title: Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance.
abstract: 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.
authors.affiliation: North Carolina Oral Health Institute, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Marnisa
authors.initials: M
authors.lastname: Sricholpech
authors.forename: Irina
authors.initials: I
authors.lastname: Perdivara
authors.forename: Megumi
authors.initials: M
authors.lastname: Yokoyama
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nagaoka
authors.forename: Masahiko
authors.initials: M
authors.lastname: Terajima
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Tomer
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Yamauchi
mesh_terms: D000818:Animals; D002113:Calcification, Physiologic / Q000502:physiology*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D003432:Cross-Linking Reagents / Q000378:metabolism; D005937:Glucaric Acid / Q000378:metabolism; D006031:Glycosylation; D006901:Hydroxylysine / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D046529:Microscopy, Electron, Transmission; D010006:Osteoblasts / Q000166:cytology / Q000201:enzymology*; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000235:genetics / Q000378:metabolism*; D008249:Protein-Lysine 6-Oxidase / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D024042:Collagen Type I; D003432:Cross-Linking Reagents; D007527:Isoenzymes; D034741:RNA, Small Interfering; D006901:Hydroxylysine; C510730:lysyl hydroxylase 3, mouse; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; D008249:Protein-Lysine 6-Oxidase; D005937:Glucaric Acid