Specificity of a soluble UDP-galactose:fucoside α1,3- galactosyltransferase that modifies the cytoplasmic glycoprotein Skp1 in dictyostelium

Skp1 is an adaptor-like protein in E3^SCF-ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Galα1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dietyostelium 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 Galα1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to α1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower Km and 35-fold higher V_max compared with a simple α-fucoside, but this of 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 trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V_max advantage, but still much less active than Skp1 itself because of a K_m difference. These findings indicate that α-GalT1 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 Galα1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.