Multiple O-glycoforms on the spore coat protein SP96 in Dictyostelium discoideum. Fuc(alpha1-3)GlcNAc-alpha-1-P-Ser is the major modification.
A decreased level of fucosylation on certain spore coat proteins of Dictyostelium discoideum alters the permeability of the spore coat. Here the post-translational modifications of a major spore coat protein, SP96, are studied in a wild type strain (X22) and a fucosylation-defective mutant (HU2470). A novel phosphoglycan structure on SP96 of the wild type strain, consisting of Fuc(alpha1-3)GlcNAc-alpha-1-P-Ser(,) was identified by electrospray ionization mass spectrometry and NMR. It was shown using monosaccharide and gas 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-deficient strains, about the nature of monoclonal antibody epitopes identified by monoclonal antibodies MUD62 and MUD166, which are known to identify O-linked glycans (Champion, A., Griffiths, K., Gooley, 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 approximately 60 sites with phosphodiester-linked N-acetylglucosamine-fucose disaccharide units and a further approximately 20 sites with fucose directly linked to the protein. Over 70% of the serine sites are modified, with less than 1% of these sites as phosphoserine. Threonine and tyrosine residues were not found to be modified.
Mreyen
M
M
Macquarie University Centre for Analytical Biotechnology, Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.
Champion
A
A
Srinivasan
S
S
Karuso
P
P
Williams
K L
KL
Packer
N H
NH
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid; D004023:Dictyostelium / Q000737:chemistry*; D005643:Fucose / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010539:Permeability; D019606:Phosphoamino Acids / Q000032:analysis; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D018629:Spectrometry, Mass, Secondary Ion; D013329:Structure-Activity Relationship
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D019606:Phosphoamino Acids; D015800:Protozoan Proteins; C061925:spore coat proteins, Dictyostelium; D005643:Fucose