Carbohydrates and activity of natural and recombinant tissue factor.
The effect of glycosylation on tissue factor (TF) activity was evaluated, and site-specific glycosylation of full-length recombinant TF (rTF) and that of natural TF from human placenta (pTF) were studied by liquid chromatography-tandem mass spectrometry. The amidolytic activity of the TF.factor VIIa (FVIIa) complex toward a fluorogenic substrate showed that the catalytic efficiency (V(max)) of the complex increased in the order rTF(1-243) (Escherichia coli) < rTF(1-263) (Sf9 insect cells) < pTF for the glycosylated and deglycosylated forms. Substrate hydrolysis was unaltered by deglycosylation. In FXase, the K(m) of FX for rTF(1-263)-FVIIa remained unchanged after deglycosylation, whereas the k(cat) decreased slightly. A pronounced decrease, 4-fold, in k(cat) was observed for pTF.FVIIa upon deglycosylation, whereas the K(m) was minimally altered. The parameters of FX activation by both rTF(1-263D)-FVIIa and pTF(D)-FVIIa were identical and similar to those for rTF(1-243)-FVIIa. In conclusion, carbohydrates significantly influence the activity of TF proteins. Carbohydrate analysis revealed glycosylation on asparagines 11, 124, and 137 in both rTF(1-263) and pTF. The carbohydrates of rTF(1-263) contain high mannose, hybrid, and fucosylated glycans. Natural pTF contains no high mannose glycans but is modified with hybrid, highly fucosylated, and sialylated sugars.
Krudysz-Amblo
Jolanta
J
Department of Biochemistry, University of Vermont, Burlington, Vermont 05405, USA.
Jennings
Mark E
ME
Mann
Kenneth G
KG
Butenas
Saulius
S
D001216:Asparagine / Q000737:chemistry; D002241:Carbohydrates / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods; D015942:Factor VIIa / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013925:Thromboplastin / Q000737:chemistry*
D002241:Carbohydrates; D011134:Polysaccharides; D011994:Recombinant Proteins; D001216:Asparagine; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa