Specificity of a soluble UDP-galactose: fucoside alpha1,3-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 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 Mn(2+) 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.
Ketcham
Catherine
C
Department of Anatomy & Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610-0235, USA.
Wang
Fei
F
Fisher
Suzanne Z
SZ
Ercan
Altan
A
van der Wel
Hanke
H
Locke
Robert D
RD
Sirajud-Doulah
K
K
Matta
Khushi L
KL
West
Christopher M
CM
D000818:Animals; D003593:Cytoplasm / Q000201:enzymology*; D004023:Dictyostelium / Q000378:metabolism*; D005643:Fucose / Q000737:chemistry / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D046911:Macromolecular Substances; D015800:Protozoan Proteins / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D014531:Uridine Diphosphate Galactose / Q000378:metabolism*
D006023:Glycoproteins; D046911:Macromolecular Substances; D015800:Protozoan Proteins; D044786:S-Phase Kinase-Associated Proteins; D005643:Fucose; D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase