Title : Human plasma and recombinant
factor VII
Abstract :
- Characterization of O-glycosylations at serine residues 52 and 60 and effects of site-directed mutagenesis of serine 52 to alanine
- Factor VII is a multidomain , vitamin K-dependent plasma glycoprotein that participates in the extrinsic pathway of blood coagulation
- Earlier studies demonstrated a novel disaccharide (Xyl-Glc) or trisaccharide (Xyl2-Glc) O-glycosidically linked to serine 52 in human plasma factor VII (Nishimura, H., Kawabata, S., Kisiel, W., Hase, S., Ikenaka, T., Shimonishi, Y., and Iwanaga, S. (1989) J. Biol
- Chem
- 264, 20320-20325)
- In the present study, human plasma and recombinant factor VII were isolated and subjected to enzymatic fragmentation
- Peptides comprising residues 48-62 of the first epidermal growth factor-like domain of each factor VII preparation were isolated for comparative analysis
- Using a combined strategy of amino acid sequencing, carbohydrate and amino acid com position analysis, and mass spectrometry, three different glycan structures consisting of either glucose, glucose-xylose, or glucose-(xylose)2 were detected O-glycosidically linked to serine 52 in plasma and recombinant factor VII
- Approximately equal amounts of the three glycan structures were observed in plasma factor VII , whereas in recombinant factor VII the glucose and the glucose-(xylose)2 structures predominated
- In addition to the O-linked glycan structures observed at serine 52 , a single fucose was found to be covalently linked at serine 60 in both human plasma and recombinant factor VII
- Carbohydrate and mass spectrometry analyses indicated that the fucosylation of serine 60 was virtually quantitative
- Metabolic labeling studies using [14C]fucose confirmed the presence of O-linked fucose at serine 60
- In order to assess whether the carbohydrate moiety at serine 52 contributes to the biological activity of factor VII , we have constructed a site-specific mutant of recombinant factor VII in which serine 52 has been replaced with an alanine residue
- Mutant factor VIIa exhibited approximately 60% of the coagulant activity of wild-type factor VIIa in a clotting assay
- The amidolytic activity of mutant factor VIIa was indistinguishable from that observed for recombinant wild-type factor VIIa
- In addition, the ability of mutant factor VIIa in complex with either purified relipidated tissue factor apoprotein or tissue factor on the surface of a human bladder carcinoma cell line (J82) to activate either factor X or factor IX was virtually identical to that observed for wild-type factor VIIa
- These results indicate that the carbohydrate moiety O-glycosidically linked to serine 52 does not appear to be involved either in the interaction of factor VIIa with tissue factor , or the expression of its proteolytic activity toward factor X or factor IX following complex formation with tissue factor
- (ABSTRACT TRUNCATED AT 400 WORDS