Purification and Characterization of the Human Cysteine-Rich S100A3 Protein and Its Pseudo Citrullinated Forms Expressed in Insect Cells

  • Kenji Kizawa
  • Masaki Unno
  • Hidenari Takahara
  • Claus W. Heizmann
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 963)

Abstract

High quantity and quality of recombinant Ca2+-binding proteins are required to study their molecular interactions, self-assembly, posttranslational modifications, and biological activities to elucidate Ca2+-dependent cellular signaling pathways. S100A3 is a unique member of the S100 protein family with the highest cysteine content (10%). This protein, derived from human hair follicles and cuticles, is characterized by an N-terminal acetyl group and irreversible posttranslational citrullination by peptidylarginine deiminase causing its homotetramer assembly. Insect cells, capable of introducing eukaryotic N-terminus and disulfide bonds, are an appropriate host in which to express this cysteine-rich protein. Four out of ten cysteines in the recombinant S100A3 form two intramolecular disulfide bridges that modulate its Ca2+-affinity. Three free thiol groups located at the C-terminus are predicted to form the high-affinity Zn2+-binding site. Citrullination of specific arginine residues in native S100A3 can be mimicked by site-directed mutagenic substitution of Arg/Ala. This chapter details our procedures used for the purification and characterization of the human S100A3 protein and its pseudo citrullinated forms expressed in insect cells.

Key words

Baculovirus Disulfide bridge Insect cell Peptidylarginine deiminase S100A3 Calcium-binding Zinc-binding EF-hand 

References

  1. 1.
    Kizawa K, Takahara H, Unno M, Heizmann CW (2011) S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles. Biochimie 93:2038–2047PubMedCrossRefGoogle Scholar
  2. 2.
    Leclerc E, Heizmann CW (2011) The importance of Ca2+/Zn2+ signaling S100 proteins and RAGE in translational medicine. Front Biosci S3:1232–1262CrossRefGoogle Scholar
  3. 3.
    Haimoto H, Kato K (1988) S100a0 (αα) protein in cardiac muscle. Isolation from human cardiac muscle and ultrastructural localization. Eur J Biochem 171:409–415PubMedCrossRefGoogle Scholar
  4. 4.
    Schaub MC, Heizmann CW (2008) Calcium, troponin, calmodulin, S100 proteins: from myocardial basics to new therapeutic strategies. Biochem Biophys Res Commun 369:247–264PubMedCrossRefGoogle Scholar
  5. 5.
    Kizawa K, Takahara H, Troxler H, Kleinert P, Mochida U, Heizmann CW (2008) Specific citrullination causes assembly of a globular S100A3 homotetramer: a putative Ca2+ modulator matures human hair cuticle. J Biol Chem 283:5004–5013PubMedCrossRefGoogle Scholar
  6. 6.
    Moore BW (1965) A soluble protein characteristic of the nervous system. Biochem Biophys Res Commun 189:739–744CrossRefGoogle Scholar
  7. 7.
    Donato R, Heizmann CW (eds) (2010) S100B protein in the nervous system and cardiovascular apparatus in normal and pathological conditions. Cardiovascular Psych Neurol 2010: 929712. doi: 10.1155/2010/929712
  8. 8.
    Marenholz I, Lovering RC, Heizmann CW (2006) An update of the S100 nomenclature. Biochim Biophys Acta 1763:1282–1283PubMedCrossRefGoogle Scholar
  9. 9.
    Brondyk WH (2009) Selecting an appropriate method for expressing a recombinant protein. Methods Enzymol 463:131–147PubMedCrossRefGoogle Scholar
  10. 10.
    Unno M, Kawasaki T, Takahara H, Heizmann CW, Kizawa K (2011) Refined crystal structure of human Ca2+/Zn2+-binding S100A3 protein characterized by two disulphide bridges. J Mol Biol 408:477–490PubMedCrossRefGoogle Scholar
  11. 11.
    Castagnola M, Inzitari R, Fanali C, Iavarone F, Vitali A, Desiderio C, Vento G, Tirone C, Romagnoli C, Cabras T, Manconi B, Sanna MT, Boi R, Pisano E, Olianas A, Pellegrini M, Nemolato S, Heizmann CW, Faa G, Messana I (2011) The surprising composition of the salivary proteome of preterm human newborn. Mol Cell Proteomics. doi: 10.1074/mcp.M110.003467
  12. 12.
    Kizawa K, Troxler H, Kleinert P, Inoue T, Toyoda M, Morohashi M, Heizmann CW (2002) Characterization of the cysteine-rich calcium-binding S100A3 protein from human hair cuticles. Biochem Biophys Res Commun 299:857–862PubMedCrossRefGoogle Scholar
  13. 13.
    Arnau J, Lauritzen C, Petersen GE, Pedersen J (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48:1–13PubMedCrossRefGoogle Scholar
  14. 14.
    Föhr UG, Heizmann CW, Engelkamp D, Schäfer BW, Cox JA (1995) Purification and cation binding properties of the recombinant human S100 calcium-binding protein A3, an EF-hand motif protein with high affinity for zinc. J Biol Chem 270:21056–21061PubMedCrossRefGoogle Scholar
  15. 15.
    Ostendorp T, Heizmann CW, Kroneck PM, Fritz G (2005) Purification, crystallization and preliminary X-ray diffraction studies on human Ca2+-binding protein S100B. Acta Crystallogr F61:673–675Google Scholar
  16. 16.
    Goch G, Vdovenko S, Kozłowska H, Bierzyñski A (2005) Affinity of S100A1 protein for calcium increases dramatically upon glutathionylation. FEBS J 272:2557–2565PubMedCrossRefGoogle Scholar
  17. 17.
    Miranda KJ, Loeser RF, Yammani RR (2010) Sumoylation and nuclear translocation of S100A4 regulates IL-1β mediated production of matrix metalloprotinase-13. J Biol Chem 285:31517–31524PubMedCrossRefGoogle Scholar
  18. 18.
    Andrassy M, Igwe J, Autschbach F, Volz C, Remppis A, Neurath MF, Schleicher E, Humpert PM, Wendt T, Liliensiek B, Morcos M, Schiekofer S, Thiele K, Chen J, Kientsch-Engel R, Schmidt AM, Stremmel W, Stern DM, Katus HA, Nawroth PP, Bierhaus A (2006) Posttranslationally modified proteins as mediators of sustained intestinal inflammation. Am J Pathol 169:1223–1237PubMedCrossRefGoogle Scholar
  19. 19.
    Lim SY, Raftery M, Cai H, Hsu K, Yan WX, Hseih HL, Watts RN, Richardson D, Thomas S, Perry M, Geczy CL (2008) S-nitrosylated S100A8: novel anti-inflammatory properties. J Immunol 181:5627–5636PubMedGoogle Scholar
  20. 20.
    Schenten V, Bréchard S, Plançon S, Melchior C, Frippiat JP, Tschirhart EJ (2010) iPLA2, a novel determinant in Ca2+- and phosphorylation-dependent S100A8/A9 regulated NOX2 activity. Biochim Biophys Acta 1803:840–847PubMedCrossRefGoogle Scholar
  21. 21.
    Sakaguchi M, Miyazaki M, Sonegawa H, Kashiwagi M, Ohba M, Kuroki T, Namba M, Huh NH (2004) PKCα mediates TGFβ-induced growth inhibition of human keratinocytes via phosphorylation of S100C/A11. J Cell Biol 164:979–984PubMedCrossRefGoogle Scholar
  22. 22.
    Cecil DL, Terkeltaub R (2008) Transamidation by transglutaminase 2 transforms S100A11 calgranulin into a procatabolic cytokine for chondrocytes. J Immunol 180:8378–8385PubMedGoogle Scholar
  23. 23.
    Kizawa K, Ito M (2005) Characterization of epithelial cells in the hair follicle with S100 proteins. Methods Mol Biol 289:209–222PubMedGoogle Scholar
  24. 24.
    Kitts PA, Possee RD (1993) A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 14:810–817PubMedGoogle Scholar
  25. 25.
    Kwon MS, Dojima T, Toriyama M, Park EY (2002) Development of an antibody-based assay for determination of baculovirus titers in 10 hours. Biotechnol Prog 18:647–651PubMedCrossRefGoogle Scholar
  26. 26.
    Hitchman RB, Siaterli EA, Nixon CP, King LA (2007) Quantitative real-time PCR for rapid and accurate titration of recombinant baculovirus particles. Biotechnol Bioeng 96:810–814PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Kenji Kizawa
    • 1
  • Masaki Unno
    • 2
  • Hidenari Takahara
    • 3
  • Claus W. Heizmann
    • 4
  1. 1.Skin Science Research Group, Innovative Beauty Science LaboratoryKanebo Cosmetics Inc.OdawaraJapan
  2. 2.Frontier Research Center for Applied Atomic SciencesIbaraki UniversityNakaJapan
  3. 3.Laboratory of Biochemistry and Molecular Biology, Department of Applied Biological Resource SciencesIbaraki UniversityInashikiJapan
  4. 4.Division of Clinical Chemistry and Biochemistry, Department of PediatricsUniversity of ZürichZürichSwitzerland

Personalised recommendations