Multiple O-glycoforms on the spore coat protein SP96 in Dictyostelium discoideum - Fuc(alpha 1-3)GlcNAc-alpha-1-P-Ser is the major modification

A decreased level of fucosylation on certain spore coat proteins of Dictyos telium discoideum alters the permeability of the spore coat. Here the post- translational modifications of a major spore coat protein, SP96, are studie d in a wild type strain (X22) and a fucosylation-defective mutant ((HU2470) . A novel phosphoglycan structure on SP96 of the wild type strain, consisti ng of Fuc(alpha 1-3)GlcNAc-alpha-1-P-Ser was identified by electrospray ion ization mass spectrometry and NMR, It was shown using monosaccharide and ga s chromatography mass spectrometry analysis that SP96 in the mutant HU2470 contained approximately 20% of wild type levels of fucose, as a result of a missing terminal fucose on the novel glycan structure. The results support previous predictions, based on inhibition studies on different fucose defi cient strains, about the nature of monoclonal antibody epitopes identified by monoclonal antibodies MUD62 and MUD166, which are known to identify O-li nked glycans (Champion, A., Griffiths, K., Cooley, A. A., Gonzalez, B. Y., Gritzali, M., West, C. M., and Williams, K. L. (1995) Microbiology 141, 785 -797). Quantitative studies on wild type SP96 indicated that there were app roximately 60 sites with phosphodiester-linked acetylglucosamine-fucose dis accharide units and a further approximately 20 sites with fucose directly l inked to the protein. Over 70% of the serine sites are modified, with less than 1% of these sites as phosphoserine. Threonine and tyrosine residues we re not found to be modified.