Carbohydrates and activity of natural and recombinant tissue factor

Abstract

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 (Vmax) of the complex increased in the order rTF 1-243 (Escherichia coli) < rTF1-263 (Sf9 insect cells) < pTF for the glycosylated and deglycosylated forms. Substrate hydrolysis was unaltered by deglycosylation. In FXase, the Km of FX for rTF 1-263-FVIIa remained unchanged after deglycosylation, whereas the kcat decreased slightly. A pronounced decrease, 4-fold, in k cat was observed for pTF·FVIIa upon deglycosylation, whereas the Km was minimally altered. The parameters of FX activation by both rTF1-263D-FVIIa and pTFD-FVIIa were identical and similar to those for rTF1-243-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 rTF1-263 contain high mannose, hybrid, and fucosylated glycans. Natural pTF contains no high mannose glycans but is modified with hybrid, highly fucosylated, and sialylated sugars. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.