Abstract
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, Hex-->Hex-->Fuc-->Hex-->HexNAc-->(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-Galpalpha1-->6-D-Galpalpha1-->L-Fucpalpha1-->2-D- Galpbeta1--> 3GlcNAc. 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.
Highlights
SKP1 is found in a multiprotein complex with cullin and an F-box-containing protein to form the SCF complex, named as an acronym of the participating proteins
The attachment site was confirmed by Edman degradation
When fraction 23 was separated on a C8 RP-HPLC column, radioactivity again eluted in a single peak, which was centered at fraction 35, did not absorb at 280 nm, and contained 30% of the original radioactivity (Fig. 1B). These results suggested that SKP1 contained only a single fucoglycopeptide
Summary
Q-TOF MS, quadrupole time-of-flight mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption time-of-flight mass spectrometry; Fuc, L-fucose; Gal, D-galactose; GlcNAc, N-acetyl-D-glucosamine; PCR, polymerase chain reaction; RPHPLC, reversed phase-high pressure liquid chromatography; GC/MS, gas chromatography mass spectrometry; cFTase, cytosolic fucosyltransferase; TMS, trimethylsilyl. Complex Cytoplasmic Glycosylation support this model (21), it has remained controversial (22). Simple glycosylation, in the form of GlcNAc O-linked to residues of Ser or Thr, is well established in this compartment (23). The sugar structures that have been described to date on cytoplasmic/nuclear proteins are generally distinctive from those produced in the secretory pathway, suggesting that there may be fundamental differences in the biogenesis and function of these modifications
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