Specific Citrullination Causes Assembly of a Globular S100A3 Homotetramer

Abstract

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. 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 a millimolar level of Ca2+ to their in (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, M. R. S. M. A. G. M. Cell. Mol. 2005; PubMed Scopus Google Scholar). Several epithelial protein barrier Ca2+-binding domains have reported to be of PAD mature profilaggrin E. G. G. Steinert P.M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google or of and R.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. N. D. R.L. PubMed Scopus Google in skin epidermis or of both these E. J. E. Steinert P.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P.M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google in hair that the Ca2+ to these proteins are to the Ca2+-dependent protein-modifying (8Kalinin A.E. Kajava A.V. Steinert P.M. BioEssays. 2002; 24: 789-800Crossref PubMed Scopus (380) Google Scholar, E.R. Zendman A.J. van Venrooij W.J. Pruijn G.J. BioEssays. 2003; 25: 1106-1118Crossref PubMed Scopus (746) Google Scholar); however, more evidence is to this previously which is unique in its high cysteine content of D. Heizmann C.W. S. A. PubMed Scopus Google Scholar) and property Heizmann C.W. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. Heizmann C.W. Kroneck P.M. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), as a protein in human hair K. Biochim. Biophys. Acta. 1996; PubMed Scopus Google Scholar). The cuticular of the but cells the and barrier function J. Biol. 2004; PubMed Scopus Google Scholar); however, is the terminal differentiation processes of this specialized with a protein envelope (8Kalinin A.E. Kajava A.V. Steinert P.M. BioEssays. 2002; 24: 789-800Crossref PubMed Scopus (380) Google Scholar). Although suggested that S100A3 is associated with the cuticular differentiation within the hair follicle K. S. K. J. Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. S. K. I. T. J. PubMed Scopus Google Scholar, K. M. M. M. J. Dermatol. 2005; PubMed Scopus Google Scholar), the affinity of S100A3 for Ca2+ Heizmann C.W. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. Heizmann C.W. Kroneck P.M. Biochim. Biophys. Acta. PubMed Scopus Google Scholar) on its role as an intracellular most S100 protein S100A3 to as a in the of Ca2+ G. M. Heizmann C.W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Fritz G. Heizmann C.W. Biol. 2002; PubMed Scopus Google Scholar). than have reported for M. S. T. M. M. W. Heizmann C.W. Kroneck P.M. Fritz G. Biochim. Biophys. Acta. 2007; PubMed Scopus Google Scholar), (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), N. Vogl T. Strupat K. Reichelt R. Sorg C. Roth J. J. Mol. Biol. 2006; 359: 961-9725Crossref PubMed Scopus (128) Google Scholar, S. Roth J. G. M. Sorg C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. N. K. Strupat K. Roth J. Biochim. Biophys. Acta. 2006; 1763: PubMed Scopus Google Scholar, A. J. Mol. Biol. 2007; PubMed Scopus Google Scholar), 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, Lukanidin E. Bronstein Biol. 2002; PubMed Scopus Google Scholar), and 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, is regarding their molecular and processes. The revealed that specific arginine conversion by a Ca2+-dependent PAD promotes S100A3 and thereby its potential Ca2+-binding The of the specifically modified in a structure of S100A3 G. M. Heizmann C.W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Fritz G. Heizmann C.W. Biol. 2002; PubMed Scopus Google Scholar) to a Ca2+-bound globular structure of the S100A3 and of S100A3 and were a using hair In a of were in of and at for were at The follicle of 20 and the of hair using of the at for 16 were 20 and a with the S100A3 was by a to S100A3 were and was to and a S100A3 was by a to S100A3 were and to using conditions in were into 4 of of and using at for with S100A3 was and to a The were with in an S100A3 was a at for by of S100A3 were and of to and to analyses in in S100A3 were by blot analyses of two-dimensional using an to citrulline residues T. K. S. A. M. J. Invest. Dermatol. Full Text PDF PubMed Scopus (128) Google Scholar). were with by was at for 20 for for and for The was with using at for residues were modified to a in and acid on the with at for 16 was to the The of S100A3 the number of of S100A3 were using The of a was signal The conversion was the for the native and the with and 4 converted and for the recombinant modified by PAD Confocal were in in for 16 and in with were and and were by to in a modified for by the peptides of PAD3 and a to PAD1 both by their affinity were with recombinant PAD2 was used with recombinant PAD1 and The were with to human PAD1 PAD2 or PAD3 were for at by with were with of to human S100A3 using an K. M. M. M. J. Dermatol. 2005; PubMed Scopus Google Scholar), was to the were with were and using an and its In of recombinant human and PAD3 were to the previously reported M. R. S. M. A. G. M. Cell. Mol. 2005; PubMed Scopus Google Scholar, T. A. J. C. A. M. M. M. T. J. Invest. Dermatol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of recombinant S100A3 was with of PAD in 20 of and at unit was as the of PAD to of in the for at T. A. J. C. A. M. M. M. T. J. Invest. Dermatol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). on a to the enzymes, recombinant S100A3 modified by recombinant PAD at for and native S100A3 the hair follicle were and with a of in for with the was a with modified S100A3 was with in at of the were on a The peptides were with the at for to in 20 to in 20 to in at for A and were acid and in The was 4 and peptides were at the were a using a were in of was to A of acid in acid in was used as was with an were in the ion with and the 20 S100A3 for substitution in human S100A3 was by using the with Heizmann C.W. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) as a to the the used were as with modified for and for and for and and for and The were for the by S100A3 and its proteins were expressed as protein in and as previously G. Heizmann C.W. Kroneck P.M. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). native molecular of S100A3 in was by chromatography using a in and concentrations of or at a of The was with the molecular proteins of titration of the of recombinant S100A3 and its proteins was in and as previously Heizmann C.W. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. Heizmann C.W. Kroneck P.M. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). at at of were using an S100A3 and the native of S100A3 protein and hair and was to S100A3 content in the since only a of the cuticular proteins were protein in the cells of was to be of the and S100A3 content was to be of This level is highest S100 proteins (e.g. of cellular brain that S100A3 is only in differentiating cuticular which for than of the this that in the cuticular could of human S100A3 follicle and hair of hair 20 of hair with recombinant S100A3 as a with recombinant S100A3 as a 20 of hair in a S100A3 both and chromatography suggested that its molecular are during the cuticular PAGE-Western by showed that the of the were for cuticular and S100A3 but not during Although several are in follicle and revealed a with for the S100A3 hair follicle and two and for the mature with the of the native by an K. T. M. M. Heizmann C.W. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar) and its The of S100A3 be by of as in hair K. T. M. Heizmann C.W. J. 2005; Google Scholar). In this we that Ca2+-dependent PAD converted more than half of the arginine residues in S100A3 to citrullines within the hair cuticular an for the modified citrulline T. K. S. A. M. J. Invest. Dermatol. Full Text PDF PubMed Scopus (128) Google Scholar), we S100A3 protein citrullines the S100A3 4 arginine and the follicle of and and whereas arginine residues in the mature S100A3 were converted to in their and A at in the follicle of arginines were conversion of S100A3 were for follicle and for the S100A3 to be during cuticular PAD S100A3 in the mammalian is reported to be by and PAD3 R. S. M. G. M. J. Invest. Dermatol. 2005; 124: Full Text Full Text PDF PubMed Scopus Google Scholar) the S. A. R. G. M. 2004; PubMed Scopus Google Scholar). In hair PAD3 is in the cuticular cells S100A3 K. S. K. J. Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. S. K. I. T. J. PubMed Scopus Google Scholar, K. M. M. M. J. Dermatol. 2005; PubMed Scopus Google Scholar) as as in the and the with T. A. J. C. A. M. M. M. T. J. Invest. Dermatol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. G. J. M. J. Invest. Dermatol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). PAD with S100A3 in order to the role of PAD in S100A3 microscopy of showed that S100A3 is in the cuticular cells but also in some cells the in PAD with cuticular differentiation were using specific of PAD to the S100A3 signal as but the of is PAD1 was in the differentiating hair cuticular and cells to a in the The with PAD1 were the and cytoplasmic of PAD1 in the differentiating cells to be associated with hair as for the epidermis M. R. S. M. A. G. M. Cell. Mol. 2005; PubMed Scopus Google Scholar, R. S. M. G. M. J. Invest. Dermatol. 2005; 124: Full Text Full Text PDF PubMed Scopus Google Scholar). PAD2 was as the differentiation of all hair epithelial however, as cellular differentiation PAD2 in the and co-localized with S100A3 in the cuticular and the differentiating cells within the and cuticular and but a PAD3 signal was the in the follicle and hair as previously reported T. A. J. C. A. M. M. M. T. J. Invest. Dermatol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of PAD3 was at an cuticular differentiation than for PAD2 and co-localized with PAD2 and PAD3 to be responsible for the of S100A3 within the cuticular of S100A3 on of human S100A3 protein was with the recombinant PAD in and the of the arginine by was The number of residues in S100A3 on the as by two-dimensional PAD1 and PAD2 are capable of converting of arginine residues in S100A3, whereas the conversion by PAD3 was PAD3 the highest for specific in PAD were to arginine at Ca2+ in S100A3 than for and recombinant M. R. S. M. A. G. M. Cell. Mol. 2005; PubMed Scopus Google Scholar). PAD2 and PAD3 that S100A3 in required higher Ca2+ concentrations for for PAD2 and for than by PAD1 suggest that millimolar Ca2+ concentrations are to PAD2 and PAD3 in cuticular The conversion of arginine in S100A3 were a of the S100A3 all peptides native and by of S100A3 revealed citrulline peptides at to their increased K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of these peptides the of the molecular of the peptides by of converted arginine The of the modified revealed that both PAD1 and PAD2 converted and Arg-51 and In with conversion was for Arg-51 and for or but not at specific conversion of Arg-51 in native S100A3 hair follicle is the of PAD3 in vivo, we were to the conversion of in the mature Ca2+-dependent S100A3 by Ca2+-dependent PAD is to structural and on the proteins by and or (11Vossenaar E.R. Zendman A.J. van Venrooij W.J. Pruijn G.J. BioEssays. 2003; 25: 1106-1118Crossref PubMed Scopus (746) Google Scholar). In this we that S100A3 the and that the follicle and recombinant S100A3 modified by PAD in have higher molecular in the of Ca2+ A chromatography using globular protein showed molecular with the for S100A3 tetramer S100A3 a either in the or of structure in a larger molecular than the the tetramer to the at Ca2+ concentrations not or the of in the is that S100A3 in a Ca2+-dependent to the N. Vogl T. Strupat K. Reichelt R. Sorg C. Roth J. J. Mol. Biol. 2006; 359: 961-9725Crossref PubMed Scopus (128) Google Scholar) and 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). In order to the of of 4 arginine residues in S100A3 on its Ca2+-binding property and we with by of the in S100A3 Heizmann C.W. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. Heizmann C.W. Kroneck P.M. Biochim. Biophys. Acta. PubMed Scopus Google Scholar) showed that the of R51A and at Ca2+ concentrations than in the but the Ca2+ concentrations for the of and were not Ca2+-binding of R51A and showed and in affinity with the type protein Gel filtration analyses in the of Ca2+ showed that and all a whereas R51A and a a tetramer the recombinant S100A3 modified by in could only assemble into to by Ca2+ the was to precisely their Ca2+-binding the Ca2+ required for the of S100A3 proteins with R51A their for Ca2+ binding are to be the S100A3 its Ca2+-binding property in conditions there is of that Arg-51 conversion in (e.g. follicle of Arg-51 in all S100A3 to be for its tetramerization. of a Ca2+ but is in vivo, the tetramer is in an in In recombinant S100A3 modified by PAD enzymes, Arg-51 was converted to by all PAD In the of of the conversion of and and substitution of these residues with alanine not either or we that citrulline residues in S100A3 converted in than are to S100A3 tetramerization. on the biological function of several S100 proteins capable of into larger than have increased in but the this not for S100 Although S100A3 to assemble as or higher at Ca2+ concentrations Heizmann C.W. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), is not there are that S100A3 tetramer formation in In this we for the that an S100A3 during human hair cuticular PAD have reported to the conversion of arginine residues in and proteins into citrulline residues in the of Ca2+ (11Vossenaar E.R. Zendman A.J. van Venrooij W.J. Pruijn G.J. BioEssays. 2003; 25: 1106-1118Crossref PubMed Scopus (746) Google Scholar). Previous that this the protein structure of such as in the skin epidermis and in the hair follicle E. G. G. Steinert P.M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), in and is into E. G. G. Steinert P.M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), whereas and to by Ca2+-dependent transglutaminase E. J. E. Steinert P.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P.M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In S100A3 into a This of S100A3 to be a to its into the of the mature T. T. S. K. I. T. J. PubMed Scopus Google Scholar). Although the functional of converted the order of the responsible PAD with we a functional role of a specific conversion of Arg-51 in S100A3 by which promotes S100A3 tetramerization. Arg-51 within the is not conserved in the acid of human S100 this is specific to type of reported with S100 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, of the of S100A3 essential for its an on its of assembly. on the structure of G. M. Heizmann C.W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Fritz G. Heizmann C.W. Biol. 2002; PubMed Scopus Google Scholar), we a prototype model of the S100A3 tetramer Arg-51 is not involved in function in the its conversion not the Ca2+-binding property of intracellular Ca2+ level to millimolar is still for S100A3 binding to In the S100A3 a of on the by III and we that the with an the of the the of Most S100 proteins have reported to III and a Ca2+ binding (1Marenholz I. Heizmann C.W. Fritz G. Biochem. Biophys. Res. Commun. 2004; 322: 1111-1122Crossref PubMed Scopus (659) Google Scholar). analyses showed that a could be with R51A at the Ca2+ that promotes its association This that the potential Ca2+-binding property of S100A3 is during its structural is to protein by S100A3, is that of S100A3 a within the The Ca2+ affinity of S100 proteins is at the intracellular Ca2+ level but be increased with their protein 37: PubMed Scopus Google Scholar). for the that the potential Ca2+-dependent role of S100A3 in could be by its a of the S100A3 Arg-51 is within its Although and on the tetramer are to by is to the structural of this a to that R51A conversion but to of the S100A3 the which a of the the of might a of within the C-terminal canonical EF-hand In the structure of the S100A3 the two are but not and are termed A and B. and is only in B. in might the of the with the Ca2+ which is in most S100 proteins, and the potential Ca2+-binding property of S100A3 tetramer and Ca2+ in the differentiating cuticular cells are to be at and millimolar showed that the millimolar of Ca2+ is for S100A3 by the PAD in In order to this is that the of Ca2+ in the S100A3 tetramer be The of cysteine residues in S100A3 the formation of complex in the cornifying cuticular cells in Although the of not an and conditions and is to the N-terminal its affinity to Ca2+ G. Heizmann C.W. Kroneck P.M. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). Ca2+ within S100A3 tetramer might be into the environment as The most this is the of the Ca2+-binding property of S100A3 during is the of Ca2+-dependent PAD is that S100A3 and Ca2+ within the Ca2+ could Ca2+-dependent protein-modifying a Ca2+ might within the cuticular cellular to its protein barrier is to the functional role of S100A3 in the epithelial S. and for and A. for of the with