Abstract
Human S100A7 (psoriasin) is a metal-chelating host-defense protein expressed by epithelial cells. S100A7 possesses two Cys residues that generate two redox isoforms of the protein. In the oxidized form (S100A7ox), Cys47 and Cys96 form an intramolecular disulfide bond, whereas these residues exist as free thiols in the reduced form (S100A7red). In this chapter, we provide a step-by-step protocol for the purification of S100A7ox and S100A7red that affords each protein in high yield and purity. In this procedure, S100A7 is expressed in Escherichia coli BL21(DE3), and the homodimer is obtained following ammonium sulfate precipitation, folding, and column chromatography. Treatment of S100A7 with 1,4-dithiothreitol (DTT) affords S100A7red. A Cu(II)-catalyzed oxidation reaction is employed to obtain S100A7ox. A RP-HPLC method that allows for baseline separation of S100A7ox and S100A7red is provided, as well as a biochemical Zn(II)-binding assay that can be employed to evaluate the functional integrity of S100A7.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Donato R, Canon BR, Sorci G et al (2013) Functions of S100 proteins. Curr Mol Med 13(1):24–57
Zackular JP, Chazin WJ, Skaar EP (2015) Nutritional immunity: S100 proteins at the host-pathogen interface. J Biol Chem 290(31):18991–18998
Cunden LS, Nolan EM (2018) Bioinorganic explorations of Zn(II) sequestration by human S100 host-defense proteins. Biochemistry 57(11):1673–1680
Jinquan T, Vorum H, Larsen CG et al (1996) Psoriasin: a novel chemotactic protein. J Invest Dermatol 107(1):5–10
Meyer JE, Harder J, Sipos B et al (2008) Psoriasin (S100A7) is a principal antimicrobial peptide of the human tongue. Mucosal Immunol 1(3):239–243
Mildner M, Stichenwirth M, Abtin A et al (2010) Psoriasin (S100A7) is a major Escherichia coli-cidal factor of the female genital tract. Mucosal Immunol 3(6):602–609
Gläser R, Harder J, Lange H et al (2005) Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection. Nat Immunol 6(1):57–64
Michalek M, Gelhaus C, Hecht O et al (2009) The human antimicrobial protein psoriasin acts by permeabilization of bacterial membranes. Dev Comp Immunol 33(6):740–746
Cunden LS, Brophy MB, Rodriguez GE et al (2017) Biochemical and functional evaluation of the intramolecular disulfide bonds in the zinc-chelating antimicrobial protein human S100A7 (Psoriasin). Biochemistry 56(43):5726–5738
Hein KZ, Takahashi H, Tsumori T et al (2015) Disulphide-reduced psoriasin is a human apoptosis-inducing broad-spectrum fungicide. Proc Natl Acad Sci U S A 112(42):13039–13044
Lee KC, Eckert RL (2007) S100A7 (Psoriasin)—mechanism of antibacterial action in wounds. J Invest Dermatol 127(4):945–957
Brodersen DE, Etzerodt M, Madsen P et al (1998) EF-hands at atomic resolution: the structure of human psoriasin (S100A7) solved by MAD phasing. Structure 6(4):477–489
Brodersen DE, Nyborg J, Kjeldgaard M (1999) Zinc-binding site of an S100 protein revealed. Two crystal structures of Ca2+-bound human psoriasin (S100A7) in the Zn2+-loaded and Zn2+-free states. Biochemistry 38(6):1695–1704
Gifford JL, Walsh MP, Vogel HJ (2007) Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs. Biochem J 405(2):199–221
Lee ISM, Suzuki M, Hayashi N et al (2000) Copper-dependent formation of disulfide-linked dimer of S100B protein. Arch Biochem Biophys 374(2):137–141
Harrison CA, Raftery MJ, Walsh J et al (1999) Oxidation regulates the inflammatory properties of the murine S100 protein S100A8. J Biol Chem 274(13):8561–8569
Smith RC, Reed VD (1992) Inhibition by thiols of copper(II)-induced oxidation of oxihemoglobin. Chem Biol Interact 82(2):209–217
Talmard C, Bouzan A, Faller P (2007) Zinc binding to amyloid-β: Isothermal titration calorimetry and Zn competition experiments with Zn sensors. Biochemistry 46(47):13658–13666
Maret W, Vallee BL (1998) Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc Natl Acad Sci U S A 95(7):3478–3482
Kocyla A, Pomorski A, Krężel A (2017) Molar absorption coefficients and stability constants of Zincon metal complexes for determination of metal ions and bioinorganic applications. J Inorg Biochem 176:53–65
Wolf R, Mirmohammadsadegh A, Walz M et al (2003) Molecular cloning and characterization of alternatively spliced mRNA isoforms from psoriatic skin encoding a novel member of the S100 family. FASEB J 17(13):1969–1971
Büchau AS, Hassan M, Kukova G et al (2007) S100A15, an antimicrobial protein of the skin: regulation by E. coli through Toll-like Receptor 4. J Invest Dermatol 127(11):2596–2604
Wolf R, Ruzicka T, Yuspa SH (2011) Novel S100A7 (psoriasin)/S100A15 (koebnerisin) subfamily: highly homologous but distinct in regulation and function. Amino Acids 41(4):789–796
Murray J, Tonkin ML, Whiting AL et al (2012) Structural characterization of S100A15 reveals a novel zinc coordination site among S100 proteins and altered surface chemistry with functional implications for receptor binding. BMC Struc Biol 12:16
Acknowledgment
Our current studies of psoriasin are supported by the National Science Foundation (CHE-1352132).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Cunden, L.S., Nolan, E.M. (2019). Preparation of the Oxidized and Reduced Forms of Psoriasin (S100A7). In: Heizmann, C. (eds) Calcium-Binding Proteins of the EF-Hand Superfamily. Methods in Molecular Biology, vol 1929. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9030-6_24
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9030-6_24
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9029-0
Online ISBN: 978-1-4939-9030-6
eBook Packages: Springer Protocols