Role of Glycosyltransferase 25 Domain 1 in Type I Collagen Glycosylation and Molecular Phenotypes.

Glycosylation in type I collagen occurs as O-linked galactosyl- (G-) lesser and glucosylgalactosyl-hydroxylysine (GG-Hyl); however, its biological significance is still not well understood. To investigate the function of this modification in bone, we have generated preosteoblast MC3T3-E1 (MC)-derived clones, short hairpin (Sh) clones, in which Glt25d1 gene expression was stably suppressed. In Sh clones, the GLT25D1 protein levels were markedly diminished in comparison to controls (MC and those transfected with the empty vector). In Sh collagen, levels of both G- and GG-Hyl were significantly diminished with a concomitant increase in the level of free-Hyl. In addition, the level of immature divalent cross-links significantly diminished while the level of the mature trivalent cross-link increased. As determined by mass spectrometric analysis, seven glycosylation sites were identified in type I collagen and the most predominant site was at the helical cross-linking site, α1-87. At all of the glycosylation sites, the relative levels of G- and GG-Hyl were markedly diminished, i.e., by ∼50-75%, in Sh collagen, and at five of these sites, the level of Lys hydroxylation was significantly increased. The collagen fibrils in Sh clones were larger, and mineralization was impaired. These results indicate that GLT25D1 catalyzes galactosylation of Hyl throughout the type I collagen molecule and that this modification may regulate maturation of collagen cross-linking, fibrillogenesis, and mineralization.

Glycosylation of Type I Collagen.

Fibrillar type I collagen is the most abundant structural protein in most tissues and organs. One of the unique and functionally important characteristics of collagen is sequential posttranslational modifications of lysine (Lys) residues. In the endoplasmic reticulum, hydroxylation of specific Lys occurs producing 5-hydroxylysine (Hyl). Then, to the 5-hydroxyl group of Hyl, a single galactose unit can be attached to form galactosyl-Hyl (Gal-Hyl) and further glucose can be added to Gal-Hyl to form glucosylgalactosyl-Hyl (GlcGal-Hyl). These are the only two O-linked glycosides found in mature type I collagen. It has been shown that this modification is critically involved in a number of biological and pathological processes likely through its regulatory roles in collagen fibrillogenesis, intermolecular cross-linking, and collagen-cell interaction. Recently, with the advances in molecular/cell biology and analytical chemistry, the molecular mechanisms of collagen glycosylation have been gradually deciphered, and the type and extent of glycosylation at the specific molecular loci can now be quantitatively analyzed. In this chapter, we describe quantitative analysis of collagen glycosylation by high-performance liquid chromatography (HPLC) and semiquantitative, site-specific analysis by HPLC-tandem mass spectrometry.

Comparative evaluation of periodontal ligament fibroblasts stored in different types of milk: effects on viability and biosynthesis of collagen.

Milk remains one of the most frequently recommended solutions for storage of avulsed teeth because it can maintain cell viability and is easily accessible. However, some negative effects of milk on avulsed teeth have been reported, just as the effects of milk on the long-term functions of cells are not clear. This study aimed to evaluate the effects of different types of milk on the viability, proliferation, and functions of periodontal ligament fibroblasts (PDLF)s in vitro. Human PDLFs were culture-medium depleted for 5 min and stored in Hanks' balanced salt solution (HBSS), whole cow's milk, low-fat cow's milk, or almond milk for 1 h at 25°C. Cell viability and proliferation were assessed using MTT assays. Expression of the genes encoding type I collagen and its modifying enzymes were analyzed using real-time PCR. Collagen matrix production was evaluated using Picrosirius red polarization. Our results showed the overall efficiency of low-fat cow's milk in maintaining the viability and proliferation of PDLFs, and in enhancing the process of collagen production. Almond milk storage resulted in the highest rate of PDLF proliferation, and comparable collagen biosynthesis ability to the control. Therefore, besides low-fat cow's milk, almond milk may potentially be an alternative tooth-storage medium for PDLF preservation and PDL tissue regeneration.

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.

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.

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.

Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture.

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.

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

UNLABELLED: The differentiation of embryonic or determined stem cell populations into adult liver fates under known conditions yields cells with some adult-specific genes but not others, aberrant regulation of one or more genes, and variations 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 the differentiation of human hepatic stem cells (hHpSCs) into adult liver fates. The 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 were used as feeders with hHpSCs. Feeders of angioblasts yielded self-replication, stellate cell precursors caused lineage restriction to hepatoblasts, mature endothelia produced differentiation into hepatocytes, and mature stellate cells and/or myofibroblasts resulted in differentiation into cholangiocytes. Paracrine signals produced by the different feeders were identified by biochemical, immunohistochemical, and quantitative reverse-transcription polymerase chain reaction analyses, and then those signals were used to replace the feeders in monolayer and three-dimensional cultures to elicit the desired biological responses from hHpSCs. The defined paracrine signals were proved to be able to yield reproducible responses from hHpSCs and to permit differentiation into fully mature and functional parenchymal cells. CONCLUSION: Paracrine signals from defined mesenchymal cell populations are important for the regulation of stem cell populations into specific adult fates; this finding is important for basic and clinical research as well as industrial investigations.

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.