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

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 [ 3 H]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, Hex3 Hex3 Fuc3 Hex3 HexNAc3(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-Galp␣136-D-Galp␣13L-Fucp␣132-DGalp␤133GlcNAc. Matrix-assisted laser-desorption timeof-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.SKP1 is found in a multiprotein complex with cullin (a cdc53 homologue) and an F-box-containing protein to form the SCF complex, named as an acronym of the participating proteins. When this complex contains an E2 enzyme, it is responsible for ubiquitinating various target proteins, depending on the identity of the F-box protein. Targets for subsequent degradation identified in Saccharomyces cerevisiae include cell cycle proteins such as the S-phase kinase inhibitor SIC1 and G 1 cyclins, and proteins specific to the nutrition of the cell (1-4). The SCF complex has also been implicated in phosphorylation of kinetochore proteins (5), and another distantly related complex affects mRNA metabolism (6). An SCF complex with Cyclin A and Cdk2 has been detected in mammalian cells, and its abundance appears increased in transformed cells (7,8). SKP1 itself is abundantly and dynamically expressed in the mouse embryo (9) and central nervous system including postmitotic neurons (10) and at very high concentrations in the inner ear organ of Corti (11,12), where it comprises up to 5% of total protein in the cytoplasm. The expression of several SKP1 genes in plants appears to be governed by morphogenetic boundaries (13). Thus SKP1 is expressed ubiquitously ...

A Non-Golgi α1,2-Fucosyltransferase That Modifies Skp1 in the Cytoplasm of Dictyostelium

“…The length of the proposed FTase catalytic domain of FT85, about 270 residues, is similar to that of the Se-FTase, about 330 residues. However, FT85 differs from the Se-and other mammalian ␣1,2-FTases in its 1) requirement for a divalent cation and a reducing agent for FTase activity in vitro, 2) greater affinities for its donor and acceptor substrates in vitro, and 3) compartmentalization in the cytoplasm rather than the Golgi lumen and absence of a membrane anchor 3 H. van der Wel and C. M. West, unpublished data. domain (see below).…”

“…Another enzyme in the Skp1 glycosylation pathway, the Skp1 GlcNAcTase (8), is also related to Family 2 GTases in its NRD2 domain. 3 Role in Cell Physiology-Glycosylation is important for nuclear accumulation of Skp1 based on immunofluorescence studies on mutant Skp1s, and either the native pentasaccharide or the disaccharide produced in GDP-Fuc Ϫ mutant cells is sufficient for localization (4). Nevertheless, FT85-mediated glycosylation is important physiologically, because FT85 mutant cells exhibit increased forward and side light scattering properties suggesting increased size and granularity (Fig.…”

“…Recently, we have obtained evidence that purified FT85 and recombinant FT85 also encode a UDP-Gal:Skp1 ␤1,3-galactosyltransferase activity, and that this activity is absent from FT85 mutant cells. 3 This activity is reconstituted by expressing the N-terminal but not the C-terminal portion of FT85. 3 Thus the C-terminal region of FT85 downstream of the polyasparagine stretch contains the catalytic domain of the FTase, but this region of the protein may act in concert with the rest of the protein for efficient processing of Skp1.…”

“…In Dictyostelium, nearly all Skp1 is modified by a pentasaccharide, Gal␣1,6Gal␣1(Fuc␣1,2Gal␤1,3GlcNAc), 1 which is attached in O-linkage to HyPro143 (3,4). The HyPro attachment site is predicted to be located between a loop and an ␣-helix near to but projecting away from the interface with the F-box protein of the SCF-complex (5).…”

Characterization of O-Phosphohydroxyproline in Rat α-Crystallin A

“…In extensins (hydroxyproline-rich glycoproteins) of the cell wall in plants, ϳ50% of protein mass is glycosylated at Hyp residues by mono-, di-, tri-(132)-␤-linked arabinoses (7,8,9). Furthermore, cytoplasmic F box-binding protein SKP1 in Dictyostelium contains a pentasaccharide linked to Hyp (10). These findings suggest that subsequent modification of Hyp is widespread and that, in analogy to all other hydroxyamino acids, phosphorylation of Hyp should be possible in native proteins.…”