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 (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.

FIGURE 1.

Immunoblot (a) and Coomassie Blue staining (b) of TF proteins. a, 2 ng of reduced TF proteins, rTF_1–243, rTF_{1–243“D”}, rTF_1–263, rTF_1–263D, pTF, and pTF_D, were electrophoresed on a 4–12% linear gradient gel and immunoblotted with anti-TF-5 and anti-TF-48 mAb. The corresponding molecular mass (kDa) as determined from the molecular mass standard is indicated to the left of the immunoblot. All proteins were treated with PNGase F enzyme and used in the FVIIa·TF amidolytic activity assay and extrinsic FXase assay. b, 3 μg of reduced TF proteins, rTF_1–263, rTF_1–263D, pTF, and pTF_D, were electrophoresed on 4–12% linear gradient gel and stained with Coomassie Blue. Both the immunoblot and staining gel demonstrate the increase in the apparent mobility of the rTF_1–263D and pTF_D relative to the glycosylated forms, rTF_1–263G and pTF_G.

FIGURE 2.

Fluorescence-linked immunoassay of TF proteins. Varying concentrations of rTF_1–263G, rTF_1–263D, pTF_G, and pTF_D were captured on anti-TF-5 mAb-coated beads and probed with biotinylated anti-TF-48 mAb and R-phycoerythrin-streptavidine. The immunoassay detects both the G and D forms of TF proteins. Each point is recorded as a mean fluorescence intensity unit (MFIU) and is a mean of two experiments ± 1 S.D.

FIGURE 3.

FVIIa·TF amidolytic activity. Varying concentrations of rTF_1–243 (●) and rTF_{1–243“D”} (○) in a, rTF_1–263G (■) and rTF_1–263D (□) in b, and pTF (♦) and pTF_D (◇) in c were incubated with FVIIa (0.5 nm) in HBS, 0.1% polyethylene glycol, 2 mm CaCl₂, pH 7.4, for 10 min. Fluorogenic substrate was added (50 μm), and the rate of substrate hydrolysis was recorded. The inset in each graph represents the calculated 1:1 stoichiometry of the TF·FVIIa complex. Each point is a mean of two experiments ± 1 S.D.

FIGURE 4.

Extrinsic FXase assay (a) and stoichiometry of relipidated TF (b). a, varying concentrations of FX were incubated with FVIIa (5 nm) in complex with rTF_1–243 (●), rTF_{1–243“D”} (○), rTF_1–263G (■), rTF_1–263D (□), pTF (♦), and pTF_D (◇) (0.1 nm). FXa generation was measured and calculated from the FXa standard curve (inset). b, the stoichiometry of relipidated TF was analyzed in the TF·FVIIa amidolytic assay. Varying concentrations of rTF_1–263D (□) and pTF_G (♦) were incubated with FVIIa (0.05 nm) in HBS, 0.1% polyethylene glycol, 2 mm CaCl₂, pH 7.4, for 10 min. Fluorogenic substrate was added (50 μm), and the rate of substrate hydrolysis was recorded. The graph represents the calculated 1:1 stoichiometry of the TF·FVIIa complex. Each point is a mean of two experiments ± 1 S.D.