Abstract
Human alpha1,3 fucosyltransferases (FucTs) contain four highly conserved cysteine (Cys) residues, in addition to a free Cys residue that lies near the binding site for GDP-fucose (Holmes, E. H., Xu, Z. , Sherwood, A. L., and Macher, B. A. (1995) J. Biol. Chem. 270, 8145-8151). The participation of the highly conserved Cys residues in disulfide bonds and their functional significance were characterized by mass spectrometry (MS) analyses and site-directed mutagenesis, respectively. Among the human FucTs is a subset of enzymes (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 containing the four conserved Cys residues were detected after reduction and alkylation, and found to be involved in disulfide bonds. The disulfide bond pattern was characterized by multiple stage MS analysis and the use of Glu-C protease and MS/MS analysis. Disulfide bonds in FucT III occur between Cys residues (Cys(81) to Cys(338) and Cys(91) to Cys(341)) at the N and C termini of the catalytic domain, bringing these ends close together in space. Mutagenesis of highly conserved Cys residues to Ser in FucT V resulted in proteins lacking enzymatic activity. Three of the four mutants have molecular weights similar to wild type enzyme and maintained an ability to bind GDP, whereas the other (Cys(104)) produced a series of lower molecular weight bands when characterized by Western blot analysis, and did not bind GDP. FucTs have highly conserved, potential N-linked sites, and our mass spectrometry analyses demonstrated that both N-linked sites are modified with oligosaccharides.
Highlights
Human ␣1,3 fucosyltransferases (FucTs) contain four highly conserved cysteine (Cys) residues, in addition to a free Cys residue that lies near the binding site for GDP-fucose
A series of protein chemistry/mass spectrometry and site-directed mutagenesis studies have been carried out to provide the first information on the structure/function relationships of these highly conserved Cys residues
Amino Acid Sequence Analysis of FucT III—The availability of a yeast expression system for FucT III provided an opportunity to obtain a sufficient quantity of enzyme for a complete characterization of the protein’s amino acid sequence, its disulfide bond pattern and the location of N-linked glycosylation
Summary
Materials—Unlabeled GDP-Fuc was purchased from Calbiochem (San Diego, CA), and GDP-[3H]Fuc was purchased from NEN Life Science Products. FucT III active fractions were eluted from the column with loading buffer containing 2 mM GDP at 0.2 ml/min. A 5-l sample of the concentrated FucT III (ϳ 5 g) was incubated with a 20-fold molar excess of PEO-maleimide-activated biotin for 30 min in the dark at room temperature (total volume 6 l). Endoproteinase Glu-C Digestion—Fractions of the tryptic digest of biotin modified FucT III eluted at 34 –38 min were collected and dried by vacuum, and dissolved in 20 l of ammonium bicarbonate buffer (100 mM, pH 7.9), and the endoproteinase Glu-C (1/10, w/w, enzyme/protein) was added. The relative collisional energy for the MS/MS and MS3 analyses was set from 30% to 43%
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