생명과학회지 (Journal of Life Science)

  • 제13권4호
  • /
  • Pages.463-472
  • /
  • 2003
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

인지질막 결합에 필요한 제5혈액응고인자 트립토판잔기들의 역할규명

Characterization of tryptophan residues of human coagulation factor V required for binding to phospholipid membranes

  • Kim, Suhng-Wook (Clinical Laboratory Science, College of Health Sciences, Korea University)
  • 발행 : 2003.08.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

제5인자와 지질막 phosphatidylserine과의 상호작용은 prothrombinase 복합체의 활성을 조절하는데 중요하다. 본 연구에서 제5인자의 지질 결합부위에 위치한 Trp2063과 Trp2064를 동시에 돌연변이 시킨 재조합 제5인자를 과발현 시키고 정제하였다. 돌연변이된 제5인자는 1-10%의 phosphatidylserine을 포함하는 지질막에서 아주낮은 활성을 보였다. surface plasmon resonance에 의해서 지질막과의 결합을 측정한 결과 돌연변이된 제5인자가 본래의 제5인자보다 고정된 지질막에의 결합이 현저하게 떨어지는 것을 관찰하였다. 제5인자가 phosphatidylserine을 포함하는 지질막에 높은 친화력으로 결합하기 위해서는 Trp2063과 Trp2064가 필수적이고 이러한 상호작용은 생리적인 phosphatidylserine 농도를 포함하는 지질막 위에서 prothrombinase 복합체의 형성에 필요하다는 결론을 내렸다.

Interactions between factor Va (HFVa) and membrane phosphatidylserine (PS) regulate the activity of the prothrombinase complex. I have previously shown that two solvent exposed hydrophobic residues located in the C2-domain, Trp2063 and Trp2064, are required for binding to immobilized PS and for expression of procoagulant activity on membranes containing 5% PS. In order to fully define the functional importance of these two residues I have expressed and isolated recombinant factor Va (rHFVa) W2063A/W2064A double mutant. In contrast to the native protein the two glycoforms resulting from alternative glycosylation of Asn2181 eluted as a single peak with rHFVa1 W2063A/W2064A eluting on the leading edge and rHFVa2 W2063A/W2064A eluting on the trailing edge. The double mutant rHFVa2 W2063A/W2064A expressed little or no procoagulant activity on membranes containing 1-10% mol % PS. In contrast, the procoagulant activity of this mutant was slightly greater than the native protein on membranes containing>18 mol % PS. The binding of rHFVa2 W2063A/W2064A to immobilized phospholipid vesicles was markedly reduced compared to the native protein in a surface plasmon resonance binding assay. I conclude that Trp2063 and Trp2064 are required for high affinity binding of factor Va to PS membranes and that this interaction is necessary for assembly of the prothrombinase complex on membranes containing physiological concentrations of PS.

키워드

참고문헌

  1. Bardelle, C., B. Furie, B. C. Furie and G. E. Gilbert. 1993. Membrane binding kinetics of factor VIII indicate a complex binding process. J. Biol. Chem. 268, 8815-8824.
  2. Higgins, D. L. and K. G. Mann. 1983. The interaction of bovine factor V and factor V-derived peptides with phospholipid vesicles. J. Biol. Chem. 258, 6503-6508.
  3. Hoekema, L., G. A. F. Nicolaes, H. C. Hemker, G. Tans and J. Rosing. 1997. Human Factor Va1 and Factor Va2: Properties in the Procoagulant and Anticoagulant Pathways. Biochemistry 36, 3331-3335. https://doi.org/10.1021/bi9623284
  4. Kalafatis, M., M. D. Rand and K. G. Mann. 1994. Factor Va-membrane interaction is mediated by two regions located on the light chain of the cofactor. Biochemistry 33, 486-493. https://doi.org/10.1021/bi00168a013
  5. Kalafatis, M., R. J. Jenny and K. G. Mann. 1990. Identification and characterization of a phospholipidbinding site of bovine factor Va. J. Biol. Chem. 265, 21580-21589.
  6. Kane, W. H. and E. W. Davie. 1988. Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders. Blood 71, 539-555.
  7. Kim, S. W., M. A. Quinn-Allen, T. Camp, S. Macedo-Ribeiro, P. Fuentes-Prior, W. Bode and W. H. Kane. 2000. Identification of functionally important amino acid residue within the C2 domain of human factor V using alanine scanning mutagenesis. Biochemistry 39, 1951-1958. https://doi.org/10.1021/bi992256r
  8. Kim, S. W., T. L. Ortel, M. A. Quinn-Allen, L. Yoo, L. Worfolk, X. Zhai, B. R. Lentz and W. H. Kane. 1999. Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex. Biochemistry 38, 11448-11454. https://doi.org/10.1021/bi991275y
  9. Koppaka, V., W. F. Talbot, X. Zhai and B. R. Lentz. 1997. Roles of factor Va heavy and light chains in protein and lipid rearrangements associated with the formation of a bovine factor Va-membrane complex. Biophys. J. 73, 2638-2652. https://doi.org/10.1016/S0006-3495(97)78293-6
  10. Krieg, U. C., B. S. Isaacs, S. S. Yemul, C. T. Esmon, H. Bayley and A. E. Johnson. 1987. Interaction of blood coagulation factor Va with phospholipid vesicles examined by using lipophilic photoreagents. Biochemistry. 26, 103-109. https://doi.org/10.1021/bi00375a015
  11. Krishnaswamy, S. and K. G. Mann. 1988. The binding of factor Va to phospholipid vesicles. J. Biol. Chem. 263, 5714-5723.
  12. Lecompte, M. F., S. Krishnaswamy, K. G. Mann, M. E. Nesheim and C. Gitler. 1987. Membrane penetration of bovine factor V and Va detected by labeling with 5-iodonaphthalene-1-azide. J. Biol. Chem. 262, 1935-1937.
  13. Macedo-Ribeiro, S., W. Bode, R. Huber, M. A. Quinn-Allen, S. W. Kim, T. L. Ortel, G. P. Bourenkov, H. D. Bartunik, M. T. Stubbs, W. H. Kane and P. Fuentes-Prior. 1999. Crystal structures of the membranebinding C2 domain of human coagulation factor V. Nature 402, 434-439. https://doi.org/10.1038/46594
  14. Mann, K. G., M. E. Nesheim, W. R. Church, P. Haley and S. Krishnaswamy. 1990. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 76, 1-16.
  15. Nicolaes, G. A., B. O. Villoutreix and B. Dahlback. 1999. Partial glycosylation of Asn2181 in human factor V as a cause of molecular and functional heterogeneity. Biochemistry 38, 13584-13591. https://doi.org/10.1021/bi991165r
  16. Nicolaes, G. A., B. O. Villoutreix and B. Dahlback. 2000. Mutations in a potential phospholipid binding loop in the C2 domain of factor V affecting the assembly of the prothrombinase complex. Blood Coagul. Fibrinolysis 11, 89-100. https://doi.org/10.1097/00001721-200001000-00010
  17. Ortel, T. L., D. Devore-Carter, M. A. Quinn-Allen and W. H. Kane. 1992. Deletion analysis of recombinant human factor V. Evidence for a phosphatidylserine binding site in the second C-type domain. J. Biol. Chem. 267, 4189-4198.
  18. Ortel, T. L., K. D. Moore, M. A. Quinn-Allen, T. Okamura, A. J. Sinclair, J. Lazarchick, R. Govindan, F. Carmagnol and W. H. Kane. 1998. Inhibitory anti-factor V antibodies bind to the factor V C2 domain and are associated with hemorrhagic manifestations. Blood 91, 4188-4196.
  19. Ortel, T. L., M. A. Quinn-Allen, F. G. Keller, J. A. Peterson, D. Larocca and W. H. Kane. 1994. Localization of functionally important epitopes within the second C-type domain of coagulation factor V using recombinant chimeras. J. Biol. Chem. 269, 15898-15905.
  20. Ortel, T. L., M. A. Quinn-Allen, L. A. Charles, D. Devore-Carter and W. H. Kane. 1992. Characterization of an acquired inhibitor to coagulation factor V. Antibody binding to the second C-type domain of factor V inhibits the binding of factor V to phosphatidylserine and neutralizes procoagulant activity. J. Clin. Invest. 90, 2340-2347. https://doi.org/10.1172/JCI116123
  21. Pellequer, J. L., A. J. Gale, J. H. Griffin and E. D. Getzoff. 1998. Homology models of the C domains of blood coagulation factors V and VIII: a proposed membrane binding mode for FV and FVIII C2 domains. Blood Cells Mol Dis 24, 448-461. https://doi.org/10.1006/bcmd.1998.0214
  22. Pratt, K. P., B. W. Shen, K. Takeshima, E. W. Davie, K. Fujikawa and B. L. Stoddard. 1999. Structure of the C2 domain of human factor VIII at 1.5 $\AA$ resolution. Nature 402, 439-442. https://doi.org/10.1038/46601
  23. Pusey, M. L. and G. L. Nelsestuen. 1984. Membrane binding properties of blood coagulation Factor V and derived peptides. Biochemistry. 23, 6202-6210. https://doi.org/10.1021/bi00320a048
  24. Pusey, M. L., L. D. Mayer, G. J. Wei, V. A. Bloomfield and G. L. Nelsestuen. 1982. Kinetic and hydrodynamic analysis of blood clotting factor V-membrane binding. Biochemistry. 21, 5262-5269. https://doi.org/10.1021/bi00264a022
  25. Rosing, J., H. M. Bakker, M. C. L. G. D. Thomassen, H. C. Hemker and G. Tans. 1993. Characterization of two forms of human factor Va with different cofactor activities. J. Biol. Chem. 268, 21130-21136.
  26. Srivastava, A., M. A. Quinn-Allen, S. W. Kim, W. H. Kane and B. R. Lentz. 2001. Soluble phosphatidylserine binds to a single identified site in the C2 domain of human factor Va. Biochemistry 40, 8246-8255. https://doi.org/10.1021/bi010449k
  27. Tracy, P. B. and K. G. Mann. 1983. Prothrombinase complex assembly on the platelet surface is mediated through the 74,000-dalton component of factor Va. Proc. Natl. Acad. Sci. U. S. A. 80, 2380-2384. https://doi.org/10.1073/pnas.80.8.2380
  28. van de Waart, P., H. Bruls, H. C. Hemker and T. Lindhout. 1983. Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles. Biochemistry. 22, 2427-2432. https://doi.org/10.1021/bi00279a019
  29. Zwaal, R. F., P. Comfurius and E. M. Bevers. 1998. Lipid-protein interactions in blood coagulation. Biochim Biophys Acta 1376, 433-453. https://doi.org/10.1016/S0304-4157(98)00018-5