title: Differences in the fractional abundances of carbohydrates of natural and recombinant human tissue factor.
abstract: BACKGROUND
  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).
  
  
  METHODS
  In this study, using mass spectrometry, we show by quantitative analysis that the extent of glycosylation varies on each protein.
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  These observations may attribute to the difference in the ability of TF-FVIIa complex to activate factor X (FX).
  
  
  CONCLUSIONS
  Structural and functional comparison of the recombinant and natural protein advances our understanding and knowledge on the biological activity of TF.
authors.affiliation: Department of Biochemistry, University of Vermont, Burlington, VT 05405, USA.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Krudysz-Amblo
authors.forename: Mark E
authors.initials: ME
authors.lastname: Jennings
authors.forename: Dwight E
authors.initials: DE
authors.lastname: Matthews
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Mann
authors.forename: Saulius
authors.initials: S
authors.lastname: Butenas
mesh_terms: D000818:Animals; D002241:Carbohydrates / Q000032:analysis*; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D007313:Insecta / Q000166:cytology; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000737:chemistry*; D013925:Thromboplastin / Q000737:chemistry*
chemical_list: D002241:Carbohydrates; D011994:Recombinant Proteins; D013925:Thromboplastin