Specificity of a soluble UDP-galactose : fucoside alpha 1,3-galactosyltransferase that modifies the cytoplasmic glycoprotein Skp1 in Dictyostelium


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KETCHAM C., WANG F., FİSHER S. Z. , Ercan A. , VAN DER WEL H., LOCKE R. D. , ...More

JOURNAL OF BIOLOGICAL CHEMISTRY, vol.279, no.28, pp.29050-29059, 2004 (Journal Indexed in SCI) identifier identifier

  • Publication Type: Article / Article
  • Volume: 279 Issue: 28
  • Publication Date: 2004
  • Doi Number: 10.1074/jbc.m313858200
  • Title of Journal : JOURNAL OF BIOLOGICAL CHEMISTRY
  • Page Numbers: pp.29050-29059

Abstract

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 Galalpha1-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 Galalpha1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to alpha1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K-m and 35-fold higher V-max compared with a simple alpha-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 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 alpha-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 Galalpha1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.