Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Apr 1;331(Pt 1):41–47. doi: 10.1042/bj3310041

The C-terminus of factor H: monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor H-related proteins.

W M Prodinger 1, J Hellwage 1, M Spruth 1, M P Dierich 1, P F Zipfel 1
PMCID: PMC1219319  PMID: 9512460

Abstract

We have generated monoclonal antibodies (mAbs) specific for the C-terminus of factor H that can be used as inhibitory antibodies for heparin binding and for the specific detection of factor H and factor H-related proteins (FHRs) in plasma and triacylglycerol-rich lipoproteins. Four distinct mAbs were established: IXF9 (IgG1), VD3 (IgG2a), VIG8 (IgG1) and IIC5 (IgG1). Each reacts specifically with FHR-1 and factor H (and also with FHR-2 in the case of VIG8), but none binds to the related FHR-3 and FHR-4 proteins nor to factor H-like protein 1. By the use of deletion mutants of factor H and by comparing the reactivity with FHR-1 and FHR-2, the binding epitopes of the mAbs were identified and localized to different short consensus repeats (SCRs): mAbs IXF9 and VD3 bind to related or even identical sites within SCR18 (factor H) and SCR3 (FHR-1) respectively. mAbs VIG8 and IIC5 bind to different epitopes located within SCRs 19 to 20 of factor H and SCRs 4 to 5 of FHR-1 respectively. Only mAb VIG8 reacts with the corresponding SCRs 3 to 4 of FHR-2. These antibodies are useful for the detection of the corresponding proteins in biological specimens such as fractions of lipoproteins. In addition, mAb VIG8 has the unique feature of inhibiting binding of factor H to heparin. Given the recent identification of a heparin- and a C3b-binding domain within the C-terminus of factor H, these mAbs should provide useful tools for functional analysis and for the precise localization of the domain(s) required for this interaction.

Full Text

The Full Text of this article is available as a PDF (506.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bayer E. A., Wilchek M. The use of the avidin-biotin complex as a tool in molecular biology. Methods Biochem Anal. 1980;26:1–45. doi: 10.1002/9780470110461.ch1. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Campbell R. D., Law S. K., Reid K. B., Sim R. B. Structure, organization, and regulation of the complement genes. Annu Rev Immunol. 1988;6:161–195. doi: 10.1146/annurev.iy.06.040188.001113. [DOI] [PubMed] [Google Scholar]
  4. Estaller C., Schwaeble W., Dierich M., Weiss E. H. Human complement factor H: two factor H proteins are derived from alternatively spliced transcripts. Eur J Immunol. 1991 Mar;21(3):799–802. doi: 10.1002/eji.1830210337. [DOI] [PubMed] [Google Scholar]
  5. Feifel E., Prodinger W. M., Mölgg M., Schwaeble W., Schönitzer D., Koistinen V., Misasi R., Dierich M. P. Polymorphism and deficiency of human factor H-related proteins p39 and p37. Immunogenetics. 1992;36(2):104–109. doi: 10.1007/BF00215286. [DOI] [PubMed] [Google Scholar]
  6. Fontaine M., Demares M. J., Koistinen V., Day A. J., Davrinche C., Sim R. B., Ripoche J. Truncated forms of human complement factor H. Biochem J. 1989 Mar 15;258(3):927–930. doi: 10.1042/bj2580927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Galfre G., Howe S. C., Milstein C., Butcher G. W., Howard J. C. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature. 1977 Apr 7;266(5602):550–552. doi: 10.1038/266550a0. [DOI] [PubMed] [Google Scholar]
  8. Gordon D. L., Kaufman R. M., Blackmore T. K., Kwong J., Lublin D. M. Identification of complement regulatory domains in human factor H. J Immunol. 1995 Jul 1;155(1):348–356. [PubMed] [Google Scholar]
  9. Hellwage J., Skerka C., Zipfel P. F. Biochemical and functional characterization of the factor-H-related protein 4 (FHR-4). Immunopharmacology. 1997 Dec;38(1-2):149–157. doi: 10.1016/s0162-3109(97)00075-1. [DOI] [PubMed] [Google Scholar]
  10. Janatova J., Reid K. B., Willis A. C. Disulfide bonds are localized within the short consensus repeat units of complement regulatory proteins: C4b-binding protein. Biochemistry. 1989 May 30;28(11):4754–4761. doi: 10.1021/bi00437a036. [DOI] [PubMed] [Google Scholar]
  11. Jokiranta T. S., Zipfel P. F., Hakulinen J., Kühn S., Pangburn M. K., Tamerius J. D., Meri S. Analysis of the recognition mechanism of the alternative pathway of complement by monoclonal anti-factor H antibodies: evidence for multiple interactions between H and surface bound C3b. FEBS Lett. 1996 Sep 16;393(2-3):297–302. doi: 10.1016/0014-5793(96)00905-2. [DOI] [PubMed] [Google Scholar]
  12. Kearney J. F., Radbruch A., Liesegang B., Rajewsky K. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol. 1979 Oct;123(4):1548–1550. [PubMed] [Google Scholar]
  13. Kristensen T., Wetsel R. A., Tack B. F. Structural analysis of human complement protein H: homology with C4b binding protein, beta 2-glycoprotein I, and the Ba fragment of B2. J Immunol. 1986 May 1;136(9):3407–3411. [PubMed] [Google Scholar]
  14. Kühn S., Skerka C., Zipfel P. F. Mapping of the complement regulatory domains in the human factor H-like protein 1 and in factor H1. J Immunol. 1995 Dec 15;155(12):5663–5670. [PubMed] [Google Scholar]
  15. Kühn S., Zipfel P. F. Mapping of the domains required for decay acceleration activity of the human factor H-like protein 1 and factor H. Eur J Immunol. 1996 Oct;26(10):2383–2387. doi: 10.1002/eji.1830261017. [DOI] [PubMed] [Google Scholar]
  16. Kühn S., Zipfel P. F. The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins. Gene. 1995 Sep 11;162(2):225–229. doi: 10.1016/0378-1119(95)00360-i. [DOI] [PubMed] [Google Scholar]
  17. Pangburn M. K., Atkinson M. A., Meri S. Localization of the heparin-binding site on complement factor H. J Biol Chem. 1991 Sep 5;266(25):16847–16853. [PubMed] [Google Scholar]
  18. Pangburn M. K., Schreiber R. D., Müller-Eberhard H. J. Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution. J Exp Med. 1977 Jul 1;146(1):257–270. doi: 10.1084/jem.146.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Park C. T., Wright S. D. Plasma lipopolysaccharide-binding protein is found associated with a particle containing apolipoprotein A-I, phospholipid, and factor H-related proteins. J Biol Chem. 1996 Jul 26;271(30):18054–18060. doi: 10.1074/jbc.271.30.18054. [DOI] [PubMed] [Google Scholar]
  20. Reid K. B., Day A. J. Structure-function relationships of the complement components. Immunol Today. 1989 Jun;10(6):177–180. doi: 10.1016/0167-5699(89)90317-4. [DOI] [PubMed] [Google Scholar]
  21. Ripoche J., Al Salihi A., Rousseaux J., Fontaine M. Isolation of two molecular populations of human complement factor H by hydrophobic affinity chromatography. Biochem J. 1984 Jul 1;221(1):89–96. doi: 10.1042/bj2210089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ripoche J., Day A. J., Harris T. J., Sim R. B. The complete amino acid sequence of human complement factor H. Biochem J. 1988 Jan 15;249(2):593–602. doi: 10.1042/bj2490593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schulz T. F., Schwäble W., Stanley K. K., Weiss E., Dierich M. P. Human complement factor H: isolation of cDNA clones and partial cDNA sequence of the 38-kDa tryptic fragment containing the binding site for C3b. Eur J Immunol. 1986 Nov;16(11):1351–1355. doi: 10.1002/eji.1830161107. [DOI] [PubMed] [Google Scholar]
  24. Schwaeble W., Schwaiger H., Brooimans R. A., Barbieri A., Möst J., Hirsch-Kauffmann M., Tiefenthaler M., Lappin D. F., Daha M. R., Whaley K. Human complement factor H. Tissue specificity in the expression of three different mRNA species. Eur J Biochem. 1991 Jun 1;198(2):399–404. doi: 10.1111/j.1432-1033.1991.tb16028.x. [DOI] [PubMed] [Google Scholar]
  25. Sharma A. K., Pangburn M. K. Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10996–11001. doi: 10.1073/pnas.93.20.10996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sim E., Palmer M. S., Puklavec M., Sim R. B. Monoclonal antibodies against the complement control protein factor H (beta 1 H). Biosci Rep. 1983 Dec;3(12):1119–1131. doi: 10.1007/BF01120205. [DOI] [PubMed] [Google Scholar]
  27. Skerka C., Hellwage J., Weber W., Tilkorn A., Buck F., Marti T., Kampen E., Beisiegel U., Zipfel P. F. The human factor H-related protein 4 (FHR-4). A novel short consensus repeat-containing protein is associated with human triglyceride-rich lipoproteins. J Biol Chem. 1997 Feb 28;272(9):5627–5634. doi: 10.1074/jbc.272.9.5627. [DOI] [PubMed] [Google Scholar]
  28. Skerka C., Kühn S., Günther K., Lingelbach K., Zipfel P. F. A novel short consensus repeat-containing molecule is related to human complement factor H. J Biol Chem. 1993 Feb 5;268(4):2904–2908. [PubMed] [Google Scholar]
  29. Skerka C., Timmann C., Horstmann R. D., Zipfel P. F. Two additional human serum proteins structurally related to complement factor H. Evidence for a family of factor H-related genes. J Immunol. 1992 May 15;148(10):3313–3318. [PubMed] [Google Scholar]
  30. Vik D. P., Muñoz-Cánoves P., Chaplin D. D., Tack B. F. Factor H. Curr Top Microbiol Immunol. 1990;153:147–162. doi: 10.1007/978-3-642-74977-3_8. [DOI] [PubMed] [Google Scholar]
  31. Whaley K., Ruddy S. Modulation of C3b hemolytic activity by a plasma protein distinct from C3b inactivator. Science. 1976 Sep 10;193(4257):1011–1013. doi: 10.1126/science.948757. [DOI] [PubMed] [Google Scholar]
  32. Zipfel P. F., Skerka C. Complement factor H and related proteins: an expanding family of complement-regulatory proteins? Immunol Today. 1994 Mar;15(3):121–126. doi: 10.1016/0167-5699(94)90155-4. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES