Human alpha 1,3/4 fucosyltransferases - Characterization of highly conserved cysteine residues and N-linked glycosylation sites

Human alpha 1,3 fucosyltransferases (FucTs) contain four highly conserved c ysteine (Cys) residues, in addition to a free Cys residue that lies near th e binding site for GDP-fucose (Holmes, E, H,, Xu, Z., Sherwood, A.L., and M acher, B, A (1995) J. Biol. Chem. 270, 8145-8151), The participation of the highly conserved Cys residues in disulfide bonds and their functional sign ificance were characterized by mass spectrometry (MS) analyses and site-dir ected mutagenesis, respectively. Among the human FucTs is a subset of enzym es (FucT III, V, and VI) having highly homologous sequences, especially in the catalytic domain, and Cys residues in FucT III and V were characterized . The amino acid sequence of FucT III was characterized. Peptides containin g the four conserved Cys residues were detected after reduction and alkylat ion, and found to be involved in disulfide bonds. The disulfide bond patter n was characterized by multiple stage MS analysis and the use of Glu-C prot ease and MS/MS analysis. Disulfide bonds in FucT III occur between Cys resi dues (Cys(81) to Cys(338) and Cys(91) to Cys(341)) at the N and C termini o f the catalytic domain, bringing these ends close together in space. Mutage nesis of highly conserved Cys residues to Ser in FucT V resulted in protein s lacking enzymatic activity. Three of the four mutants have molecular weig hts similar to wild type enzyme and maintained an ability to bind GDP, wher eas the other (Cys(104)) produced a series of lower molecular weight bands when characterized by Western blot analysis, and did not bind GDP. FucTs ha ve highly conserved, potential N-linked sites, and our mass spectrometry an alyses demonstrated that both N-linked sites are modified with oligosacchar ides.