Molecular characterization of a novel UDP-galactose:fucoside α3-galactosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium

Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Galα1,Galα1,3Fucα1,2Gal-β1, 3GlcNAcα1O-, at a 4-hydroxylated derivative of Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Galα1,3Fuc linkage by transfer of Gal from UDP-αGal to Fucα1,2Galβ1,3GlcNAcα1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal β-propeller domain. Overexpression of GT78 with a His₆-tag resulted in a 120-fold increase in GalT-activity in cytosolic extracts, and purified His₆-GT78 exhibited α3GalT-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 α3GalT, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.