Identification and structural analysis of the tetrasaccharide NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Fuc alpha 1-->O-linked to serine 61 of human factor IX.
O-Linked fucose has been found attached to Thr/Ser residues within the sequence Cys-X-X-Gly-Gly-Thr/Ser-Cys in the N-terminal EGF domains of several coagulation/fibrinolytic proteins. Carbohydrate composition and mass spectrometric analyses of tryptic and thermolytic peptides containing the corresponding site (Ser-61) in the first EGF domain of human factor IX indicated the presence of a tetrasaccharide containing one residue each of sialic acid, galactose, N-acetylglucosamine, and fucose. The Ser-61 tetrasaccharide was not susceptible to alpha-fucosidase digestion. Fragments generated during mass spectrometric analysis indicated that fucose was the attachment sugar residue. The involvement of fucose in the carbohydrate-peptide linkage was confirmed by two-dimensional 1H NMR spectroscopic analysis of the glycopeptide containing factor IX residues 57-65. The complete structure of the tetrasaccharide was obtained by methylation analysis and two-dimensional 1H TOCSY and ROESY experiments as NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Fuc alpha 1-->O-Ser61.
Harris
R J
RJ
Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080.
van Halbeek
H
H
Glushka
J
J
Basa
L J
LJ
Ling
V T
VT
Smith
K J
KJ
Spellman
M W
MW
D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D005164:Factor IX / Q000737:chemistry*; D005643:Fucose / Q000032:analysis; D006020:Glycopeptides / Q000302:isolation & purification; D006026:Glycoside Hydrolases; D006801:Humans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification; D012694:Serine; D013912:Threonine
D006020:Glycopeptides; D009844:Oligosaccharides; D010446:Peptide Fragments; D005643:Fucose; D013912:Threonine; D012694:Serine; D005164:Factor IX; D006026:Glycoside Hydrolases