Human alpha 1,3/4 fucosyltransferases. Characterization of highly conserved cysteine residues and N-linked glycosylation sites.
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.
Holmes
E H
EH
Department of Cell Surface Biochemistry, Molecular Medicine, Northwest Hospital, Seattle, Washington 98134, USA.
Yen
T Y
TY
Thomas
S
S
Joshi
R
R
Nguyen
A
A
Long
T
T
Gallet
F
F
Maftah
A
A
Julien
R
R
Macher
B A
BA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D017124:Conserved Sequence; D003545:Cysteine; D004220:Disulfides; D005647:Fucosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D004220:Disulfides; D009844:Oligosaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase; D003545:Cysteine