Abstract
A novel fucosyltransferase (cFTase) activity has been enriched over 10(6)-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galbeta1,3 GlcNAc beta-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent Mr of 95 X 10(3), was Mg2+-dependent with a neutral pH optimum, and exhibited a Km for GDP-fucose of 0.34 microM, a Km for pNP-LNB of 0.6 mM, and a Vmax for pN-P-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent Mr of 85 X 10(3), which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-125I-salicylate. Based on substrate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucalpha1, 2Galbeta1,3GIcNAcbeta-pNP. The cFTase preferred substrates with a Galbeta1,3linkage, and thus its acceptor substrate specificity resembles the human Secretor-type alpha1,2- FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K(m) of 0.21 microM and an apparent V(max) of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucalpha1,2Galbeta1,3GlcNAcbeta-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.
Highlights
The glycosylation of proteins traversing the secretory pathway of eukaryotic cells has been well studied (1–3)
FP21 has been detected in the cyclin A/cdcK2 complex associated with the G1 checkpoint of the human HeLa cell cycle (13), the kinetechore complex (14) of budding yeasts,1 and in association with certain membranes of Dictyostelium
Since a GDP-Fuc synthesis mutant in Dictyostelium exhibits slow growth which can be partially rescued by exogenous Fuc,2 we have investigated the fucosylation of FP21 for its possible involvement in this phenotype
Summary
The glycosylation of proteins traversing the secretory pathway of eukaryotic cells has been well studied (1–3). Fucosylation mutant, as if it were the primary acceptor substrate for this enzyme This activity was able to attach Fuc to lacto-N-biose (Gal1,3GlcNAc-) attached to a hydrophobic aglycone moiety, as determined by cross-competition studies. This enzyme activity appeared to mediate attachment of a peripheral Fuc, and was novel in terms of its apparent cytosolic, rather than Golgi, compartmentalization, and its submicromolar Km for GDP-Fuc. As shown here, a 1.2 million-fold purified preparation of the enzyme activity retains its high affinity for GDP-Fuc, displays a high affinity for afucosyl FP21, links Fuc in a ␣1,2 linkage to a 1,3-linked Gal on type 1 and 3 acceptors, and copurifies with a polypeptide which can be photoaffinity-labeled with a donor substrate analog
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
