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.
Van Der Wel
Hanke
H
Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610-0235, USA.
Fisher
Suzanne Z
SZ
West
Christopher M
CM
D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism*; D004023:Dictyostelium / Q000201:enzymology / Q000378:metabolism*; D004187:Disaccharides / Q000378:metabolism*; D016695:Glycosyltransferases / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018797:Cell Cycle Proteins; D004187:Disaccharides; D011994:Recombinant Proteins; D044786:S-Phase Kinase-Associated Proteins; D016695:Glycosyltransferases