O-Linked N,N′-Diacetyllactosamine (LacdiNAc)-modified Glycans in Extracellular Matrix Glycoproteins Are Specifically Phosphorylated at Subterminal N-Acetylglucosamine

• Published in: The Journal of biological chemistry 2012-05, Vol.287 (22), p.18275-18286
• Main Authors: Breloy, Isabelle, Pacharra, Sandra, Ottis, Philipp, Bonar, David, Grahn, Ammi, Hanisch, Franz-Georg
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - chemistry; Acetylglucosamine - metabolism; Amino Acid Sequence; Animals; Blotting, Western; Carbohydrate Glycoprotein; Carbohydrate Structure; Cattle; Cell Line; Egg Proteins - chemistry; Egg Proteins - metabolism; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix Proteins; Extracellular Matrix Proteins - chemistry; Extracellular Matrix Proteins - metabolism; Glycobiology and Extracellular Matrices; Glycosylation; Humans; LacdiNAc; Lactose - analogs & derivatives; Lactose - chemistry; Lactose - metabolism; Mass Spectrometry - methods; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Molecular Sequence Data; Mucin-type O-Linked Glycans; Phosphoglycans; Phosphorylation; Polysaccharides - chemistry; Polysaccharides - metabolism; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - metabolism; Zona Pellucida Glycoproteins; ZP3; α-Dystroglycan
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.280297

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 MS2 analysis of tetralysine complexes and by MSn 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. LacdiNAc and sulfo-LacdiNAc modification of N-glycoproteins/peptides is well documented. O-Linked LacdiNAc and the novel phospho-LacdiNAc modification were detected and structurally characterized on core 2-glycans of six ECM-related proteins. LacdiNAc and phospho-LacdiNAc are expressed on ECM proteins. This novel modification opens new aspects in the posttranslational control of protein function.