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J Biol Chem, Vol. 273, Issue 29, 18242-18249, July 17, 1998

The Cytoplasmic F-box Binding Protein SKP1 Contains a Novel Pentasaccharide Linked to Hydroxyproline in Dictyostelium

Patana Teng-umnuay, Howard R. Morris, Anne Dell, Maria Panico, Thanai Paxton, and Christopher M. West

From the Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida 32610-0235 and the  Department of Biochemistry, Imperial College, London SW7 2AY, United Kingdom

SKP1 is involved in the ubiquitination of certain cell cycle and nutritional regulatory proteins for rapid turnover. SKP1 from Dictyostelium has been known to be modified by an oligosaccharide containing Fuc and Gal, which is unusual for a cytoplasmic or nuclear protein. To establish how it is glycosylated, SKP1 labeled with [³H]Fuc was purified to homogeneity and digested with endo-Lys-C. A single radioactive peptide was found after two-dimensional high performance liquid chromatography. Analysis in a quadrupole time-of-flight mass spectrometer revealed a predominant ion with a novel mass. Tandem mass spectrometry analysis yielded a set of daughter ions which identified the peptide and showed that it was modified at Pro-143. A second series of daughter ions showed that Pro-143 was hydroxylated and derivatized with a potentially linear pentasaccharide, HexHexFucHexHexNAc(HyPro). The attachment site was confirmed by Edman degradation. Gas chromatography-mass spectrometry analysis of trimethylsilyl-derivatives of overexpressed SKP1 after methanolysis showed the HexNAc to be GlcNAc. Exoglycosidase digestions of the glycopeptide from normal SKP1 and from a fucosylation mutant, followed by matrix-assisted laser desorption time-of-flight mass spectrometry analysis, showed that the sugar chain consisted of D-Galp16-D-Galp1L-Fucp12-D-Galp13GlcNAc. Matrix-assisted laser-desorption time-of-flight mass spectrometry analysis of all SKP1 peptides resolved by reversed phase-high performance liquid chromatography showed that SKP1 was only partially hydroxylated at Pro-143 and that all hydroxylated SKP1 was completely glycosylated. Thus SKP1 is variably modified by an unusual linear pentasaccharide, suggesting the localization of a novel glycosylation pathway in the cytoplasm.

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