Neutral Cysteine Protease Bleomycin Hydrolase Is Essential for the Breakdown of Deiminated Filaggrin into Amino Acids
Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH2 terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from β-naphthylamide derivatives and hydrolyzed citrulline-β-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin. Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH2 terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from β-naphthylamide derivatives and hydrolyzed citrulline-β-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin. The mammalian epidermal keratinocytes arise from proliferating basal cells and move outward through a series of distinct differentiation events to form the stratum corneum (1Watt F.M. Curr. Opin. Cell Biol. 1989; 1: 1107-1115Crossref PubMed Scopus (232) Google Scholar, 2Rawlings A.V. Harding C.R. Dermatol. Ther. 2004; 17: 43-48Crossref PubMed Google Scholar). During this progressive epidermal differentiation, keratinocytes express different proteins such as keratins, profilaggrin/filaggrin, involucrin, small proline-rich proteins, loricrin, cystatin A, and elafin, which form the cornified envelope of mature corneocytes (3Wiedow O. Schroder J.M. Gregory H. Young J.A. Christophers E. J. Biol. Chem. 1990; 265: 14791-14795Abstract Full Text PDF PubMed Google Scholar, 4Hohl D. Mehrel T. Lichti U. Turner M.L. Roop D.R. Steinert P.M. J. Biol. Chem. 1991; 266: 6626-6636Abstract Full Text PDF PubMed Google Scholar, 5Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E. Welter J.F. J. Investig. Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 6Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 7Takahashi M. Tezuka T. Katunuma N. Arch. Biochem. Biophys. 1996; 329: 123-126Crossref PubMed Scopus (36) Google Scholar). Profilaggrin is synthesized as a large, extremely insoluble phosphoprotein that consists of a unique NH2-terminal Ca2+-binding protein of the S-100 family, linked to 10–20 tandem filaggrin monomer repeats (8Harding C.R. Scott I.R. J. Mol. Biol. 1983; 170: 651-673Crossref PubMed Scopus (202) Google Scholar, 9Dale B.A. Resing K.A. Lonsdale-Eccles J.D. Ann. N. Y. Acad. Sci. 1985; 455: 330-342Crossref PubMed Scopus (144) Google Scholar, 10McGrath J.A. Uitto J. Trends Mol. Med. 2008; 14: 20-27Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Each individual filaggrin repeat is completely removed by proteolysis to generate the mature filaggrin monomer (a molecular mass of 37 kDa in human). Then, filaggrin is completely degraded in the uppermost layer of the stratum corneum to produce a mixture of free and modified hygroscopic amino acids that are important for maintaining epidermal hydration (2Rawlings A.V. Harding C.R. Dermatol. Ther. 2004; 17: 43-48Crossref PubMed Google Scholar, 11Scott I.R. Harding C.R. Dev. Biol. 1986; 115: 84-92Crossref PubMed Scopus (282) Google Scholar, 12Rawlings A.V. Scott I.R. Harding C.R. Bowser P.A. J. Investig. Dermatol. 1994; 103: 731-741Abstract Full Text PDF PubMed Scopus (365) Google Scholar, 13Verdier-Sevrain S. Bonte F. J. Cosmet. Dermatol. 2007; 6: 75-82Crossref PubMed Scopus (371) Google Scholar). In addition, a number of proteins are subjected to various post-translational modifications such as disulfide bonding, N-(γ-glutamyl)-lysine isopeptide cross-linking, and deimination during the terminal differentiation of epidermal keratinocytes (4Hohl D. Mehrel T. Lichti U. Turner M.L. Roop D.R. Steinert P.M. J. Biol. Chem. 1991; 266: 6626-6636Abstract Full Text PDF PubMed Google Scholar, 6Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 14Eckert R.L. Rorke E.A. Environ. Health Perspect. 1989; 80: 109-116Crossref PubMed Scopus (128) Google Scholar, 15Lee S.C. Kim I.G. Marekov L.N. O'Keefe E.J. Parry D.A. Steinert P.M. J. Biol. Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar). Deimination is catalyzed by peptidylarginine deiminase (PAD), 2The abbreviations used are: PAD, peptidylarginine deiminase; NMF, natural moisturizing factor; Cit-MCA, citrulline-4-methylcoumaryl-7-amide; Cit-β-NA, citrulline-β-naphthylamide; BH, bleomycin hydrolase; DTT, dithiothreitol; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; FRETS, fluorescence resonance energy transfer substrate; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; MS, mass spectrometry; HPLC, high pressure liquid chromatography. which converts arginine to citrulline in proteins (17Tarcsa 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 (316) Google Scholar, 18Vossenaar E.R. Zendman A.J. Van Venrooji W.J. Pruijin G.J. BioEssays. 2003; 25: 1106-1118Crossref PubMed Scopus (801) Google Scholar, 19Chavanas S. Mechin M.C. Nachat R. Adoue V. Coudane F. Serre G. Simon M. J. Dermatol. Sci. 2006; 44: 63-72Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The modification seems essential for the processing into free amino acids including citrulline. Several proteases reportedly participate in the processing of profilaggrin. Furin, a member of the proprotein convertase family, has been proposed to cleave the NH2 terminus of profilaggrin, facilitating the release of the NH2-terminal S-100 protein (20Presland R.B. Kimball J.R. Kautsky M.B. Lewis S.P. Lo C.Y. Dale B.A. J. Investig. Dermatol. 1997; 108: 170-178Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 21Pearton D.J. Nirunsuksiri W. Rehemtulla A. Lewis S.P. Presland R.B. Dale B.A. Exp. Dermatol. 2001; 10: 193-203Crossref PubMed Scopus (84) Google Scholar). In contrast, calpain I and profilaggrin endopeptidase I (PEP-I) were implicated in the processing of the linker regions between the filaggrin monomer repeats to generate the filaggrin monomer (22Resing K.A. Walsh K.A. Haugen-Scofield J. Dale B.A. J. Biol. Chem. 1989; 264: 1837-1845Abstract Full Text PDF PubMed Google Scholar, 23Resing K.A. al-Alawi N. Blomquist C. Fleckman P. Dale B.A. J. Biol. Chem. 1993; 268: 25139-25145Abstract Full Text PDF PubMed Google Scholar, 24Resing K.A. Thulin C. Whiting K. al-Alawi N. Mostad S. J. Biol. Chem. 1995; 270: 28193-28198Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 25Yamazaki M. Ishidoh K. Suga Y. Saido T.C. Kawashima S. Suzuki K. Kominami E. Ogawa H. Biochem. Biophys. Res. Com. 1997; 235: 652-656Crossref PubMed Scopus (51) Google Scholar). Recently, significant results regarding the conversion of profilaggrin to filaggrin have been obtained with the knock-out of matriptase/MT-SP1, prostasin/channel-activating serine protease 1/Prss 8, and caspase 14 in mice (26List K. Szabo R. Wertz P.W. Segre J. Haudenschild C.C. Kim S.Y. Bugge T.H J. Cell Biol. 2003; 163: 901-910Crossref PubMed Scopus (183) Google Scholar, 27Leyvraz C. Charles R.P. Rubera I. Guitard M. Rotman S. Breiden B. Sandhoff K. Hummler E. J. Cell Biol. 2005; 170: 486-496Crossref Scopus (239) Google Scholar, 28Denecker G. Hoste E. Gilbert B. Hochepied T. Ovaere P. Lippens S. Van Den Broecke C. Van Damme P. D'Herde K. Hachem J.P. Borgonie G. Presland R.B. Schoonjans L. Libert C. Vandekerckhove J. Gevaert K. Vandenabeele P. Declercq W. Nat. Cell Biol. 2007; 9: 666-674Crossref PubMed Scopus (239) Google Scholar). These proteases were a key component of the profilaggrin-processing pathway in terminal epidermal differentiation. However, although the signal initiating the degradation of profilaggrin at a defined stage of the maturation of the stratum corneum was found to be the water gradient within the stratum corneum itself (11Scott I.R. Harding C.R. Dev. Biol. 1986; 115: 84-92Crossref PubMed Scopus (282) Google Scholar), the proteases for the processing of filaggrin and/or the deiminated form into peptides following the breakdown of these peptides to amino acids including citrulline remain unknown. In this study, we have purified a novel aminopeptidase using a deiminated substrate from rat skin homogenate and identified it as a neutral cysteine protease, bleomycin hydrolase (BH). Furthermore, we investigated the processing of the deiminated filaggrin by calpain I or caspase 14. Based on these results, we proposed that calpain I participated preferentially in the processing of deiminated filaggrin into peptides and then BH appeared essential for the breakdown of the peptides into amino acids. Materials—Recombinant human filaggrin and mouse PAD type 3 (mPAD3) were produced and purified according to the methods reported by Kanno et al. (29Kanno T. Kawada A. Yamanouchi J. Yoshida-Noro C. Yoshiki A. Shiraiwa M. Kusakabe M. Manabe M. Tezuka T. Takahara H. J. Investig. Dermatol. 2000; 115: 813-823Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and by Ohsugi et al. (30Ohsugi I. Takahara H. Shiraiwa M. Sugawara K. Arch. Biochem. Biophys. 1995; 317: 62-68Crossref PubMed Scopus (5) Google Scholar), respectively. BH was purified from the homogenate of newborn rat epidermis as described previously (31Takeda A. Higuchi D. Yamamoto T. Nakamura Y. Masuda Y. Hirabayashi T. Nakaya K. J. Biochem. (Tokyo). 1996; 119: 29-36Crossref PubMed Scopus (26) Google Scholar). Human calpain I was purchased from Calbiochem (Darmstadt, Germany). Human caspase 14 was from BIOMOL International LP (Plymouth, PA). Citrulline-β-naphthylamide (Cit-β-NA) and citrulline-4-methylcoumaryl-7-amide (Cit-MCA) were obtained from Bachem Bioscience (Bubendorf, Switzerland). Fluorescence resonance energy transfer substrate (FRETS)-25Cit libraries were commercially synthesized at Peptide Institute Inc. (Osaka, Japan). All other chemicals used were of reagent grade. Detection of Citrulline Aminopeptidase Activity by Double Layer Fluorescent layer fluorescent zymography was as described by Katunuma et al. N. R. S. Biochem. 2005; PubMed Scopus Google Scholar). The rat extract was with an of a of and and then to a was at at The was then with water and with or and at 37 for to the pH. was with in for and The was in of or DTT, and Then, the was with the fluorescent substrate to a double layer. at 37 for fluorescent activity in the double layer were using a Japan). Fluorescent were using a Japan). of an with Citrulline Aminopeptidase Activity from rat skin was with of and in a and then a in a The was by with the of insoluble the was to a of the from the with the was to a and the was with B. The proteins were with a gradient of from to in B. The were by and B. The was to a The protein was with the activity was detected by aminopeptidase activity using during the of The purified enzyme a of molecular mass 48 kDa on Deimination of Filaggrin by was with at an of at 37 for different in a of and deimination for different filaggrin was at 37 for with in The was at a of The were by according to the methods of PubMed Scopus Google Scholar) using The were with of Filaggrin with I or was deiminated at 37 at of The degradation of and deiminated filaggrin was at an of for I was at with filaggrin in and DTT, caspase 14 was at 37 with filaggrin in CHAPS, DTT, and as a for an the degradation in the mixture was detected by analysis of was by using the on a The were with the in and to the for was using a in of from with I or 14 by of were using with N. F. W. T. Full Text Full Text PDF PubMed Scopus Google Scholar). the of and deiminated filaggrin with calpain I or caspase 14 for at an of the with caspase 14 were by with a for the of and then the The were with BH in DTT, and at 37 were removed at different and the was by were with of and of of The mixture was and of was Fluorescence was The amino were using of as a a degradation with calpain I or caspase and filaggrin were removed by and the were with BH at 37 amino acids were using a amino with by gradient with according to the analysis acids with were detected by the at and were obtained from the the and The was by and by the on Human was with of human in at were in a for with and and then at with the BH and filaggrin in the in were for at with and in and in were in an a laser with at and by and respectively. were of the enzyme with citrulline aminopeptidase activity was it was with (31Takeda A. Higuchi D. Yamamoto T. Nakamura Y. Masuda Y. Hirabayashi T. Nakaya K. J. Biochem. (Tokyo). 1996; 119: 29-36Crossref PubMed Scopus (26) Google Scholar). The peptides were by a on an using a gradient of in for at a of detected by the at were in and The NH2-terminal amino were using an Detection of Citrulline Aminopeptidase Activity with Double Layer Fluorescent and endopeptidase using and in newborn rat epidermal extract by double layer fluorescent in strong citrulline aminopeptidase activity was detected in rat epidermal extract at and and rat extract was a was detected at the at as as strong activity with at at and However, endopeptidase activity libraries was detected These results the of a novel enzyme with strong citrulline aminopeptidase activity at neutral in rat epidermal and of an with Citrulline Aminopeptidase Activity from was using and in the of an enzyme with citrulline aminopeptidase activity from newborn rat epidermal The purified enzyme revealed a with a molecular mass of 48 kDa on and as a with a molecular mass of 280 kDa by These results that the enzyme with citrulline aminopeptidase activity is a We the aminopeptidase of the enzyme by activity various The enzyme released different amino acids from the a it released Pro and was the substrate for the aminopeptidase of the a between and and a of and of However, activity was found protein including deiminated filaggrin, and cystatin A. that the enzyme in the degradation of peptides in We have a and the NH2-terminal amino of tryptic peptides of the purified The of the enzyme obtained with was by as in in which six were as in of The NH2-terminal amino of the six peptides are in that tryptic and with the amino and of the sequence of rat BH L. A.J. J.F. of and Scholar). double layer fluorescent zymography was using rat BH, strong activity was detected at the the was in newborn rat epidermal extract at In addition, citrulline aminopeptidase activity was with activity to bleomycin the of from newborn rat was found that the enzyme with citrulline aminopeptidase activity was identical to BH in rat which aminopeptidase activity bleomycin hydrolase activity in the normal mammalian Furthermore, with of normal human skin to an enzyme with citrulline aminopeptidase activity using and filaggrin in the granular layer of human epidermis and Double using revealed a co-localization of BH and filaggrin in the granular layer of BH and filaggrin. for BH and filaggrin in of human skin was A, were with and of human skin with were subjected to BH and filaggrin in the granular layer. The and the of the stratum corneum and the between the epidermis and of of of Filaggrin by I or filaggrin was purified from extract of which arginine (29Kanno T. Kawada A. Yamanouchi J. Yoshida-Noro C. Yoshiki A. Shiraiwa M. Kusakabe M. Manabe M. Tezuka T. Takahara H. J. Investig. Dermatol. 2000; 115: 813-823Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). was completely deiminated with that the of arginine to the of in a of of the filaggrin on to The deiminated filaggrin an was by to the conversion from arginine to citrulline The deiminated filaggrin was to was at as from the of with a molecular mass Then, we degradation by calpain I or caspase 14. and the of the in the filaggrin and deiminated filaggrin on with calpain I or caspase 14. The proteolysis of was and of the substrate was degraded within that the proteases to have a high of and deiminated Furthermore, the deiminated filaggrin was to calpain I the filaggrin. These results calpain I to be the protease for the degradation of deiminated of the deiminated filaggrin by calpain I or caspase 14. The degradation of the and deiminated filaggrin by calpain I or caspase 14 was at an filaggrin of for in vitro. I was at with filaggrin in and DTT, caspase 14 was at 37 with it in CHAPS, DTT, and as a The of degradation was by by a the degradation of deiminated filaggrin by calpain we the at of the from and and deiminated and filaggrin degraded by calpain I. In obtained at the for and from be and for filaggrin and and for deiminated as as a few other and These and many appeared in the of the of for by calpain I that calpain I degraded the into many peptides of different mass. Furthermore, the that deiminated filaggrin was degraded into filaggrin by calpain I. the at of from and deiminated degraded by caspase 14. 14 degraded into a few peptides of limited mass with different These results that calpain I preferentially on deiminated filaggrin with caspase 14. Detection of from of Filaggrin with I or investigated the free amino acids released from calpain or caspase filaggrin peptides by BH using a and amino amino were released from the calpain filaggrin and deiminated filaggrin peptides with BH in a The peptides from deiminated filaggrin were to BH those from filaggrin. Furthermore, and deiminated filaggrin were released by BH On the other hand, few of free amino or amino acids were detected from the caspase peptides by These results suggest that calpain I and BH are essential for the breakdown of deiminated filaggrin peptides into amino acids. We identified and purified a novel enzyme with citrulline aminopeptidase activity in a rat epidermal of the is that the enzyme is identical with rat BH, a enzyme natural and substrate are unknown. that BH an important role in the synthesis of free amino acids acting as from deiminated filaggrin peptides in the stratum corneum. BH is to a and to a of proteases L. A.J. J.F. of and Scholar, P. J. Mol. Biol. 1995; PubMed Scopus Google Scholar). The and of BH that the enzyme has a However, this has for many Recently, BH was to be in the post-translational processing of peptides for L. M. G. C. H. S. H. Nat. 2000; 1: PubMed Scopus Google Scholar) and a J. M. A. H. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, BH has been to with proteins, such as in I. G. G. W. Curr. 1997; PubMed Scopus (5) Google Scholar), the human of enzyme R.P. Mol. PubMed Scopus Google Scholar), human proteins R.P. C. S.C. PubMed Scopus Google Scholar), and precursor protein R.P. J. 2000; 14: PubMed Scopus Google Scholar). In the we that BH participated in the degradation of from precursor protein A. H. T. A. 2006; PubMed Scopus Google Scholar). et al. D.R. E. J. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) that BH newborn mice a and that the of the mouse and the R.B. D. Lewis S.P. C. Fleckman P. J.P. J. Investig. 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These results that BH participated in the release of free amino acids including citrulline in peptides from deiminated filaggrin in mammalian We proposed a for the degradation of filaggrin as in are at high within corneocytes and to of the of the stratum corneum Scholar). water and in hygroscopic as (2Rawlings A.V. Harding C.R. Dermatol. Ther. 2004; 17: 43-48Crossref PubMed Google Scholar). of amino acids or derivatives such as and with and and E.J. A. J. Cosmet. Sci. PubMed Scopus Google Scholar, I.R. Harding C.R. Biophys. PubMed Scopus (239) Google Scholar). The is critical to epidermal differentiation and implicated in the and modification of proteins in the epidermis S. 1993; 14: Google Scholar, P.M. P. K. C. D. G. E. T. M.L. J. Investig. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). PAD activity was by the during epidermal differentiation, and/or participated in the deimination of filaggrin of co-localization by T. S. Ogawa H. Manabe M. H. Biochem. Biophys. Res. Com. 1996; PubMed Scopus Google Scholar, M.C. M. Nachat R. M. A. Serre G. Takahara H. Simon M. Cell Mol. Sci. 2005; PubMed Scopus Google Scholar). However, it is which is in the degradation of deiminated filaggrin into peptides and then into free amino acids. that calpain a cysteine protease in preferentially the deiminated filaggrin to peptides of molecular with filaggrin. those peptides were degraded into free amino acids by The that caspase 14 is in the processing of filaggrin, in the profilaggrin-processing pathway G. Ovaere P. Vandenabeele P. Declercq W. J. Cell Biol. 2008; PubMed Scopus Google Scholar). The revealed that caspase 14 the deiminated filaggrin into limited of at a cleavage However, was in to caspase 14 with and few of amino acids were released from caspase peptides by These results suggest that calpain I a role in the processing of deiminated filaggrin into peptides. In the results of these have identified BH as essential for the degradation of the peptides from deiminated filaggrin into amino although is to the role of BH during terminal epidermal differentiation. with