Abstract
We have recently discovered unusual sugar chains (xylose (Xyl)-glucose (Glc) and (Xyl)2-Glc) linked to a serine residue in the epidermal growth factor (EGF)-like domains of human and bovine clotting factors VII (Ser-52), IX (Ser-53), and protein Z (Ser-53), in addition to bovine platelet glycoprotein thrombospondin. We now have evidence of another modification in the first EGF-like domain of human factor IX, which proved to be a tetrasaccharide O-fucosidically linked to Ser-61. Two large peptides containing Ser-61 (positions 44-63), named hIX-GP1 and hIX-GP2, were first isolated from the lysyl endopeptidase-digest of human factor IX, by reversed-phase high performance liquid chromatography (HPLC). Data on the component sugar analysis after pyridylamination (PA) and sialic acid analysis of the isolated peptides indicated that they contained 1 mol each of galactose (Gal), fucose (Fuc), N-acetylglucosamine (GlcNAc), and N-acetylneuraminic acid (NeuAc), in addition to Glc and Xyl. hIX-GP1 was further digested with asparaginyl endopeptidase, and two glycopeptides containing Ser-61, named N-3 (positions 59-63) and N-9 (positions 55-63), were isolated, respectively. These glycopeptides were both composed of 1 mol each of Gal, Fuc, GlcNAc, and NeuAc but did not contain Xyl and Glc. Moreover, the data on beta-elimination for N-9 and of the fast atom bombardment mass spectrometric analysis on peptide N-3 suggested the presence of a tetrasaccharide linked to Ser-61. An analysis of the PA-oligosaccharide released from hIX-GP1 by hydrazinolysis followed by pyridylamination revealed that the reducing end was PA-Fuc. All the results support the proposal that human factor IX has a novel tetrasaccharide consisting of 1 mol each of Gal, Fuc, GlcNAc, and NeuAc, which is O-glycosidically linked to Ser-61 through the Fuc residue.
Highlights
We have recently discovered unusual sugar chains and the deduced amino acid sequence revealed that human (xylose (Xy1)-glucose (Glc) and (Xyl)z-Glc)linked to a factor IX (415 amino acid residues) consists of a y-carboxyserine residue in the epidermal growth factor (EGF)- glutamic acid (G1a)-richdomain, two epidermal growth factor like domains of human andbovine clotting factorsVI1 (EGF)’-like domains, an activation peptide region, and a (Ser-52), IX (Ser-53), and proteinZ (Ser-53), in addi- serineprotease domain
high performance liquid chromatography (HPLC) on a Cosmosil 5C4-300column (Fig. lA).A peak that eluted at 37.2 min contained the peptide corresponding to positions 44-63 in the first EGFlike domain, as judged from the data obtained with amino acid analyses
With retention times of44.6 and 45.8 min, named hIX-GP1 and hIX-GP2, had amino acid compositions corresponding to positions 44-63 (Table I)
Summary
Isolation and Identification of hIX-GPl and hIX-GP2"To isolate a glycopeptide containing Ser-61, human factor IX was reduced, S-pyridylethylated, and digested with lysyl endopeptidase. HPLC on a Cosmosil 5C4-300column (Fig. lA).A peak that eluted at 37.2 min (indicatedby an arrow in Fig. A ) contained the peptide corresponding to positions 44-63 in the first EGFlike domain, as judged from the data obtained with amino acid analyses. Electrospray ionization mass spectrometric analysis of hIX-GP1 indicated that theobserved mass of 3529.1agreed well with the theoretical mass value of 3529.7, as calculated from the peptide sequence and the sugar composition (data not shown) These results suggested that hIXGP1 andhIX-GP2 were glycopeptidesderived from the region corresponding to the sequence from Gln-44 to Lys-63 in the first EGF-like domain and thatthey contained adisaccharide (Xyl-Glc) chain linked to Ser-53. An intense peak at m/z = 1358.8 agreed well with the theoretical mass value of N-3, as calculated from the peptide sequence and the sugar composition (Fig. 3) These data strongly suggest the existence of a tetrasaccharide linked to Ser-61 and consisting of 1 mol each of Gal, Fuc, GlcNAc, and NeuAc (Fig. 4).
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