Biochemical characterization of plasma-derived tissue factor pathway inhibitor: post-translational modification of free, full-length form with particular reference to the sugar chain.
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 lipoproteins; however, the most functionally active form is thought to be the free, full-length form (f-pTFPI). 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.


OBJECTIVE
To purify f-pTFPI and analyze the sugar chain structures.


CONCLUSIONS
f-pTFPI 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-pTFPI 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.
Mori
Y
Y
Therapeutic Protein Products Research Department, The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, Kumamoto, Japan.
Hamuro
T
T
Nakashima
T
T
Hamamoto
T
T
Natsuka
S
S
Hase
S
S
Iwanaga
S
S
D002241:Carbohydrates / Q000032:analysis*; D002845:Chromatography; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D008074:Lipoproteins / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D010766:Phosphorylation; D011480:Protease Inhibitors; D011499:Protein Processing, Post-Translational
D002241:Carbohydrates; D006023:Glycoproteins; D008074:Lipoproteins; D011480:Protease Inhibitors; C051928:lipoprotein-associated coagulation inhibitor