title: Site-specific quantitative analysis of overglycosylation of collagen in osteogenesis imperfecta using hydrazide chemistry and SILAC.
abstract: We recently developed a novel method for analysis of collagen O-glycosylations, which include galactosyl-hydroxylysine (GHL) and glucosyl-galactosyl-hydroxylysine (GGHL), using hydrazide chemistry (Taga, Y., Mol. Cell. Proteomics 2012, 11 (6), M111.010397). Here we investigated an overglycosylation model of collagen produced by cultured skin fibroblasts from osteogenesis imperfecta (OI) patients using this method. Many GHL/GGHL sites were identified in normal and OI type I collagens by LC-MS analysis after the glycopeptide purification procedure. Further, relative quantification was performed on each identified glycopeptide using stable isotope labeling by amino acids in cell culture (SILAC). Significant increases of GGHL were observed at respective glycosylation sites of type I collagen in OI, whereas an OI-specific glycosylation site was not found. These results demonstrated that the overglycosylation of type I collagen proceeds only at specific sites, resulting in accumulation of GGHL, rather than because of an increase of nonspecific glycosylation. Although the roles of collagen O-glycosylations in OI and even in normal conditions are still incompletely understood, the location of GHL/GGHL in the collagen sequence is suggested to be important for their functions.
authors.affiliation: Nippi Research Institute of Biomatrix, Toride, Ibaraki 302-0017, Japan. y-taga@nippi-inc.co.jp
authors.forename: Yuki
authors.initials: Y
authors.lastname: Taga
authors.forename: Masashi
authors.initials: M
authors.lastname: Kusubata
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Ogawa-Goto
authors.forename: Shunji
authors.initials: S
authors.lastname: Hattori
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry / Q000378:metabolism; D024042:Collagen Type I / Q000737:chemistry / Q000378:metabolism*; D024061:Collagen Type III / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006834:Hydrazines / Q000737:chemistry*; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010013:Osteogenesis Imperfecta / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry
chemical_list: D024042:Collagen Type I; D024061:Collagen Type III; D006834:Hydrazines; D010446:Peptide Fragments; C022814:glycosylated collagen; D003094:Collagen