Title : Biochemical characterization of plasma-derived
tissue factor pathway inhibitor : post-translational modification of free, full-length form with particular reference to the sugar chain
Abstract :
- BACKGROUND: Tissue factor pathway inhibitor ( TFPI ) is a physiological protease inhibitor that inhibits the initial reactions of the extrinsic blood coagulation pathway
- Most TFPI in human plasma is associated with lipo proteins ; however, the most functionally active form is thought to be the free, full-length form (f-p TFPI )
- Cell culture derived TFPI and recombinant TFPI ( rTFPI ) exhibit variations in their respective anticoagulant activity, which may be caused by post-translational modifications, such as the frequent differences in sugar chain structures among recombinant proteins
- Sugar chain structures in rTFPI expressed in Chinese hamster ovary (CHO) cells have been reported previously, but those of plasma TFPI have not been
- OBJECTIVES: To purify f-p TFPI and analyze the sugar chain structures
- RESULTS AND CONCLUSION: f-p TFPI was purified to homogeneity from blood plasma using a combination of anion-exchange, heparin affinity, immunoaffinity, and reversed-phase chromatographies, resulting in a yield of 76%
- f-p TFPI showed a partially phosphorylated glycoprotein comprising a total of 276 amino acids by peptide mapping
- The sugar chain structures were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion of the pyridylamino sugar chains and the following results were obtained
- (Sialyl) Galbeta1-3GalNAc was linked to Thr(175) , partially to Thr(14) and Ser(174) ; sialyl complex-type sugar chains to Asn(117) and Asn(167) , whereas Asn(228) was not glycosylated
- Neuraminidase-resistant acidic sugar chains including sulfated sugar chains were not observed significantly
- The protease inhibitory activities of f-pTFPI towards activated factor (F) X and tissue factor-activated FVII complex were identical to those of full-length rTFPI expressed in CHO cells
Output (sent_index, trigger,
protein,
sugar,
site):
- 4. structures, , rTFPI, structures, -
- 7. glycoprotein, , glycoprotein, -, -
- 9. glycosylated, , -, -, Asn(228)
- 9. linked, , -, (Sialyl) Galbeta1-3GalNAc, Asn(117) and Asn(167)
- 9. linked, , -, (Sialyl) Galbeta1-3GalNAc, Thr(14) and Ser(174)
- 9. linked, , -, (Sialyl) Galbeta1-3GalNAc, Thr(175)
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 9. tissue factor pathway inhibitor, (Sialyl) Galbeta1-3GalNAc, Asn(117) and Asn(167)
- 9. tissue factor pathway inhibitor, (Sialyl) Galbeta1-3GalNAc, Thr(14) and Ser(174)
- 9. tissue factor pathway inhibitor, (Sialyl) Galbeta1-3GalNAc, Thr(175)
- 9. tissue factor pathway inhibitor, -, Asn(228)