Subscribe to RSS
DOI: 10.1055/s-0038-1646566
In Vitro Carboxylation of a Blood Coagulation Factor IX Precursor Produced by Recombinant-DNA Technology
Publication History
Received 18 July 1988
Accepted after revision 14 October 1988
Publication Date:
30 June 2018 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/198902/lookinside/thumbnails/10-1055-s-0038-1646566-1.jpg)
Summary
Blood coagulation factor IX (Christmas factor) is a plasma protein which is required for normal haemostasis. A functional deficiency of factor IX results in haemophilia B, a bleeding disorder which is generally treated by infusions of factor IX concentrates prepared from pooled human plasma. The use of human blood products is connected with the risk of transmitting viral agents responsible for diseases such as hepatitis B and AIDS. Recombinant DNA techniques may provide the means to produce the required proteins without exposing the patients to these risks and at lower costs. One of the problems which has to be overcome before recombinant factor IX can be used for therapeutical purposes is related to the vitamin K-dependent carboxylation of its 12 NH2-terminal glutamate residues. In cell cultures this carboxylation, which is required to render the protein its procoagulant activity, is far from complete, especially at high expression levels. In this paper we describe the in vitro carboxylation of non and/or partly carboxylated recombinant factor IX produced by transformed Chinese hamster ovary cells. The identity of the newly formed Gla residues was verified and it could be demonstrated that all carboxyl groups had been incorporated into the recombinant factor IX.
-
References
- 1 Busby S, Kumar A, Joseph M, Halfpap L, Insley M, Berkner K, Kurachi K, Woodbury R. Expression of active human factor IX in transfected cells. Nature 1985; 316: 271-273
- 2 Kaufman RJ, Wasley LC, Furie BC, Furie B, Shoemaker ChB. Expression, purification, and characterization of recombinant gamma- carboxylated factor IX synthesized in Chinese hamster ovary cells. J Biol Chem 1986; 261: 9622-9628
- 3 De la Salle H, Altenburger W, Elkaim R, Dott K, Dieterlé A, Drillien R, Cazenave JP, Tolstoshev P, Lecocq JP. Active gamma- carboxylated human factor IX expressed using recombinant DNA. Nature 1985; 316: 268-270
- 4 Suttie JW. Mechanism of action of vitamin K: synthesis of gamma- carboxyglutamic acid. CRC Crit Rev Biochem 1980; 8: 191-223
- 5 Femlund P, Stenflo S. Beta-hydroxyaspartic acid in vitamin K- dependent proteins. J Biol Chem 1983; 258: 12509-12512
- 6 Kurachi K, Davie EW. Isolation and characterization of a cDNA coding for human factor IX. Proc Natl Acad Sci 1982; USA 79: 6461-6464
- 7 Bentley AK, Rees DJ G, Rizza C, Brownlee GG. Defective propeptide processing of blood clotting factor IX caused by mutation of arginine to glutamine at position-4. Cell 1986; 45: 343-348
- 8 Diuguid DL, Rabiet MJ, Furie BC, Liebman HA, Furie B. Molecular basis of hemophilia B: a defective enzyme due to an unprocessed propeptide is caused by a point mutation in the factor IX precursor. Proc Natl Acad Sci USA 1986; 83: 5803-5807
- 9 Jorgensen MJ, Cantor AB, Furie BC, Brown CL, Shoemaker CB, Furie B. Recognition site directing vitamin K-dependent gamma- carboxylation residues on the propeptide of factor IX. Cell 1987; 48: 185-191
- 10 Suttie JW, Hoskins JA, Engelke J, Hopfgartner A, Ehrlich H, Bang NV, Belagase RM, Schoner B, Long GL. Vitamin K-dependent carboxylase: possible role of the substrate “propeptide” as an intracellular recognition site. Proc Natl Acad Sci USA 1987; 84: 634-637
- 11 De Metz M, Soute BA M, Hemker HC, Vermeer C. Stimulation of the vitamin K-dependent carboxylase from bovine liver. Biochem J 1983; 209: 719-724
- 12 Meunier L, Allain JP, Frommel D. Performances of an artificial reagent for one-stage factor IX assay. Thromb Diath Haemostas 1975; 33: 547-552
- 13 Balland A, Faure T, Carvallo D, Cordier P, Ulrich P, Fournet B, de la Salle H, Lecocq JP. Characterisation of two differently processed forms of human recombinant factor IX synthetisized in CHO cells transformed with a polycystronic vector. Eur J Biochem 1988; 172: 565-572
- 14 Soute BA M, Ulrich MM W, Vermeer C. Vitamin K-dependent carboxylase: increased efficiency of the carboxylation reaction. Thromb Haemostas 1987; 57: 77-81
- 15 De Metz M, Vermeer C, Soute BA M, Hemker HC. Identification of phospholipid as an essential part of bovine vitamin K-dependent carboxylase. J Biol Chem 1981; 256: 10843-10846
- 16 Kuwada M, Katayama K. An inproved method for the determination of gamma-carboxyglutamic acid in proteins, bone and urine. Analyt Biochem 1983; 141: 173-179
- 17 Sedmak JJ, Grossberg SE. A rapid, sensitive and versatile assay for protein using Comassie Brilliant Blue G250. Analyt Biochem 1977; 79: 544-552
- 18 Di Scipio RG, Hermadson MA, Yates SG, Davie EW. A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor) and protein S. Biochemistry 1977; 16: 698-706
- 19 Friezner Degen SJ, MacGillivray RT, Davie EW. Characterization of the complementary deoxyribonucleic acid and gene coding for human prothrombin. Biochemistry 1983; 22: 2087-2097
- 20 Fung MR, Campbell RM, MacGillivray RT. Blood coagulation factor X mRNA encodes a single polypeptide chain containing a prepro leader sequence. Nucleic Acids Res 1984; 12: 4481-4492
- 21 Hagen FS, Gray CC, O’Hare P, Grant FJ, Saari GC, Woodbury RG, Hart CE, Insley M, Kisiel W, Kurachi K, Davie EW. Characterization of cDNA coding for human factor VII. Proc Natl Acad Sci USA 1986; 83: 2412-2416
- 22 Foster PC, Yoshitake S, Davie EW. The nucleotide sequence of the gene for human protein C. Proc Natl Acad Sci USA 1985; 82: 4673-4677
- 23 Beckmann RJ, Schmidt RJ, Santerre RF, Plutzky J, Crabtree GR, Long GL. The structure and evaluation of a 461 amino acid human protein C precursor and its messenger RNA, based upon the DNA sequence of cloned human liver cDNAs. Nucleic Acids Res 1985; 13: 5233-5247
- 24 Soute BA M, Vermeer C, De Metz M, Hemker HC, Lijnen HR. In vitro prothrombin synthesis from a purified precursor protein. III. Preparation of an acid-soluble substrate for vitamin K-dependent carboxylase by limited proteolysis of bovine descarboxyprothrombin. Biochim Biophys Acta 1981; 676: 101-107
- 25 Van Haarlem LJ M, Soute BA M, Vermeer C. Vitamin K-dependent carboxylase: possible role for thioredoxin inthe reduction of vitamin K-metabolites in liver. FEBS Lett 1987; 222: 353-357