Identification and structural analysis of the tetrasaccharide NeuAc.alpha.(2.fwdarw.6)Gal.beta.(1.fwdarw.4)GlcNAc.beta.(1.fwdarw.3)Fuc.alpha.1.fwdarw.O-linked to serine 61 of human factor IX

Abstract

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.

Protein O -fucosyltransferase 1 is an essential component of Notch signaling pathways

“…Fucose is attached to Ser or Thr in EGF repeats containing the consensus sequence Cys-(Xaa) [3][4][5] -Ser(Thr)-Cys between the second and third Cys residues (6,7). It is found on urokinase-type plasminogen activator (uPA) (8), on Notch receptors and their ligands (6,9), on Cripto (10,11), and on other proteins, including blood clotting factors. O-Fucose is required for signaling by uPA through the uPA receptor (12), for Fringe modulation of Notch receptor signaling in Drosophila (4,5) and in coculture assays (4,(13)(14)(15)(16), and for Cripto to mediate Nodal signaling (10,11).…”

Interaction of Factor IXa with Factor VIIIa

“…The first 46 amino acids constitute the prepro leader sequence that is removed before secretion of the molecule. Also during biosynthesis, the protein undergoes several posttranslational modifications that include ␥-carboxylation of first 12 Glu residues, partial hydroxylation of Asp 64 , and glycosylation at residues Ser 53 , Ser 61 , Asn 157 , Asn 167 , Thr 159 , and Thr 169 (1)(2)(3)(4)(5). The resulting mature protein of 415 amino acids (M r 57,000) is a zymogen of serine protease factor IXa and contains 17% carbohydrate by weight (6).…”

“…Finally, the protease domain is thought to play a primary role in binding to factor VIIIa (20,21). Although attempts have been made to investigate the role of the protease domain Ca 2ϩ binding site by mutational analysis, the data do not provide mechanistic details as to the inability of these mutants to function in clotting (22,23 4 in the autolysis loop of factor IXa leads to a reduction in its affinity for factor VIIIa binding, and this proteolysis is prevented by binding of Ca 2ϩ to the protease domain. An account of this work has been presented in abstract form (24).…”

Analysis of the N‐linked oligosaccharides of human C1s using electrospray ionisation mass spectrometry

“…Considering that Cls contains in its N-terminal region (positions 115-160) an epidermal growth factor-like domain, it should be mentioned that several cases of O-glycosylation have been reported on serine residues located in such domains, e.g. in coagulation Factor IX [18][19][20]. To our knowledge, however, no case of a single Olinked pentose residue has ever been described.…”