, Volume 14, Issue 2, pp 97–107 | Cite as

Identification and characterization of major bovine serum tyrosine-O-sulfate-binding protein as a complement factor H

  • Yoichi Sakakibara
  • Masahito Suiko
  • P. H. P. Fernando
  • Tomio Ohashi
  • Ming-Cheh Liu
Regular Research Papers


A major tyrosine-O-sulfate (TyrS)-binding protein present in bovine serum was purified to electrophoretic homogeneity using a combination of TyrS-Affi-Gel 10 affinity chromatographyy, DEAE-Bio-Gel A ion-exchange chromatography, and hydroxylapatite chromatography. The purified TyrS-binding protein migrated as doublet protein bands with apparent molecular weights of ca. 160, 000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. N-termini of the two forms of purified TyrS-binding protein contain most likely identical sequence for the first fifteen amino acids residues, which displays a high degree of homology to those of human and mouse complement factor H. Furthermore, the purified TyrS-binding protein exhibited immunologic cross-reactivity with anti-human complement factor H. These results indicate the identity of the purified TyrS-binding protein being bovine complement factor H. The two forms of the purified bovine factor H were investigated with respect to the sensitivity to limited trypsin digestion. The high-molecular weight form was cleaved into two fragments with apparent molecular masses of, respectively, 45 kD and 125 kD. The low-molecular weight form was cleaved in a different manner to generate three major fragments with molecular masses of 25 kD, 45 kD and 100 kD, respectively. Limited V8 protease mapping of the two forms yielded similar, yet unidentical, peptide band patterns. Purified bovine factor H appeared to bind agarose-bonded heparin through its anion-binding domain and the binding was inhibited by the presence of free heparin or dextran sulfate.

Key Words

Tyrosine-O-sulfate protein-protein interaction complement factor H heparin 



N-2-hydroxylpiperazine-N′-2-ethanesulfonic acid


Nonidet P-40


phosphate-buffered saline


sodium dodecyl sulfate-polyacrylamide gel electrophoresis




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  1. Bettelheim FR (1954) Tyrosine-O-sulfate in a peptide from fibrinogen. J. Am. Chem. Soc. 76: 2838–2839.Google Scholar
  2. Bolton AE & Hunter WM (1973) The labelling of proteins to high specific radioactivities by conjugation to a125I-containing acylating agent. Biochem. J. 133: 529–539.Google Scholar
  3. Cleveland DW, Fischer SG, Kirschner MW & Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252: 1102–1106Google Scholar
  4. Gardner WD, White PJ & Hoch SO (1980) Identification of a major human serum DNA-binding protein as β1H of the alternative pathway of complement of complement activation. Biochem. Biophys. Res. Commun. 94: 61–67.Google Scholar
  5. Harada Y, Bonhomme F, Natsuume-Sakai S, Tomita T & Moriwaki K (1989) Serological survey of complement factor H in common laboratory and wild mice: a new third allotype. Immunogenetics 29: 148–154.Google Scholar
  6. Harlow E & Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, New York.Google Scholar
  7. Hunkapiller MW, Lujan E, Ostrander F & Hood E (1983) Isolation of microgram quantities of proteins from polyacrylamide gel for amino acid analysis. Meth. Enzymol. 91: 227–236.Google Scholar
  8. Huttner WB & Baeuerle PA (1988) Protein Sulfation on Tyrosine. In: Satir B (ed.) Modern Cell Biology. Vol. 6 (pp. 97–140) Alan Liss, New York.Google Scholar
  9. Jevons FR (1963) Tyrosine-O-sulfate in fibriogen and fibrin. Biochem. J. 89: 621–624.Google Scholar
  10. Koistinen V (1993) Effects of Sulfated polyanions on functions of complement factor H. Mol. Immunol. 30: 113–118.Google Scholar
  11. Kristensen T & Tack BF (1986) Murine protein H is comprised of 20 repeating units, 61 amino acids in length. Proc. Natl. Acad. Sci. U.S.A. 83: 3963–3967.Google Scholar
  12. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.Google Scholar
  13. Liu J, Han JR, Liu C-C, Suilo M & Liu M-C (1993) Identification of a putative tyrosine-O-sulphate (TyrS) receptor possibly functioning in the biosynthetic transport of tyrosine-sulfated proteins in Madin-Darby canine kidney cells. Biochem. J. 294: 407–417.Google Scholar
  14. Liu M-C & Lipmann F (1985) Isolation of tyrosine-O-sulfate by pronase hydrolysis from fibronectin secreted by Fujinami sarcoma virus-infected rat fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 82: 34–37.Google Scholar
  15. Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membanes. J. Biol. Chem. 262: 10035–10038.Google Scholar
  16. Meri S & Pangburn MK (1990) Discrimination between activators and nonactivators of the alternative pathway of complement Regulation via a sialic acid/polyanion-bindins site on factor H. Proc. Natl. Acad. Sci. U.S.A. 87: 3982–3986.Google Scholar
  17. Nakano Y, Tobe T, Matsuda T, Sakamoto T & Tomita M (1984) Isolation and characterization of rabbit H of the alternative complement pathway. 95: 1469–1475.Google Scholar
  18. Pangburn MK, Atkinson MAL & Meri S (1991) Localization of the heparin-binding site on complement factor H. J. Biol. Chem. 266: 16847–16853.Google Scholar
  19. Pangburn MK & Müller-Eberhard HJ (1978) Complement C3 convertase: cell surface restriction of β1H control and generation of restriction on neuraminidase-treated cells. Proc. Natl. Acad. Sci. U.S.A. 75: 2416–2420.Google Scholar
  20. Ripoche J, Al Salihi A, Rousseaux J & Fontaine M (1984) Isolation of two molecular populations of human complement factor H by hydrophobic affinity chromatography. Biochem. J. 221: 89–96.Google Scholar
  21. Ripoche J, Day AJ, Harris TJR & Sim RB (1988) The complete amino acid sequence of human complement factor H. Biochem. J. 249: 593–602.Google Scholar
  22. Ripoche J, Erdei A, Gilbert D, Al Salihi A, Sim RB & Fontaine M (1988) Two populations of complement factor H differ in their ability to bind to cell surfaces. Biochem. J. 253: 475–480.Google Scholar
  23. Sim RB & DiScipio RG (1982) Purfication and structural studies on the complement-system control protein β1H (factor H). Biochem. J. 205: 285–293.Google Scholar
  24. Whaley K & Ruddy S (1976) Modulation of the alternative complement pathway by β1H globulin. J. Exp. Med. 144: 1147–1163Google Scholar
  25. Whaley K & Ruddy S (1976) Modulation of C3b hemolytic activity by a plasma protein distinct from C3b inactivator. Science 193: 1011–1013.Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Yoichi Sakakibara
    • 1
    • 2
  • Masahito Suiko
    • 2
  • P. H. P. Fernando
    • 2
  • Tomio Ohashi
    • 2
  • Ming-Cheh Liu
    • 1
  1. 1.Department of BiochemistryThe University of Texas Health Center at TylerTylerUSA
  2. 2.Department of Biological Resource SciencesMiyazaki UniversityMiyazaki, MiyazakiJapan

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