Thromb Haemost 1992; 67(05): 526-532
DOI: 10.1055/s-0038-1648487
Original Articles
Schattauer GmbH Stuttgart

Expression and Characterization of Recombinant Human Protein S in Heterologous Cells - Studies of the Interaction of Amino Acid Residues Leu-608 to Glu-612 with Human C4b-Binding Protein

Glenn T G Chang
1   The Department of Haematology, University Hospital Utrecht, Utrecht, The Netherlands
,
Hans K Ploos van Amstel
2   Haemostasis and Thrombosis Research Unit, Leiden University Hospital, Leiden, The Netherlands
,
Martin Hessing
1   The Department of Haematology, University Hospital Utrecht, Utrecht, The Netherlands
,
Pieter H Reitsma
2   Haemostasis and Thrombosis Research Unit, Leiden University Hospital, Leiden, The Netherlands
,
Rogier M Bertina
2   Haemostasis and Thrombosis Research Unit, Leiden University Hospital, Leiden, The Netherlands
,
Bonno N Bouma
1   The Department of Haematology, University Hospital Utrecht, Utrecht, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 25 July 1991

Accepted after revision 07 October 1991

Publication Date:
03 July 2018 (online)

Summary

Mouse C127 epithelioid cells were genetically engineered to produce biologically active ³-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of ³-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully ³-carboxylated and fully p-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gin, respectively and this variant was designated C variant. The binding of these variants to C4BP was the same as wild type recombinant protein S. This suggests that amino acid residues leu-608, asp-609, asp-611 and glu-612 are not essential for binding of the intact full length protein to C4BP.

 
  • References

  • 1 DiScipio RG, Hermodsson MA, Yates SG, Davie EW. A comparison of human prothrombin, factor IX, factor X and protein S. Biochemistry 1977; 16: 698-706
  • 2 DiScipio RG, Davie EW. Characterization of protein S, a gamma-carboxyglutamic containing protein from bovine and human plasma. Biochemistry 1979; 18: 899-904
  • 3 Walker FJ. Regulation of activated protein C by a new protein. A possible function for bovine protein S. J Biol Chem 1980; 255: 5521-5524
  • 4 Gardiner JE, McGann MA, Berridge CW, Fulcher CA, Zimmerman TS, Griffin JH. Protein S is a cofactor for activated protein C in plasma and in the inactivation of purified factor VIII :C. Circulation 1984; 70: 205 (suppl II)
  • 5 Koedam JA, Meijers JCM, Sixma JJ, Bouma BN. Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor. J Clin Invest 1988; 82: 1236-1243
  • 6 Comp PC, Nixon RR, Cooper MR, Esmon CT. Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 1984; 74: 2082-2088
  • 7 Schwartz HP, Fisher M, Hopmeier P, Batard MA, Griffin JH. Plasma protein S deficiency in familiar thrombotic disease. Blood 1984; 64: 1297-1300
  • 8 Engesser L, Broekmans AW, Briet E, Brommer EJP, Bertina RM. Hereditary protein S deficiency: clinical manifestations. Ann Intern Med 1987; 106: 677-682
  • 9 Dahlback B. Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S. Biochem J 1983; 209: 847-856
  • 10 Bertina RM, van Wijngaarden A, Reinalda-Poot J, Poort SR, Bom VJJ. Determination of plasma protein S, the protein cofactor of activated protein C. Thromb Haemostas 1985; 53: 268-272
  • 11 Dahlback B, Lundwall A, Stenflo J. Primary structure of bovine vitamin K-dependent protein S. Proc Natl Acad Sci USA 1986; 78: 2512-2516
  • 12 Lundwall A, Dackowski W, Cohen E, Shaffer M, Mahr A, Dahlback B, Stenflo J, Wydro R. Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation. Proc Natl Acad Sci USA 1986; 83: 6716-6720
  • 13 Hoskins J, Norman DK, Beckman RJ, Long G. Cloning and characterization of human liver cDNA encoding a protein S precursor. Proc Natl Acad Sci USA 1987; 84: 349-353
  • 14 Ploos van Amstel HK, van der Zanden AL, Reitsma PH, Bertina RM. Human protein S cDNA encodes phe-16 and tyr-222 in consensus sequences for the post translational processing. FEBS Lett 1987; 222: 186-190
  • 15 Walker FJ. Characterization of a synthetic peptide that inhibits the interaction between protein S and C4b-binding protein. J Biol Chem 1989; 264: 17645-17648
  • 16 Dahlback B. Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein. J Biol Chem 1986; 261: 12022-12027
  • 17 Hessing M, Vlooswijk RAA, Hackeng TM, Kanters D, Bouma BN. The localization of heparin-binding fragments on human C4b-binding protein. J Immunol 1990; 144: 204-208
  • 18 Hessing M, Kanters D, Hackeng TM, Bouma BN. Identification of different forms of human C4b-binding protein lacking p-chain and protein S binding ability. Thromb Haemostas 1990; 64: 245-250
  • 19 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci USA 1977; 76: 5463-5467
  • 20 Sarver N, Ricca GA, Hood M, Link J, Tarr GC, Drohan WN. Subtained high level expression of recombinant human gamma interferon using a bovine papilloma virus vector. In: Papilloma Viruses: Molecular and Clinical Aspects. Howley PM, Broker TR. (eds) Alan R Liss Inc; New York: 1985. pp 515-527
  • 21 Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory; New York: 1982
  • 22 Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 1985; 82: 488-492
  • 23 Graham F, van der Eb A. A new technique for the assay of infectivity of human adenovirus. Virology 1983; 52: 456-467
  • 24 Deutz-Terlouw PP, Ballering L, Van Wijngaarden A, Bertina RM. Two ELISAs for measurement of protein S and their use in the laboratory diagnosis of protein S deficiency. Clin Chim Acta 1989; 186: 321-334
  • 25 Hessing M, Meijers JCM, van Mourik JA, de Groot PH, Bouma BN. Monoclonal antibodies against human protein S inhibit both cofactor function and binding to cultured endothelial cells. Thromb Haemostas. 1989 62. SY-XXII-5 (abstr)
  • 26 Grinnell BW, Walls JD, Marks C, Glasebrook AL, Berg DT, Yan BS, Bang NU. Gamma-carboxylated isoforms of recombinant human protein S with different biologic properties. Blood 1990; 76: 2546-2554
  • 27 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature (London) 1970; 227: 680-685
  • 28 Morrisey JH. Silverstain for proteins in polyacrylamide gels. A modified procedure with enhanced uniform sensitivity. Anal Biochem 1981; 117: 307-310
  • 29 Towbin HJ, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamidegels to nitrocellulose sheets. Proc Natl Acad Sci USA 1979; 76: 4350-4354
  • 30 Kuwada M, Katayama K. An improved method for the determination of gamma-carboxylic acids in proteins, bone and urine. Anal Biochem 1983; 131: 173-179
  • 31 Stenflo J, Lundwall A, Dahlback B. Beta-hydroxyasparagine in domains homologous to the epidermal growth factor precursor in vitamin K dependent protein S. Proc Natl Acad Sci USA 1987; 84: 368-372
  • 32 Bertina RM, Ploos van Amstel HK, van Wijngaarden A, Coenen J, Leemhuis MP, Deutz-Terlouw PP, van der Linden IK, Reitsma PH. Heerlen polymorphism of protein S, an immunologic polymorphism due to diphormism of residue 460. Blood 1990; 76: 538-548
  • 33 Ohlin A-K, Landes G, Bourdon P, Oppenheimer C, Wydro R, Stenflo J. Beta-hydroxyaspartic acid in the first epidermal growth factor-like domain of protein C. J Biol Chem 1988; 263: 19240-19248
  • 34 Malm J, Cohen E, Dackowski W, Dahlback B, Wydro R. Expression of completely gamma-carboxylated and beta-hydroxylated recombinant human vitamin K-dependent protein S with full biological activity. Eur J Biochem 1990; 187: 737-743
  • 35 Dahlback B, Frohm B, Nelsetuen G. High affinity interaction between C4b-binding protein and vitamin K dependent protein S in the presence of calcium. Suggestion of a third component in blood regulating the interaction. J Biol Chem 1990; 265: 16082-16087
  • 36 Nelson RM, Long GL. Solution-phase equilibrium binding interaction of human protein S with C4b-binding protein. Biochemistry 1991; 30: 2384-2390
  • 37 Fernandez JA, Griffin JH. Identification of regions of protein S essential for binding to C4b-binding protein. Thromb Haemostas 1991; 65-711 (abstr)