Mechanisms and functions of collagen glycosylations in bone

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

Lysine post-translational modifications of collagen

Type I collagen is the most abundant structural protein in vertebrates. It is a heterotrimeric molecule composed of two α1 chains and one α2 chain, forming a long uninterrupted triple helical structure with short non-triple helical telopeptides at both the N- and C-termini. During biosynthesis, collagen acquires a number of post-translational modifications, including lysine modifications, that are critical to the structure and biological functions of this protein. Lysine modifications of collagen are highly complicated sequential processes catalysed by several groups of enzymes leading to the final step of biosynthesis, covalent intermolecular cross-linking. In the cell, specific lysine residues are hydroxylated to form hydroxylysine. Then specific hydroxylysine residues located in the helical domain of the molecule are glycosylated by the addition of galactose or glucose-galactose. Outside the cell, lysine and hydroxylysine residues in the N- and C-telopeptides can be oxidatively deaminated to produce reactive aldehydes that undergo a series of non-enzymatic condensation reactions to form covalent intra- and inter-molecular cross-links. Owing to the recent advances in molecular and cellular biology, and analytical technologies, the biological significance and molecular mechanisms of these modifications have been gradually elucidated. This chapter provides an overview on these enzymatic lysine modifications and subsequent cross-linking

Lysyl Hydroxylase 3-mediated Glucosylation in Type I Collagen: MOLECULAR LOCI AND BIOLOGICAL SIGNIFICANCE

Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846–8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization

Unusual Fragmentation Pathways in Collagen Glycopeptides

Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids –(X—Y—Gly)n. Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides, i.e. in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed. The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways – amide bond and glycosidic bond cleavage – are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e. Arg, Lys, HyK and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations, to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides

Glycosylation and Cross-linking in Bone Type I Collagen

Fibrillar type I collagen is the major organic component in bone, providing a stable template for mineralization. During collagen biosynthesis, specific hydroxylysine residues become glycosylated in the form of galactosyl- and glucosylgalactosyl-hydroxylysine. Furthermore, key glycosylated hydroxylysine residues, α1/2-87, are involved in covalent intermolecular cross-linking. Although cross-linking is crucial for the stability and mineralization of collagen, the biological function of glycosylation in cross-linking is not well understood. In this study, we quantitatively characterized glycosylation of non-cross-linked and cross-linked peptides by biochemical and nanoscale liquid chromatography-high resolution tandem mass spectrometric analyses. The results showed that glycosylation of non-cross-linked hydroxylysine is different from that involved in cross-linking. Among the cross-linked species involving α1/2-87, divalent cross-links were glycosylated with both mono- and disaccharides, whereas the mature, trivalent cross-links were primarily monoglycosylated. Markedly diminished diglycosylation in trivalent cross-links at this locus was also confirmed in type II collagen. The data, together with our recent report (Sricholpech, M., Perdivara, I., Yokoyama, M., Nagaoka, H., Terajima, M., Tomer, K. B., and Yamauchi, M. (2012) Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance. J. Biol. Chem. 287, 22998–23009), indicate that the extent and pattern of glycosylation may regulate cross-link maturation in fibrillar collagen

Decorin modulates collagen matrix assembly and mineralization

Decorin (DCN) is one of the major matrix proteoglycans in bone. To investigate the role of DCN in matrix mineralization, the expression of DCN in MC3T3-E1 (MC) cell cultures and the phenotypes of MC-derived clones expressing higher (sense; S-DCN) or lower (antisense; AS-DCN) levels of DCN were characterized. DCN expression was significantly decreased as the mineralized nodules were formed and expanded in vitro. In S-DCN clones, in vitro matrix mineralization was inhibited, whereas in AS-DCN clones, mineralization was accelerated. At the microscopic level, collagen fibers in S-DCN clones were thinner while those of AS-DCN clones were thicker and lacked directionality compared to the controls. At the ultrastructural level, the collagen fibrils in S-DCN clones were markedly thinner, whereas those of AS-DCN clones were larger and irregular in shape. The results from Fourier transform infrared spectroscopy analysis demonstrated that in AS-DCN cultures the mineral content was greater but the crystallinity of mineral was poorer than that of the controls at early stage of mineralization. The in vivo transplantation assay demonstrated that no mineralized matrices were formed in S-DCN transplants, whereas they were readily detected in AS-DCN transplants at 3 weeks of transplantation. The areas of bone-like matrices in AS-DCN transplants were significantly greater than the controls at 3 weeks but became comparable at 5 weeks. The bone-like matrices in AS-DCN transplants exhibited woven bone-like non-lamellar structure while the lamellar bone-like structure was evident in the control transplants. These results suggest that DCN regulates matrix mineralization by modulating collagen assembly

Paracrine signals from mesenchymal cell populations govern the expansion and differentiation of human hepatic stem cells to adult liver fates

Differentiation of embryonic or determined stem cell populations to adult liver fates under known conditions yields cells with some but not other adult-specific genes, aberrant regulation of one or more genes, and variation in the results from experiment to experiment. We tested the hypothesis that sets of signals produced by freshly isolated, lineage-dependent mesenchymal cell populations would yield greater efficiency and reproducibility in driving differentiation of human hepatic stem cells (hHpSCs) to adult liver fates. Subpopulations of liver-derived mesenchymal cells, purified by immunoselection technologies, included 1) angioblasts; 2) mature endothelia; 3) hepatic stellate cell precursors; 4) mature stellate cells (pericytes) and 5) myofibroblasts. Freshly immunoselected cells of each of these subpopulations were established in primary cultures under wholly defined (serum-free) conditions that we developed for short-term cultures and used them as feeders with hHpSCs. Feeders of angioblasts yielded self-replication; stellate cell precursors caused lineage restriction to hepatoblasts; mature endothelia produced differentiation to hepatocytes; and mature stellate cells and/or myofibroblasts resulted in differentiation to cholangiocytes. Paracrine signals, produced by the different feeders, were identified by biochemical, immunohistochemical, and qRT-PCR analyses and then those signals were used to replace the feeders in monolayer and 3-D cultures to elicit the desired biological responses from the hHpSCs. The defined paracrine signals proved able to yield reproducible responses from the hHpSCs and to permit differentiation to fully mature and functional parenchymal cells

Glycosylation and Cross-linking in Bone Type I Collagen

Fibrillar type I collagen is the major organic component in bone, providing a stable template for mineralization. During collagen biosynthesis, specific hydroxylysine residues become glycosylated in the form of galactosyl- and glucosylgalactosyl-hydroxylysine. Furthermore, key glycosylated hydroxylysine residues, α1/2-87, are involved in covalent intermolecular cross-linking. Although cross-linking is crucial for the stability and mineralization of collagen, the biological function of glycosylation in cross-linking is not well understood. In this study, we quantitatively characterized glycosylation of non-cross-linked and cross-linked peptides by biochemical and nanoscale liquid chromatography-high resolution tandem mass spectrometric analyses. The results showed that glycosylation of non-cross-linked hydroxylysine is different from that involved in cross-linking. Among the cross-linked species involving α1/2-87, divalent cross-links were glycosylated with both mono- and disaccharides, whereas the mature, trivalent cross-links were primarily monoglycosylated. Markedly diminished diglycosylation in trivalent cross-links at this locus was also confirmed in type II collagen. The data, together with our recent report (Sricholpech, M., Perdivara, I., Yokoyama, M., Nagaoka, H., Terajima, M., Tomer, K. B., and Yamauchi, M. (2012) Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance. J. Biol. Chem. 287, 22998–23009), indicate that the extent and pattern of glycosylation may regulate cross-link maturation in fibrillar collagen