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

doi:10.1074/jbc.275.16.12164

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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^*

Marcus Mreyen, Alan Champion, Supriya Srinivasan, Peter Karuso^§, Keith L. Williams, and Nicolle H. Packer^¶

From Macquarie University Centre for Analytical Biotechnology, Department of Biological Sciences and ^§ Department of Chemistry, Macquarie University, Sydney, New South Wales 2109, Australia

A decreased level of fucosylation on certain spore coat proteins of Dictyostelium discoideum alters the permeability of thespore coat. Here the post-translational modifications of a majorspore coat protein, SP96, are studied in a wild type strain (X22)and a fucosylation-defective mutant (HU2470). A novel phosphoglycanstructure on SP96 of the wild type strain, consisting of Fuc( $alpha$ 1-3)GlcNAc- $alpha$ -1-P-Ser_,was identified by electrospray ionization mass spectrometry andNMR. It was shown using monosaccharide and gas chromatographymass spectrometry analysis that SP96 in the mutant HU2470 containedapproximately 20% of wild type levels of fucose, as a result ofa missing terminal fucose on the novel glycan structure. The resultssupport previous predictions, based on inhibition studies on differentfucose-deficient strains, about the nature of monoclonal antibodyepitopes 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). Quantitativestudies on wild type SP96 indicated that there were approximately60 sites with phosphodiester-linked N-acetylglucosamine-fucosedisaccharide units and a further approximately 20 sites with fucosedirectly linked to the protein. Over 70% of the serine sites aremodified, with less than 1% of these sites as phosphoserine. Threonineand tyrosine residues were not found to bemodified.

^* This work was also supported by the Australian Research Council (through a Research Infrastructure (Equipment and Facilities)Program grant) and the Wellcome Foundation for establishment ofthe Macquarie NMR Facility.The costs of publication of this articlewere defrayed in part by the payment of page charges. The articlemust therefore be hereby marked "advertisement" in accordancewith 18 U.S.C. Section 1734 solely to indicate thisfact.

Supported by the Deutschen Akademischen Austauschdienst from a Hochschulsonderprogramm IIIscholarship.

^¶ To whom correspondence should be addressed: Proteome Systems Ltd., Locked Bag 2073, North Ryde, Sydney, New South Wales, Australia1670. Tel.: 61-2-9889-1830; Fax: 61-2-9889-1805; E-mail: nicolle.packer@proteomesystems.com.

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Multiple O-Glycoforms on the Spore Coat Protein SP96 in Dictyostelium discoideum Fuc(1-3)GlcNAc--1-P-Ser IS THE MAJOR MODIFICATION*

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^*