Genetic analysis has implicated the cell surface glycoprotein gp130 in cell interactions of the social amoeba Dictyostelium, and information about the utilization of the 18 N-glycosylation sequons present in gp130 is needed to identify critical molecular determinants of its activity. Various glycomics strategies, including mass spectrometry of native and derivatized glycans, monosaccharide analysis, exoglycosidase digestion, and antibody binding, were applied to characterize a nonanchored version secreted from Dictyostelium. s-gp130 is modified by a predominant Man(8)GlcNAc(4) species containing bisecting and intersecting GlcNAc residues and additional high-mannose N-glycans substituted with sulfate, methyl-phosphate, and/or core alpha 3-fucose. Site mapping confirmed the occupancy of 15 sequons, some variably, and glycopeptide analysis confirmed 14 sites and revealed extensive heterogeneity at most sites. Glycopeptide glycoforms ranged from Man(6) to Man(9), GlcNAc(0-2) (peripheral), Fuc(0-2) (including core alpha 3 and peripheral), (SO(4))(0-1), and (MePO(4))(0-1), which represented elements of virtually the entire known cellular N-glycome as inferred from prior metabolic labeling and mass spectrometry studies. gp130, and a family of 14 related predicted glycoproteins whose polypeptide sequences are rapidly diverging in the Dictyostelium lineage, may contribute a functionally important shroud of high-mannose N-glycans at the interface of the amoebae with each other, their predators and prey, and the soil environment.
mass spectrometry, glycobiology, N-glycan, Dictyostelium, glycopeptidome, gp130
NCBI PubMed ID: 20443635Publication DOI: 10.1021/pr901195cJournal NLM ID: 101128775Publisher: Washington, DC: American Chemical Society
Correspondence: Cwest2@ouhsc.edu
Institutions: Department of Biochemistry & Molecular Biology and Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA
Methods: gel filtration, SDS-PAGE, sugar analysis, Western blotting, amino acid analysis, MALDI-TOF MS, genetic methods, enzymatic digestion, HPAEC-PAD, permethylation, cloning, RP-HPLC, MALDI-TOF/TOF MS, reductive amination, nanoLC-MS/MS
Found