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Differences in the fractional abundances of carbohydrates of natural and recombinant human tissue factor.


ABSTRACT: Tissue factor (TF) is a single polypeptide integral membrane glycoprotein composed of 263 residues and is essential to life in its role as the initiator of blood coagulation. Previously we have shown that the activity of the natural placental TF (pTF) and the recombinant TF (rTF) from Sf9 insect cells is different (Krudysz-Amblo, J. et al (2010) J. Biol. Chem. 285, 3371-3382).In this study, using mass spectrometry, we show by quantitative analysis that the extent of glycosylation varies on each protein.Fractional abundance of each glycan composition at each of the three glycosylation sites reveals the most pronounced difference to be at asparagine (Asn) 11. This residue is located in the region of extensive TF-factor VIIa (FVIIa) interaction. Carbohydrate fractional abundance at Asn11 revealed that glycosylation in the natural placental TF is much more prevalent (~76%) than in the recombinant protein (~20%). The extent of glycosylation on Asn124 and Asn137 is similar in the two proteins, despite the pronounced differences in the carbohydrate composition. Additionally, 77% of rTF exists as TF des-1, 2 (missing the first two amino acids from the N-terminus). In contrast, only 31% of pTF is found in the des-1, 2 form.These observations may attribute to the difference in the ability of TF-FVIIa complex to activate factor X (FX).Structural and functional comparison of the recombinant and natural protein advances our understanding and knowledge on the biological activity of TF.

SUBMITTER: Krudysz-Amblo J 

PROVIDER: S-EPMC3048926 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Differences in the fractional abundances of carbohydrates of natural and recombinant human tissue factor.

Krudysz-Amblo Jolanta J   Jennings Mark E ME   Matthews Dwight E DE   Mann Kenneth G KG   Butenas Saulius S  

Biochimica et biophysica acta 20101221 4


<h4>Background</h4>Tissue factor (TF) is a single polypeptide integral membrane glycoprotein composed of 263 residues and is essential to life in its role as the initiator of blood coagulation. Previously we have shown that the activity of the natural placental TF (pTF) and the recombinant TF (rTF) from Sf9 insect cells is different (Krudysz-Amblo, J. et al (2010) J. Biol. Chem. 285, 3371-3382).<h4>Methods</h4>In this study, using mass spectrometry, we show by quantitative analysis that the exte  ...[more]

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