lysine (Lys) residues, glycosylation of specific hydroxylysine (Hyl) 2 residues, and the formation of covalent intermolecular cross-links. Although several functions have been proposed for collagen glycosylation, such as control of collagen fibrillogenesis (3-7), cross-linking (8 -14), remodeling (15)(16)(17)(18)(19)(20)(21)(22), and collagen-cell interaction (23, 24), the function is still not well defined due in part to the lack of clear understanding in the mechanism of this modification.In fibrillar collagens, glycosylation occurs at specific Hyl residues by hydroxylysine galactosyltransferase (GT) (EC 2.4.1.50) and galactosylhydroxylysine-glucosyltransferase (GGT) (EC 2.4.1.66) resulting in the formation of galactosylhydroxylysine (G-Hyl) and glucosylgalactosylhydroxylysine (GGHyl), respectively. Recently, these enzymatic activities were found in the multi...

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Collagen is one of the most widely used biomaterials for tissue engineering and regenerative medicine. Fish collagen peptides (FCP) have been used as a dietary supplement, but their effects on the cellular function are still poorly understood. The objective of this study was to investigate the effects of FCP on collagen synthesis, quality and mineralization using an osteoblastic MC3T3-E1 cell culture system. Cells treated with FCP significantly upregulated the gene expression of several collagen modifying enzymes and more collagen was deposited in the cultures. Collagen in the treated group showed a greater extent of lysine hydroxylation, higher levels of hydroxylysine-aldehyde derived cross-links and accelerated cross-link maturation compared with the untreated group. Furthermore, the treated group showed accelerated matrix mineralization. These results indicate that FCP exerts a positive effect on osteoblastic cells in terms of collagen synthesis, quality and mineralization, thereby suggesting the potential utility of FCP for bone tissue engineering.