O-linked N,N'-diacetyllactosamine (LacdiNAc)-modified glycans in extracellular matrix glycoproteins are specifically phosphorylated at subterminal N-acetylglucosamine |
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Authors: | Breloy Isabelle Pacharra Sandra Ottis Philipp Bonar David Grahn Ammi Hanisch Franz-Georg |
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Institution: | From the ‡Institute of Biochemistry II, Medical Faculty, University of Cologne, 50931 Köln, Germany.;the ¶Center for Molecular Medicine Cologne, University of Cologne, 50931 Köln, Germany, and ;the §Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, 413 45 Göteborg, Sweden |
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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. |
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Keywords: | Carbohydrate Glycoprotein Carbohydrate Structure Extracellular Matrix Proteins Glycosylation Phosphorylation LacdiNAc ZP3 α-Dystroglycan Mucin-type O-Linked Glycans Phosphoglycans |
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