Specific Citrullination Causes Assembly of a Globular S100A3 Homotetramer
2007; Elsevier BV; Volume: 283; Issue: 8 Linguagem: Inglês
10.1074/jbc.m709357200
ISSN1083-351X
AutoresKenji Kizawa, Hidenari Takahara, Heinz Troxler, P. Kleinert, Urara Mochida, Claus W. Heizmann,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoS100A3 is a unique member of the Ca2+-binding S100 protein family with the highest cysteine content and affinity for Zn2+. This protein is highly expressed in the differentiating cuticular cells within the hair follicle and organized into mature hair cuticles. Previous studies suggest a close association of S100A3 with epithelial differentiation, leading to hair shaft formation, but its molecular function is still unknown. By two-dimensional PAGE-Western blot analyses using a modified citrulline antibody, we discovered that more than half of the arginine residues of native S100A3 are progressively converted to citrullines by Ca2+-dependent peptidylarginine deiminases. Confocal immunofluorescent microscopy showed that the cytoplasmic S100A3 within the cuticular layer is mostly co-localized with the type III isoform of peptidylarginine deiminase (PAD3) but not with PAD1. Recombinant PAD1 and PAD2 are capable of converting all 4 arginines in recombinant S100A3, whereas PAD3 specifically converts only Arg-51 into citrulline. Gel filtration analyses showed that either enzymatic conversion of Arg-51 in S100A3 to citrulline or its mutational substitution with alanine (R51A) promotes a homotetramer assembly. Fluorescent titration of R51A suggested that its potential Ca2+ binding property increased during tetramerization. A prototype structural model of the globular Ca2+-bound S100A3 tetramer with citrulline residues is presented. High concentrations of S100A3 homotetramer might provide the millimolar level of Ca2+ required for hair cuticular barrier formation. S100A3 is a unique member of the Ca2+-binding S100 protein family with the highest cysteine content and affinity for Zn2+. This protein is highly expressed in the differentiating cuticular cells within the hair follicle and organized into mature hair cuticles. Previous studies suggest a close association of S100A3 with epithelial differentiation, leading to hair shaft formation, but its molecular function is still unknown. By two-dimensional PAGE-Western blot analyses using a modified citrulline antibody, we discovered that more than half of the arginine residues of native S100A3 are progressively converted to citrullines by Ca2+-dependent peptidylarginine deiminases. Confocal immunofluorescent microscopy showed that the cytoplasmic S100A3 within the cuticular layer is mostly co-localized with the type III isoform of peptidylarginine deiminase (PAD3) but not with PAD1. Recombinant PAD1 and PAD2 are capable of converting all 4 arginines in recombinant S100A3, whereas PAD3 specifically converts only Arg-51 into citrulline. Gel filtration analyses showed that either enzymatic conversion of Arg-51 in S100A3 to citrulline or its mutational substitution with alanine (R51A) promotes a homotetramer assembly. Fluorescent titration of R51A suggested that its potential Ca2+ binding property increased during tetramerization. A prototype structural model of the globular Ca2+-bound S100A3 tetramer with citrulline residues is presented. High concentrations of S100A3 homotetramer might provide the millimolar level of Ca2+ required for hair cuticular barrier formation. Several EF-hand type Ca2+-binding proteins are involved in a multitude of Ca2+-dependent cellular processes. The S100 protein family is the largest subgroup of more than 20 members characterized by two highly conserved Ca2+-binding domains: a classical C-terminal EF-hand with a canonical Ca2+-binding loop and a S100 specific N-terminal EF-hand motif (1Marenholz I. Heizmann C.W. Fritz G. Biochem. Biophys. Res. Commun. 2004; 322: 1111-1122Crossref PubMed Scopus (659) Google Scholar, 2Marenholz I. Lovering R.C. Heizmann C.W. Biochim. Biophys. Acta. 2006; 1763: 1282-1283Crossref PubMed Scopus (111) Google Scholar). Extensive studies have revealed diverse functional roles of several S100 proteins in variety of cellular processes, such as cell growth and differentiation, cell cycle regulation, transcription, and signal transduction receptor activities. They are also associated with human diseases, including inflammation, brain disorders, cancer, diabetes, heart failure, and pathological conditions of the skin and hair follicle. The basic structural and functional unit of most S100 proteins was previously thought to be a noncovalently associated antiparallel dimer; however, there is increasing evidence that some members assemble into higher order oligomers, thereby conferring their biological function (3Novitskaya V. Grigorian M. Kriajevska M. Tarabykina S. Bronstein I. Berezin V. Bock E. Lukanidin E. J. Biol. Chem. 2000; 275: 41278-41286Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 4Kiryushko D. Novitskaya V. Soroka V. Klingelhofer J. Lukanidin E. Berezin V. Bock E. Mol. Cell. Biol. 2006; 26: 3625-3638Crossref PubMed Scopus (117) Google Scholar, 5Leukert N. Vogl T. Strupat K. Reichelt R. Sorg C. Roth J. J. Mol. Biol. 2006; 359: 961-9725Crossref PubMed Scopus (128) Google Scholar, 6Xie J. Burz D.S. He W. Bronstein I.D. Lednev I. Shekhtman A. J. Biol. Chem. 2007; 282: 4218-42316Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 7Ostendorp T. Leclerc E. Galichet A. Koch M. Demling N. Weigle B. Heizmann C.W. Kroneck P.M. Fritz G. EMBO J. 2007; 26: 3868-3878Crossref PubMed Scopus (201) Google Scholar). In terrestrial animals, Ca2+ is an essential divalent cation in the formation of the epithelial protective barrier by superficial tissues that are exposed to the external environment (8Kalinin A.E. Kajava A.V. Steinert P.M. BioEssays. 2002; 24: 789-800Crossref PubMed Scopus (380) Google Scholar). A specialized protein structure termed the cornified cell envelope (CE) 2The abbreviations used are: CEcornified envelopePADpeptidylarginine deiminaseMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightDTTdithiothreitolBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolMES4-morpholineethanesulfonic acidHPLChigh pressure liquid chromatographyCitcitrulline. 2The abbreviations used are: CEcornified envelopePADpeptidylarginine deiminaseMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightDTTdithiothreitolBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolMES4-morpholineethanesulfonic acidHPLChigh pressure liquid chromatographyCitcitrulline. encapsulates the corneocytes of mammalian skin epidermis and hair fiber cuticle. A number of the genes encoding the CE precursor proteins are clustered in the epidermal differentiation complex on human chromosome 1q21 (9Marenholz I. Volz A. Ziegler A. Davies A. Ragoussis I. Korge B.P. Mischke D. Genomics. 1996; 37: 295-302Crossref PubMed Scopus (103) Google Scholar) with genes for EF-hand type Ca2+-binding proteins, including both 16 S100 proteins (2Marenholz I. Lovering R.C. Heizmann C.W. Biochim. Biophys. Acta. 2006; 1763: 1282-1283Crossref PubMed Scopus (111) Google Scholar) and more than four larger peptides with an S100-like domain at the N terminus (e.g. profilaggrin and trichohyalin) (10Huber M. Siegenthaler G. Mirancea N. Marenholz I. Nizetic D. Breitkreutz D. Mischke D. Hohl D. J. Invest. Dermatol. 2005; 124: 998-1007Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Most of these genes were reported to be coordinately expressed during epithelial terminal differentiation, and the translation products were incorporated to the CE (8Kalinin A.E. Kajava A.V. Steinert P.M. BioEssays. 2002; 24: 789-800Crossref PubMed Scopus (380) Google Scholar); however, the functional role of S100 proteins and S100-like domains responsible for regulating the intracellular Ca2+ ion in the epithelial cornifying processes is poorly understood. cornified envelope peptidylarginine deiminase matrix-assisted laser desorption/ionization time-of-flight dithiothreitol 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol 4-morpholineethanesulfonic acid high pressure liquid chromatography citrulline. cornified envelope peptidylarginine deiminase matrix-assisted laser desorption/ionization time-of-flight dithiothreitol 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol 4-morpholineethanesulfonic acid high pressure liquid chromatography citrulline. Two Ca2+-dependent protein-modifying enzymes, peptidylarginine deiminase (PAD; EC 3.5.3.15), which converts peptidylarginine residues to citrulline (11Vossenaar E.R. Zendman A.J. van Venrooij W.J. Pruijn G.J. BioEssays. 2003; 25: 1106-1118Crossref PubMed Scopus (746) Google Scholar), and transglutaminase (EC 2.3.2.13), which introduces Nϵ-(γ-glutamyl)lysine isopeptide cross-links (12Eckert R.L. Sturniolo M.T. Broome A.M. Ruse M. Rorke E.A. J. Invest. Dermatol. 2005; 124: 481-492Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), are involved in the epithelial barrier formation. Although this biochemical process is believed to be precisely regulated by the intracellular Ca2+ concentration, there remains controversy regarding the mechanism by which PAD and transglutaminase could be activated in vivo, since both require a nearly millimolar level of Ca2+ to exhibit their full activities in vitro (12Eckert R.L. Sturniolo M.T. Broome A.M. Ruse M. Rorke E.A. J. Invest. Dermatol. 2005; 124: 481-492Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 18Méchin M.C. Enji M. Nachat R. Chavanas S. Charveron M. Ishida-Yamamoto A. Serre G. Takahara H. Simon M. Cell. Mol. Life Sci. 2005; 62: 1984-1995Crossref PubMed Scopus (67) Google Scholar). Several epithelial protein barrier components containing Ca2+-binding domains have been reported to be natural substrates of PAD (the mature filaggrin subunit, proteolytically processed from profilaggrin (13Tarcsa E. Marekov L.N. Mei G. Melino G. Lee S.C. Steinert P.M. J. Biol. Chem. 1996; 271: 30709-30716Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar)) or of transgutaminase (S100A7, S100A10, and S100A11 (14Robinson N.A. Eckert R.L. J. Biol. Chem. 1998; 273: 2721-2728Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 15Ruse M. Lambert A. Robinson N. Ryan D. Shon K.J. Eckert R.L. Biochemistry. 2001; 40: 3167-3173Crossref PubMed Scopus (85) Google Scholar)) in skin epidermis or of both these enzymes (trichohyalin (16Tarcsa E. Marekov L.N. Andreoli J. Idler W.W. Candi E. Chung S.I. Steinert P.M. J. Biol. Chem. 1997; 272: 27893-27901Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 17Steinert P.M. Parry D.A. Marekov L.N. J. Biol. Chem. 2003; 278: 41409-41419Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar)) in hair follicles. It has been proposed that the Ca2+ ions bound to these proteins are presented to the Ca2+-dependent protein-modifying enzymes (8Kalinin A.E. Kajava A.V. Steinert P.M. BioEssays. 2002; 24: 789-800Crossref PubMed Scopus (380) Google Scholar, 11Vossenaar E.R. Zendman A.J. van Venrooij W.J. Pruijn G.J. BioEssays. 2003; 25: 1106-1118Crossref PubMed Scopus (746) Google Scholar); however, more direct evidence is necessary to support this notion. We previously identified S00A3, which is unique in its high cysteine content (10 of 101 amino acids) (19Engelkamp D. Schäfer B.W. Mattei M.G. Erne P. Heizmann C.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6547-6551Crossref PubMed Scopus (174) Google Scholar) and Zn2+-binding property (Kd = 1.5-11 μm) (20Föhr U.G. Heizmann C.W. Engelkamp D. Schäfer B.W. Cox J.A. J. Biol. Chem. 1995; 270: 21056-21061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 21Fritz G. Heizmann C.W. Kroneck P.M. Biochim. Biophys. Acta. 1998; 1448: 264-276Crossref PubMed Scopus (39) Google Scholar), as a predominant protein present in human hair cuticles (22Kizawa K. Uchiwa H. Murakami U. Biochim. Biophys. Acta. 1996; 1312: 94-98Crossref PubMed Scopus (42) Google Scholar). The flattened cuticular layers composed of the mechanically tough but dead cells confer the physical and chemical barrier function (23Rogers G.E. Int. J. Dev. Biol. 2004; 48: 163-170Crossref PubMed Scopus (146) Google Scholar); however, little is known about the terminal differentiation processes of this specialized epithelium equipped with a thick protein envelope (8Kalinin A.E. Kajava A.V. Steinert P.M. BioEssays. 2002; 24: 789-800Crossref PubMed Scopus (380) Google Scholar). Although our previous investigation suggested that S100A3 is closely associated with the cuticular differentiation within the hair follicle (24Kizawa K. Tsuchimoto S. Hashimoto K. Uchiwa H. J. Invest. Dermatol. 1998; 111: 879-886Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 25Takizawa T. Takizawa T. Arai S. Kizawa K. Uchiwa H. Sasaki I. Inoue T. J. Histochem. Cytochem. 1999; 47: 525-532Crossref PubMed Scopus (35) Google Scholar, 26Kizawa K. Toyoda M. Ito M. Morohashi M. Br. J. Dermatol. 2005; 152: 314-320Crossref PubMed Scopus (22) Google Scholar), the very low affinity of S100A3 for Ca2+ (Kd = 4-30 mm) (20Föhr U.G. Heizmann C.W. Engelkamp D. Schäfer B.W. Cox J.A. J. Biol. Chem. 1995; 270: 21056-21061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 21Fritz G. Heizmann C.W. Kroneck P.M. Biochim. Biophys. Acta. 1998; 1448: 264-276Crossref PubMed Scopus (39) Google Scholar) cast doubt on its supposed role as an intracellular Ca2+-modulating protein. Like most S100 protein members, S100A3 has been considered to exist as a symmetric dimer even in the absence of Ca2+ (27Fritz G. Mittl P.R. Vasak M. Grütter M.G. Heizmann C.W. J. Biol. Chem. 2002; 277: 33092-33098Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 28Mittl P.R. Fritz G. Sargent D.F. Richmond T.J. Heizmann C.W. Grütter M.G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1255-1261Crossref PubMed Scopus (19) Google Scholar). Larger assemblies than dimers have been reported for S100A2 (29Koch M. Bhattacharya S. Kehl T. Gimona M. Vasak M. Chazin W. Heizmann C.W. Kroneck P.M. Fritz G. Biochim. Biophys. Acta. 2007; 1773: 457-470Crossref PubMed Scopus (45) Google Scholar), S100A4 (3Novitskaya V. Grigorian M. Kriajevska M. Tarabykina S. Bronstein I. Berezin V. Bock E. Lukanidin E. J. Biol. Chem. 2000; 275: 41278-41286Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 4Kiryushko D. Novitskaya V. Soroka V. Klingelhofer J. Lukanidin E. Berezin V. Bock E. Mol. Cell. Biol. 2006; 26: 3625-3638Crossref PubMed Scopus (117) Google Scholar), S100A8/S100A9 (5Leukert N. Vogl T. Strupat K. Reichelt R. Sorg C. Roth J. J. Mol. Biol. 2006; 359: 961-9725Crossref PubMed Scopus (128) Google Scholar, 30Teigelkamp S. Bhardwaj R.S. Roth J. Meinardus-Hager G. Karas M. Sorg C. J. Biol. Chem. 1991; 266: 13462-13467Abstract Full Text PDF PubMed Google Scholar, 31Vogl T. Leukert N. Barczyk K. Strupat K. Roth J. Biochim. Biophys. Acta. 2006; 1763: 1298-1306Crossref PubMed Scopus (136) Google Scholar, 32Korndorfer I.P. Brueckner F. Skerra A. J. Mol. Biol. 2007; 370: 887-898Crossref PubMed Scopus (198) Google Scholar), S100A12 (6Xie J. Burz D.S. He W. Bronstein I.D. Lednev I. Shekhtman A. J. Biol. Chem. 2007; 282: 4218-42316Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 33Moroz O.V. Antson A.A. Dodson E.J. Burrell H.J. Grist S.J. Lloyd R.M. Maitland N.J. Dodson G.G. Wilson K.S. Lukanidin E. Bronstein I.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 407-413Crossref PubMed Scopus (93) Google Scholar), and S100B (7Ostendorp T. Leclerc E. Galichet A. Koch M. Demling N. Weigle B. Heizmann C.W. Kroneck P.M. Fritz G. EMBO J. 2007; 26: 3868-3878Crossref PubMed Scopus (201) Google Scholar); however, little information is available regarding their molecular entities and natural assembly processes. The present investigation has revealed that specific arginine conversion catalyzed by a Ca2+-dependent PAD enzyme promotes S100A3 tetramerization and thereby confers its potential Ca2+-binding property. The location of the specifically modified site in a known three-dimensional structure of apodimeric S100A3 (27Fritz G. Mittl P.R. Vasak M. Grütter M.G. Heizmann C.W. J. Biol. Chem. 2002; 277: 33092-33098Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 28Mittl P.R. Fritz G. Sargent D.F. Richmond T.J. Heizmann C.W. Grütter M.G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1255-1261Crossref PubMed Scopus (19) Google Scholar) allowed us to hypothesize a Ca2+-bound globular structure of the S100A3 homotetramer. Extraction and Purification of S100A3 from Hair Follicles and Cuticles–About 200 beard follicles were plucked from a Japanese male using hair tweezers. In a mortar, nonkeratinized parts of follicles were crushed in 2 ml of 0.2 m Tris-HCl buffer (pH 7.6) containing 0.1 m DTT, 1 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride. After centrifugation at 14,000 × g for 15 min, supernatants were stored at -70 °C. The combined follicle extract of 20 preparations (i.e. 4000 follicles) and the extract of hair cuticles (200 mg) using 10 ml of the above extraction buffer at 37 °C for 16 h were dialyzed against 20 mm Tris, 1 mm EDTA buffer (pH 7.6) containing 10 mm DTT and then loaded onto a Q-Sepharose™ (GE Healthcare) column (1 ml) preequilibrated with the same buffer. Bound S100A3 was eluted by a linear gradient from 0 to 0.6 m NaCl. Fractions containing S100A3 were combined, and (NH4)2SO4 was added to 1.5 m and then loaded onto a Macro-Prep® t-butyl hydrophobic interaction (Bio-Rad) column (1 ml). Bound S100A3 was eluted by a linear gradient from 1.5 to 0 m (NH4)2SO4. Resultant crude S100A3 fractions were centrifugally desalted and condensed to 100 μl using Microcon® YM-3 filters (Millipore Corp., Bedford, MA). Samples preheated under reducing conditions in NuPAGE® LDS sample buffer (Invitrogen) were loaded into 4 of 10 wells of 4-12% Bis-Tris gel (7.8 × 6.3 × 0.15 cm; Invitrogen) and then electrophoresed using MES-SDS buffer at 200 V for 35 min. After staining with Rapidstain™ reagent (Calbiochem), each S100A3 band was excised and transferred to a D-tube™ 3.5 kDa cut-off dialyzer (Calbiochem). The tubes were submerged with 25 mm Tris, 200 mm glycine (pH 8.6) in an AE6100 electrophoretic apparatus (Atto, Tokyo, Japan). S100A3 was eluted from a gel slice at 100 V for 45 min, followed by 1 min of reversed electric current. Purified S100A3 aliquots were combined and condensed after supplementation of DTT to 5 mm and then subjected to electrospray ionization-mass spectrometric analyses (see supplemental method). Citrulline Contents in S100A3–Peptidylcitrullines in S100A3 were detected by Western blot analyses of two-dimensional polyacrylamide gels using an antibody to citrulline residues (34Senshu T. Akiyama K. Kan S. Asaga H. Ishigami A. Manabe M. J. Invest. Dermatol. 1995; 105: 163-169Abstract Full Text PDF PubMed Scopus (128) Google Scholar). Samples were loaded onto immobilized pH gradient strips with pH range 3-5 (Sigma) by in-gel rehydration. Isoelectric focusing was performed at 200 V for 20 min, 450 V for 15 min, 750 V for 15 min, and 2000 V for 30 min. The second dimension was performed with precast NuPAGE® 4-12% Bis-Tris Zoom gel (7.8 × 6.3 × 0.1 cm; Invitrogen) using MES-SDS buffer at 200 V for 35 min. Citrulline residues were chemically modified to form a ureido group adduct in 0.0125% FeCl3, 2.3 m H2SO4, 1.5 m H3PO4, 0.25% 2,3-butanedione monoxime, 0.125% antipyrine, and 0.25% acetic acid on the protein-transferred polyvinylidene difluoride membrane prefixed with 1% glutaraldehyde at 37 °C for 16 h. An anti-citrulline (modified) detection kit (Upstate Biotechnology, Inc., Lake Placid, NY) was employed according to the supplier's manual. The pI of each S100A3 spot reflects the exact number of peptidylcitrulline residues. Relative intensities of silver-stained S100A3 spots were measured using image-analyzing software (Scion). The background of a nonspot area was subtracted from each signal intensity (I) value. The conversion rate was calculated via the equation for the acetylated native form (conversion (%) = (N0 × IpI 4.5/Itotal + N1 × IpI 4.3/Itotal + N2 × IpI 4.1/Itotal + N3 × IpI 3.9/Itotal + N4 × IpI 3.8/Itotal)/4 × 100 (where N0, N1, N2, N3, and N4 represent the forms with 0, 1, 2, 3, and 4 converted arginines, respectively)) and for the recombinant modified by PAD enzymes (conversion (%) = (N0 × IpI 4.7/Itotal + N1 × IpI 4.5/Itotal + N2 × IpI 4.3/Itotal + N3 × IpI 4.1/Itotal + N4 × IpI 4.0/Itotal)/4 × 100). Confocal Immunofluorescent Microscopy–Plucked beard follicles were fixed in 4% paraformaldehyde in phosphate-buffered saline for 16 h and embedded in paraffin. Sections with 6-μm thickness were deparaffinized and rehydrated, and then antigens were retrieved by heating to 95 °C in a modified citrate buffer (S1700; Dakocytomation, Carpinteria, CA) for 30 min. Polyclonal rabbit antibodies raised by us against the multiple antigen peptides of PAD3 (a partial sequence, 233DKVSYEVPRLHGDEER248) and a commercially available antibody to PAD1 from Covalab (Lyon, France), both purified by their own antigen-bound affinity columns, were employed. Commercially available rabbit antibody immunized with recombinant PAD2 from Shima Laboratory (Tokyo, Japan) was used after preabsorption with recombinant PAD1 and PAD3. The pretreated sections were reacted with diluted primary rabbit antibody to human PAD1 (5 μg/ml), PAD2 (15 μg/ml), or PAD3 (5 μg/ml). All reaction steps were performed for 45 min at room temperature, followed by three rinses with phosphate-buffered saline. Sections were then incubated with 10 μg/ml of Alexa Fluor® 488-labeled goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR). Subsequently, biotinylated antibody to human S100A3 (5 μg/ml), prepared using an EZ-link® NHS-PEO solid phase biotinylation kit (Pierce) (26Kizawa K. Toyoda M. Ito M. Morohashi M. Br. J. Dermatol. 2005; 152: 314-320Crossref PubMed Scopus (22) Google Scholar), was applied to detect the second target antigen. Finally, sections were incubated with 10 μg/ml Alexa Fluor® 594-streptavidin conjugate (Molecular Probes). Images were acquired and processed using an LSM510 fluorescent confocal microscope and its accompanying software (Carl Zeiss AG, Göttingen, Germany). In Vitro Modification of S100A3–Active recombinant human PAD1, PAD2, and PAD3 enzymes were prepared according to the previously reported procedures (18Méchin M.C. Enji M. Nachat R. Chavanas S. Charveron M. Ishida-Yamamoto A. Serre G. Takahara H. Simon M. Cell. Mol. Life Sci. 2005; 62: 1984-1995Crossref PubMed Scopus (67) Google Scholar, 35Kanno T. Kawada A. Yamanouchi J. Yosida-Noro C. Yoshiki A. Shiraiwa M. Kusakabe M. Manabe M. Tezuka T. Takahara H. J. Invest. Dermatol. 2000; 115: 813-823Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Conventionally, 1 μg of recombinant S100A3 was reacted with 25 milliunits of PAD enzymes in 20 μl of 100 mm Tris-HCl buffer (pH 7.5) containing 10 mm CaCl2 and 5 mm DTT at 37 °C. One unit was defined as the amount of PAD able to citrullineate 1 μmol of benzoyl-l-arginine ethyl ester in the same reaction buffer for 1 h at 55 °C (35Kanno T. Kawada A. Yamanouchi J. Yosida-Noro C. Yoshiki A. Shiraiwa M. Kusakabe M. Manabe M. Tezuka T. Takahara H. J. Invest. Dermatol. 2000; 115: 813-823Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Peptide Mapping–After purification on a Superdex 75 column to remove the enzymes, recombinant S100A3 modified by three recombinant PAD enzymes (25 milliunits/μg S100A3) at 37 °C for 24 h and native S100A3 purified from the hair follicle were reduced and alkylated with a half-volume of 1 m iodoacetamide dissolved in 3 m Tris-HCl (pH 8.4) for 15 min. After blocking unreacted reagent with DTT, the reaction mixture was passed through a NAP™ 5 column preequilibrated with 5 mm Tris-HCl (pH 8.0). Concentrated modified S100A3 was digested with endoproteinase Lys-C (Roche Applied Science) in 25 mm Tris-HCl containing 1 mm EDTA (pH 8.0) at 37 °C overnight. 6 μl of the Lys-C digest were loaded on a reversed-phase capillary HPLC column (PepMap C18, 0.3 × 150 mm; LC Packings). The peptides were separated with the following HPLC program: (i) isocratic flow at 5% B (v/v) for 5 min; (ii) linear gradient from 5 to 20% B in 5 min; (iii) linear gradient from 20 to 60% B in 20 min; (iv) linear gradient from 60 to 100% B in 5 min; (v) isocratic flow at 100% B for 10 min. Solvents A and B were 0.1% trifluoroacetic acid and 80% CH3CN in 0.07% trifluoroacetic acid, respectively. The flow rate was 4 μl/min, and peptides were detected at 220 nm. Peptides eluting from the HPLC column were collected onto a 600-μm AnchorChip™ target (Bruker Daltonics, Leipzig, Germany), using a Probot micro-fraction collector (LC Packings). Fractions were collected in 20-s intervals. 1.3 μl of matrix solution was added to each fraction. A 1:5 dilution of saturated α-cyano-4-hydroxycinnamic acid in 33% CH3CN, 0.1% trifluoroacetic acid in ethanol/acetone (2:1) was used as matrix solution. Mass mapping was performed with an Autoflex® MALDI-TOF mass spectrometer (Bruker Daltonics). Peptides were analyzed in the positive ion mode with delayed extraction (70 ns) and the following voltages: source, 19 kV; extraction, 16.55 kV; lens, 8.35 kV; reflector, 20 kV. Mutated S100A3 Production–The point mutation for Arg/Ala substitution in human S100A3 was introduced by PCR using the QuikChange® II mutagenesis system (Stratagene) with pMal-c2-S100A3 (20Föhr U.G. Heizmann C.W. Engelkamp D. Schäfer B.W. Cox J.A. J. Biol. Chem. 1995; 270: 21056-21061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) as a template according to the instruction manual. Briefly, the mutagenic primer sets used were as listed below, with modified bases shown underlined: for R3A, 5′-GAGTGAGGATGGCCGCGCCTCTGGAGCAGG-3′ and 5′-CCTGCTCCAGAGGCGCGGCCATCCTCACTC-3′; for R22A, 5′-CCAGGAATACGCAGGGGCCTGTGGGGACAAATAC-3′ and 5′-GTATTTGTCCCCACAGGCCCCTGCGTATTCCTGG-3′; for R51A, 5′-ACCTGGACCCCGACTGAGTTTGCGGAATGTGACT-3′ and 5′-AGTCACATTCCGCAAACTCAGTCGGGGTCCAGGT-3′; and for R77A, 5′-CTTTGTGGAGTATGTGGCCTCACTTGCCTGCCTC-3′ and 5′-GAGGCAGGCAAGTGAGGCCACATACTCCACAAAG-3′. The resulting mutated plasmids containing staggered nicks were processed for transformation after the template plasmid digestion by DpnI. S100A3 and its mutated proteins were expressed as maltose-binding fusion protein in Escherichia coli and purified under anaerobic conditions, as previously described (21Fritz G. Heizmann C.W. Kroneck P.M. Biochim. Biophys. Acta. 1998; 1448: 264-276Crossref PubMed Scopus (39) Google Scholar). Apparent Molecular Mass Analyses–The native apparent molecular mass of S100A3 in solution was determined by size exclusion chromatography using a Superdex™ 75 10/300 GL column (GE Healthcare) in 50 mm Tris buffer containing 150 mm KCl, 1 mm DTT, and various concentrations of CaCl2 or EGTA (pH 7.5) at a flow rate of 0.5 ml/min. The column was standardized with the low molecular mass calibration proteins (GE Healthcare). Titration of Ca2+-induced Conformational Change–Fluorescent titration of the single Trp residue of recombinant S100A3 and its mutated proteins (2 μm) was carried out in 50 mm Tris buffer containing 150 mm KCl and 1 mm DTT (pH 7.5), as previously described (20Föhr U.G. Heizmann C.W. Engelkamp D. Schäfer B.W. Cox J.A. J. Biol. Chem. 1995; 270: 21056-21061Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 21Fritz G. Heizmann C.W. Kroneck P.M. Biochim. Biophys. Acta. 1998; 1448: 264-276Crossref PubMed Scopus (39) Google Scholar). Emission fluorescent intensities at 340 nm (5-nm slit) excited at 295 nm (10-nm slit) after cumulative additions of CaCl2 were recorded using an RF5000 spectrofluorophotometer (Shimazu, Kyoto, Japan). Citrullinated S100A3 Derived from Hair Follicles and Cuticles–We purified the native form of S100A3 protein derived from beard follicles and matured hair cuticles (Fig. 1 and Table 1). It was difficult to estimate S100A3 content in the matured cuticles, since only a small percentage of the cuticular proteins were extracted. Total soluble protein in the nonkeratinized cells of plucked follicles was estimated to be 10% of the wet mass, and S100A3 content was found to be about 3% of this. This tissue expression level is highest among known abundant S100 proteins (e.g. S100B represents about 0.5% of total cellular brain protein). Given that S100A3 is found almost only in differentiating cuticular cells, which account for less than 10% of the follicular tissue, this indicates that in the condensed (dehydrated) cuticular cells, it could reach high, even millimolar, concentrations.TABLE 1Purification of human S100A3 from beard follicle and hair cuticlePurification stepFrom 4000 beard folliclesFrom 200 mg of hair cuticlesbSeparated from about 20 g of hair fiber.AmountaMeasured densitometrically with recombinant S100A3 as a standard.YieldAmountYieldμg%μg%Crude extract307294Anion exchange chromatography2247317359Hydrophobic interaction chromatography1685410636Electric elution6621238a Measured densitometrically with recombinant S100A3 as a standard.b Separated from about 20 g of hair fiber. Open table in a new tab Different S100A3 elution profiles from both anion exchange and hydrophobic interaction chromatography (Fig. 1B) suggested that its molecular characteristics are altered during the cuticular differentiation. Two-dimensional PAGE-Western blots probed by anti-S100A3 antibody showed that the proportions of the isoelectric variants were different for cuticular and follicular S100A3 but did not change during purification steps (Fig. 2A). Although several spots are found in follicle and cuticle derivatives, electrospray ionization-mass spectrometry revealed a single peak with mass 11,624 Da for the S100A3 purified from hair follicle and two peaks (11,624 and 11,640 Da) for the mature cuticle derivative, consistent with the calculated mass of the native form (
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