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Papers In Press, published online ahead of print May 3, 2004
J. Biol. Chem, 10.1074/jbc.M313858200
Submitted on December 18, 2003
Revised on May 3, 2004
Accepted on May 3, 2004

Specificity of a soluble UDP-galactose:fucoside a1,3 galactosyltransferase that modifies the cytoplasmic glycoprotein Skp1 in dictyostelium

Catherine Ketcham, Fei Wang, S. Z. Fisher, Altan Ercan, Hanke van der Wel, R. D. Locke, S. ud-Doulah.k, Khushi L. Matta, and Christopher M. West

Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104

Corresponding Author: christopher-west{at}ouhsc.edu

Skp1 is an adaptor-like protein in E3SCF-ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Gala1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc-conjugates in the presence of Mn2+ and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Gala1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to a1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower Km and 35-fold higher Vmax compared to a simple a-fucoside, but this advantage was abolished by denaturation or alkylation of Cys-residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di- and tri-saccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a Vmax advantage, but still much less active than Skp1 itself owing to a Km difference. These findings indicate that aGalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Gala1,3Fuc-linkage is deficient in expressed mutant Skp1 proteins.


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