title: O-linked N,N'-diacetyllactosamine (LacdiNAc)-modified glycans in extracellular matrix glycoproteins are specifically phosphorylated at subterminal N-acetylglucosamine.
abstract: The terminal modification of glycans by β4 addition of N-acetylgalactosamine to N-acetylglucosamine with formation of the N,N-diacetyllactosediamine (LacdiNAc) moiety has been well documented for a number of N-linked glycoproteins and peptides, like neurohormones. Much less is known about O-glycoproteins in this regard because only human zona pellucida glycoprotein 3 (ZP3) and bovine proopiomelanocortin were reported to be LacdiNAc-modified. In searching for mammalian proteins modified with O-linked LacdiNAc we identified six positive species among nine endogenous and recombinant O-glycoproteins, which were extracellular matrix, or matrix-related proteins. These are ZP3 and the five novel LacdiNAc-positive species ECM1, AMACO, nidogen-1, α-dystroglycan, and neurofascin. The mass spectrometric analyses revealed a core 2-based tetrasaccharide as the common structural basis of O-linked LacdiNAc that could be further modified, similar to the type 2 LacNAc termini, with fucose, sialic acid, or sulfate. Here, we provide structural evidence for a novel type of mucin-type O-glycans that is strictly specific for LacdiNAc termini: sugar phosphorylation with formation of GalNAcβ1-4(phospho-)GlcNAc. The structural details of the phosphatase-labile compound were elucidated by MS(2) analysis of tetralysine complexes and by MS(n) measurements of the permethylated glycan alditols. Phospho-LacdiNAc was detected in human HEK-293 as well as in mouse myoblast cells and in bovine brain tissue.
authors.affiliation: Institute of Biochemistry II, Medical Faculty, University of Cologne, Köln, Germany.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Breloy
authors.forename: Sandra
authors.initials: S
authors.lastname: Pacharra
authors.forename: Philipp
authors.initials: P
authors.lastname: Ottis
authors.forename: David
authors.initials: D
authors.lastname: Bonar
authors.forename: Ammi
authors.initials: A
authors.lastname: Grahn
authors.forename: Franz-Georg
authors.initials: FG
authors.lastname: Hanisch
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D004527:Egg Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007785:Lactose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
chemical_list: D004527:Egg Proteins; D016326:Extracellular Matrix Proteins; D008562:Membrane Glycoproteins; D011134:Polysaccharides; D011956:Receptors, Cell Surface; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse; C093701:N-acetylgalactosaminyl-1-4-N-acetylglucosamine; D007785:Lactose; D000117:Acetylglucosamine