Glycopeptidome of a Heavily N-Glycosylated Cell Surface Glycoprotein of Dictyostelium Implicated in Cell Adhesion

Abstract

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.

Specific Glycoforms of MUC5AC and Endorepellin Accurately Distinguish Mucinous from Nonmucinous Pancreatic Cysts

“…Glycomic Analyses-N-linked glycans from total cyst fluid glycoproteins were released according to previously described methods (27,28). Briefly, samples (ϳ125 g protein, 20 l total volume) were thawed, diluted 2-fold with 8 M urea, 25 mM ammonium bicarbonate (pH 7.8), and clarified via centrifugation at 4°C at 12,000 ϫ g for 20 min.…”

Evolutionary diversity of social amoebae N-glycomes may support interspecific autonomy

“…Classical studies documented that Dd glycans derive from the canonical Glc 3 Man 9 GlcNAc 2 -PP-Dol precursor, and consist of a family of Man 6–9 structures variably modified by bisecting and intersecting β4-GlcNAc residues, core α3-linked fucose (Fuc), and peripheral Man-linked Fuc, SO 4 and CH 3 -PO 4 [5], as recently confirmed by mass spectrometry (MS) studies [6–8]. During development, these structures undergo major changes in terms of the number of substituents and extent of Man-trimming [5, 9].…”

“…For example, a global fucosylation mutant exhibits slow growth and forms abnormal cell aggregates in suspension [13], mucin-type O-glycosylation mutants exhibit abnormal sorting of prespore and prestalk cells [14] and abnormal spore coat assembly [15, 16], anionic N-glycan processing mutants exhibit altered kinetics of protein compartmentalization [17, 18], and cytoplasmic glycosylation mutants exhibit abnormal O 2 -sensing [19, 20]. Studies from our laboratory and others have begun to use MS to explore the N-glycomes of two cellular slime molds [6, 3, 7, 8, 21, 22] used as model organisms for interspecific relations, Dd and Dictyostelium purpureum ( Dp ). Recently, the genomes of additional social amoebae have been sequenced [23, 2], opening the door to apply genomics to glycogene identification to enable a genetic approach to addressing these questions.…”

“…Though all CSM N-glycans appear to derive from the same Glc 3 Man 9 GlcNAc 2 precursor, their glycomic profiles as revealed by MALDI-TOF-MS are highly distinctive and do not converge during development. Based on a recent analysis of the prominent cell surface glycoprotein gp130 [6], this repertoire is likely to be reflective of cell surface glycocalyx differences that have the potential to influence interspecific and kin recognition, predator-prey relationships, and commensal interactions with other bacteria.…”

N‐glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by ‘‘off‐line’’ liquid chromatography and mass spectrometry

“…Some mutants with defects in N-glycosylation are known, but much of the available information on the glycans of wild-type and mutant strains was based on radiolabelling studies and not on mass spectrometry. Recently, though, studies from two laboratories have used a combination of HPLC and MALDI-TOF MS to elucidate the structures of many N-glycans from D. discoideum including some from glycosylation mutants (21)(22)(23) . Key to the workflow in our own laboratory has been the use of a number of separation techniques, including solid-phase extraction early on in the analyses followed by either reversed phase (RP) and hydrophilic-interaction/anionic-exchange (HIAX) HPLC in order to yield fractions with lower complexity, thus avoiding suppression of a number of the glycans in the subsequent mass spectrometric (MS and MS/MS) analyses.…”