A bifunctional diglycosyltransferase forms the Fucalpha1,2Galbeta1,3-disaccharide on Skp1 in the cytoplasm of dictyostelium.

Skp1 is a subunit of the Skp1 cullin-1 F-box protein (SCF) family of E3 ubiquitin ligases and of other regulatory complexes in the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by a pentasaccharide with the type I blood group H antigen (Fucalpha1,2Galbeta1,3GlcNAc-) at its core. Addition of the Fuc is catalyzed by FT85, a 768-amino acid protein whose fucosyltransferase activity maps to the C-terminal half of the protein. A strain whose FT85 gene is interrupted by a genetic insertion produces a truncated, GlcNAc-terminated glycan on Skp1, suggesting that FT85 may also have beta-galactosyltransferase activity. In support of this model, highly purified native and recombinant FT85 are each able to galactosylate Skp1 from FT85 mutant cells. Site-directed mutagenesis of predicted key amino acids in the N-terminal region of FT85 abolishes Skp1 beta-galactosyltransferase activity with minimal effects on the fucosyltransferase. In addition, a recombinant form of the N-terminal region exhibits beta-galactosyltransferase but not fucosyltransferase activity. Kinetic analysis of FT85 suggests that its two glycosyltransferase activities normally modify Skp1 processively but can have partial function individually. In conclusion, FT85 is a bifunctional diglycosyltransferase that appears to be designed to efficiently extend the Skp1 glycan in vivo.