PURIFICATION AND CHARACTERIZATION OF AN ALPHA-1,2-L-FUCOSYL-TRANSFERASE, WHICH MODIFIES THE CYTOSOLIC PROTEIN FP21, FROM THE CYTOSOL OF DICTYOSTELIUM

A novel fucosyltransferase (cFTase) activity has been enriched over 10 (6)-fold from the cytosolic compartment of Dictyostelium based on tran sfer of [H-3]fucose from GDP-[H-3]fucose to Gal beta 1,3GlcNAc beta-pa ranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activi ty behaved as a single component during purification over DEAE-, pheny l-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex ge l filtration resins. The purified activity possessed an apparent M(r) of 95 x 10(3), was Mg2+-dependent with a neutral pH optimum, and exhib ited a K-m for GDP-fucose of 0.34 mu M, a K-m for pNP-LNB of 0.6 mM, a nd a V-max for pNP-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identifie d a polypeptide with an apparent M(r) of 85 x 10(3), which coeluted wi th the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-I-125-salicylate. B ased on substate analogue studies, exoglycosidase digestions, and co-c hromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fuc alpha 1,2Gal beta 1,3GlcNAc beta-pNP. The cFTase preferred substrates with a Gal beta 1,3 linkage, and thus its accepto r substrate specificity resembles the human Secretor-type alpha 1,2-FT ase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, w ere purified from the cytosol of a Dictyostelium mutant and found to b e substrates for the cFTase, which exhibited an apparent K-m of 0.21 m u M and an apparent V-max of 460 nmol/min/mg protein toward GP21-II. T he highly purified cFTase was inhibited by the reaction products Fuc a lpha 1,2Gal beta 1,3GlcNAc beta-pNP and FP21-II. FP21-I and recombinan t FP21 were not inhibitory, suggesting that acceptor substrate specifi city is based primarily on carbohydrate recognition. A cytosolic locat ion for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its sub strates, and its failure to be detected in crude membrane preparations .