An integral membrane glycoprotein, tissue factor (TF), in coordination with factor VIIa (FVIIa), plays an important role in initiating the process in which blood coagulates. The recombinant form of tissue factor (rTF) has been used as a surrogate for the natural glycoprotein but has limitations in this capacity, based on both its structure and function. The opportunity to compare the natural and recombinant forms of TF offers insight into the relationship between the structure of TF/rTF and its role in blood coagulation.
Because of their enhanced protein-folding ability and complex glycosylation process, Sf9 insect cell lines are preferred for determining differences in glycosylation and fractional abundances of glycans in both natural and recombinant TF. Researchers acknowledge that differences exist between the proteins derived from mammalian versus insect cell lines, but in general, this cell line offers enhanced access to the information that may elucidate the role of differential glycosylation, particularly the abundant asparagine (Asn-linked) glycans, in the structure and function of TF.
To accomplish this comparison, Krudysz-Amblo et al. used liquid chromatography/mass spectrometry (Thermo Scientific) to induce dissociation of the glycopeptides and identify specific carbohydrate composition based on specific mass losses. Each sample was run three times, and the LC/MS peak areas were used to determine the average relative abundance for each glycan.
The results reveal that the glyscolylation sites Asn 9/11, Asn 124, and Asn 137 play a likely role in TF function. Fractional abundance at Asn 11, the mature protein in comparison to the truncated version at Asn 9, indicates that glycosylation is more abundant in natural TF (76%) when compared to recombinant TF (20%). At both Asn 124 and Asn 137, the degree of glycosylation is similar in both natural and recombinant TF, but the specific carbohydrate composition varies. At these sites, 31% of natural TF exists in the des-1,2 form, whereas 77% of recombinant TF exists in the des-1,2 form.
Researchers note that the proximity of glycosylation sites Asparagine 9, Asn 11, and Asn 137 to areas of increased FVIIa interaction and the proximity of Asn 124 to FX binding with the TF-FVIIa complex likely point to a specific glycan role in the function of TF. Indeed, the difference in glycosylation at Asn9 and Asn11 indicate a distinction between natural and recombinant TF. Further studies are needed for these sites, as well as for the glycosylation of Asn 124 with its specific location in the membrane and proximity to the FX region.
It is highly likely that the extent of glycosylation and the specific carbohydrates added during this process both play an important role in the biological function of TF as a blood coagulant. Further research into the impact of posttranslational modifications, such as glycosylation, on biological structure and function may have clinical, diagnostic, and research implications.
Krudysz-Amblo, J., et al. (2011) ‘Differences in the fractional abundances of carbohydrates of natural and recombinant human tissue factor’, Biochimica et Biophysica Acta, 1810 (4), (pp. 398-405)