Title: A Potential Role for Multiple Tissue Kallikrein Serine Proteases in Epidermal Desquamation
Abstract: Desquamation of the stratum corneum is a serine protease-dependent process. Two members of the human tissue kallikrein (KLK) family of (chymo)tryptic-like serine proteases, KLK5 and KLK7, are implicated in desquamation by digestion of (corneo)desmosomes and inhibition by desquamation-related serine protease inhibitors (SPIs). However, the epidermal localization and specificity of additional KLKs also supports a role for these enzymes in desquamation. This study aims to delineate the probable contribution of KLK1, KLK5, KLK6, KLK13, and KLK14 to desquamation by examining their interactions, in vitro, with: 1) colocalized SPI, lympho-epithelial Kazal-type-related inhibitor (LEKTI, four recombinant fragments containing inhibitory domains 1–6 (rLEKTI(1–6)), domains 6–8 and partial domain 9 (rLEKTI(6–9′)), domains 9–12 (rLEKTI(9–12)), and domains 12–15 (rLEKTI(12–15)), secretory leukocyte protease inhibitor, and elafin and 2) their ability to digest the (corneo)desmosomal cadherin, desmoglein 1. KLK1 was not inhibited by any SPI tested. KLK5, KLK6, KLK13, and KLK14 were potently inhibited by rLEKTI(1–6), rLEKTI(6–9′), and rLEKTI(9–12) with Ki values in the range of 2.3–28.4 nm, 6.1–221 nm, and 2.7–416 nm for each respective fragment. Only KLK5 was inhibited by rLEKTI(12–15) (Ki = 21.8 nm). No KLK was inhibited by secretory leukocyte protease inhibitor or elafin. Apart from KLK13, all KLKs digested the ectodomain of desmoglein 1 within cadherin repeats, Ca2+ binding sites, or in the juxtamembrane region. Our study indicates that multiple KLKs may participate in desquamation through cleavage of desmoglein 1 and regulation by LEKTI. These findings may have clinical implications for the treatment of skin disorders in which KLK activity is elevated. Desquamation of the stratum corneum is a serine protease-dependent process. Two members of the human tissue kallikrein (KLK) family of (chymo)tryptic-like serine proteases, KLK5 and KLK7, are implicated in desquamation by digestion of (corneo)desmosomes and inhibition by desquamation-related serine protease inhibitors (SPIs). However, the epidermal localization and specificity of additional KLKs also supports a role for these enzymes in desquamation. This study aims to delineate the probable contribution of KLK1, KLK5, KLK6, KLK13, and KLK14 to desquamation by examining their interactions, in vitro, with: 1) colocalized SPI, lympho-epithelial Kazal-type-related inhibitor (LEKTI, four recombinant fragments containing inhibitory domains 1–6 (rLEKTI(1–6)), domains 6–8 and partial domain 9 (rLEKTI(6–9′)), domains 9–12 (rLEKTI(9–12)), and domains 12–15 (rLEKTI(12–15)), secretory leukocyte protease inhibitor, and elafin and 2) their ability to digest the (corneo)desmosomal cadherin, desmoglein 1. KLK1 was not inhibited by any SPI tested. KLK5, KLK6, KLK13, and KLK14 were potently inhibited by rLEKTI(1–6), rLEKTI(6–9′), and rLEKTI(9–12) with Ki values in the range of 2.3–28.4 nm, 6.1–221 nm, and 2.7–416 nm for each respective fragment. Only KLK5 was inhibited by rLEKTI(12–15) (Ki = 21.8 nm). No KLK was inhibited by secretory leukocyte protease inhibitor or elafin. Apart from KLK13, all KLKs digested the ectodomain of desmoglein 1 within cadherin repeats, Ca2+ binding sites, or in the juxtamembrane region. Our study indicates that multiple KLKs may participate in desquamation through cleavage of desmoglein 1 and regulation by LEKTI. These findings may have clinical implications for the treatment of skin disorders in which KLK activity is elevated. As the outermost layer of the skin, the stratum corneum functions as the body's main protective barrier against physical and chemical damage, dehydration, and microbial pathogens. Inter-corneocyte cohesion within the stratum corneum depends primarily on corneodesmosomes, structurally modified desmosomes (1Brody I. Acta Derm. Venereol. 1968; 48: 290-302PubMed Google Scholar, 2Skerrow C.J. Clelland D.G. Skerrow D. J. Cell Sci. 1989; 92: 667-677PubMed Google Scholar, 3Serre G. Mils V. Haftek M. Vincent C. Croute F. Reano A. Ouhayoun J.P. Bettinger S. Soleilhavoup J.P. J. Invest. Dermatol. 1991; 97: 1061-1072Abstract Full Text PDF PubMed Google Scholar). Akin to classical desmosomes, corneodesmosomes maintain tissue integrity and mediate cell adhesion via calcium-dependent interactions between two families of desmosomal cadherins, the desmogleins (DSG1–4) 2The abbreviations used are: DSG, desmoglein; KLK, human tissue kallikrein gene; KLK, human tissue kallikrein protein; LEKTI, lymphoepithelial Kazal-type related inhibitor; NE, neutrophil elastase; NS, Netherton syndrome; SKALP, skin-derived antileukoproteinase; SLPI, secretory leukocyte proteinase inhibitor; SPI, serine protease inhibitor; SPINK5, serine protease inhibitor kazal-type 5; TBS-T, Tris-buffered saline-Tween; WAP, whey acid protein; trappin, transglutaminase substrate and wap domain containing protein; AMC, 7-Amino-4-methylcoumarin; Boc, t-butoxycarbonyl; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. and desmocollins 1–3 (4Green K.J. Gaudry C.A. Nat. Rev. Mol. 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During normal stratum corneum desquamation, the most superficial corneocytes are shed from the skin surface. This process requires proteolysis of the corneodesmosomal adhesion molecules DSG1 (8Lundstrom A. Egelrud T. J. Invest. Dermatol. 1990; 94: 216-220Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 9Suzuki Y. Koyama J. Moro O. Horii I. Kikuchi K. Tanida M. Tagami H. Br. J. Dermatol. 1996; 134: 460-464Crossref PubMed Scopus (74) Google Scholar), desmocollin-1 (10King I.A. Tabiowo A. Fryer P.R. J. Cell Biol. 1987; 105: 3053-3063Crossref PubMed Scopus (16) Google Scholar), and corneodesmosin (11Simon M. Jonca N. Guerrin M. Haftek M. Bernard D. Caubet C. Egelrud T. Schmidt R. Serre G. J. Biol. Chem. 2001; 276: 20292-20299Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) likely mediated by both trypsin-like and chymotrypsin-like serine proteases (9Suzuki Y. Koyama J. Moro O. Horii I. Kikuchi K. Tanida M. Tagami H. Br. J. 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Once active, both enzymes have been shown to concertedly digest DSG1, desmocollin-1, and corneodesmosin, in vitro (11Simon M. Jonca N. Guerrin M. Haftek M. Bernard D. Caubet C. Egelrud T. Schmidt R. Serre G. J. Biol. Chem. 2001; 276: 20292-20299Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 24Caubet C. Jonca N. Brattsand M. Guerrin M. Bernard D. Schmidt R. Egelrud T. Simon M. Serre G. J. Invest. Dermatol. 2004; 122: 1235-1244Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 25Descargues P. Deraison C. Prost C. Fraitag S. Mazereeuw-Hautier J. D'Alessio M. Ishida-Yamamoto A. Bodemer C. Zambruno G. Hovnanian A. J. Invest. Dermatol. 2006; 126: 1622-1632Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In addition to KLKs 5 and 7, accumulating reports suggest that other kallikreins are candidate desquamation-related enzymes, based on their epidermal localization and substrate specificity. For instance, kallikrein 1, 4, 6, 8, 9, 10, 11, 13, and 14 transcripts and/or proteins are also expressed in the stratum granulosum (26Komatsu N. Takata M. Otsuki N. Toyama T. Ohka R. Takehara K. Saijoh K. J. Invest. Dermatol. 2003; 121: 542-549Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 27Kuwae K. Matsumoto-Miyai K. Yoshida S. Sadayama T. Yoshikawa K. Hosokawa K. Shiosaka S. Mol. Pathol. 2002; 55: 235-241Crossref PubMed Scopus (38) Google Scholar, 28Komatsu N. Saijoh K. Toyama T. Ohka R. Otsuki N. Hussack G. Takehara K. Diamandis E.P. Br. J. Dermatol. 2005; 153: 274-281Crossref PubMed Scopus (121) Google Scholar), and kallikrein 6, 8, 10, 11, 13, and 14 protein levels have been quantified in the stratum corneum (29Komatsu N. Saijoh K. Sidiropoulos M. Tsai B. Levesque M.A. Elliott M.B. Takehara K. Diamandis E.P. J. Invest. Dermatol. 2005; 125: 1182-1189Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Interestingly, Stefansson et al. (30Stefansson K. Brattsand M. Ny A. Glas B. Egelrud T. Biol. Chem. 2006; 387: 761-768Crossref PubMed Scopus (61) Google Scholar) have recently shown that KLK14 is responsible for ∼50% of the total trypsin-like serine protease activity in the stratum corneum. Moreover, because KLK14 can activate and be activated by KLK5, it may also participate in the cascade pathway (23Brattsand M. Stefansson K. Lundh C. Haasum Y. Egelrud T. J. Invest. Dermatol. 2005; 124: 198-203Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Because desquamation is a serine protease-dependent process (9Suzuki Y. Koyama J. Moro O. Horii I. Kikuchi K. Tanida M. Tagami H. Br. J. Dermatol. 1996; 134: 460-464Crossref PubMed Scopus (74) Google Scholar, 12Lundstrom A. Egelrud T. J. Invest. Dermatol. 1988; 91: 340-343Abstract Full Text PDF PubMed Scopus (91) Google Scholar), it is regulated by serine protease inhibitors (SPIs). 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The predicted activity of LEKTI against trypsin-like serine proteases based on the predominance of basic P1 residues within its inhibitory domains, the colocalization of LEKTI with multiple trypsin-like kallikrein serine proteases within the stratum granulosum and stratum corneum (22Ishida-Yamamoto A. Deraison C. Bonnart C. Bitoun E. Robinson R. O'Brien T.J. Wakamatsu K. Ohtsubo S. Takahashi H. Hashimoto Y. Dopping-Hepenstal P.J. McGrath J.A. Iizuka H. Richard G. Hovnanian A. J. Invest. Dermatol. 2005; 124: 360-366Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 31Komatsu N. Takata M. Otsuki N. Ohka R. Amano O. Takehara K. Saijoh K. J. Invest. Dermatol. 2002; 118: 436-443Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 33Hachem J.P. Wagberg F. Schmuth M. Crumrine D. Lissens W. Jayakumar A. Houben E. Mauro T.M. Leonardsson G. Brattsand M. Egelrud T. Roseeuw D. Clayman G.L. Feingold K.R. Williams M.L. Elias P.M. J. Invest. 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Although the (chymo)trypsin-like substrate specificity and epidermal localization of many KLKs other than KLK5 and KLK7 are compatible with a function in the terminal stages of epidermal turnover, their role as desquamatory proteases has not been investigated. Thus, in an attempt to delineate the possible involvement of multiple KLKs in desquamation, this study examines the interactions between KLK1, KLK5, KLK6, KLK13, and KLK14 with: 1) epidermal SPI (LEKTI, SLPI, and elafin/SKALP) and 2) the (corneo)desmosomal cadherin DSG1, in vitro. Materials—7-Amino-4-methylcoumarin (AMC) was purchased from Sigma-Aldrich. AMC peptide substrates Boc-Val-Pro-Arg-AMC (VPR-AMC), H-Pro-Phe-Arg-AMC (PFR-AMC), and Boc-Gln-Ala-Arg-AMC (QAR-AMC) were purchased from Bachem Bioscience (King of Prussia, PA), and methoxysuccinyl-Ala-Ala-Pro-Val-AMC (AAPV-AMC) was obtained from Calbiochem. All substrates were diluted to a final concentration of 80 mm in Me2SO and stored at –20 °C. Recombinant mature KLK1 and KLK6 were expressed and purified from a baculovirus/insect cell line system as previously described (43Laxmikanthan G. Blaber S.I. Bernett M.J. Scarisbrick I.A. Juliano M.A. Blaber M. Proteins. 2005; 58: 802-814Crossref PubMed Scopus (58) Google Scholar, 44Bernett M.J. Blaber S.I. Scarisbrick I.A. Dhanarajan P. Thompson S.M. Blaber M. J. Biol. Chem. 2002; 277: 24562-24570Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) (kind gifts from Dr. M. Blaber, Florida State University). Recombinant pro-KLK5, pro-KLK13, and mature KLK14 were produced using the Easyselect™ Pichia pastoris expression system (Invitrogen) as described in detail elsewhere (45Michael I.P. Sotiropoulou G. Pampalakis G. Magklara A. Ghosh M. Wasney G. Diamandis E.P. J. Biol. Chem. 2005; 280: 14628-14635Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 46Sotiropoulou G. Rogakos V. Tsetsenis T. Pampalakis G. Zafiropoulos N. Simillides G. Yiotakis A. 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El Naggar A.K. Bebok Z. Clayman G.L. Arch. Biochem. Biophys. 2005; 435: 89-102Crossref PubMed Scopus (18) Google Scholar, 49Jayakumar A. Kang Y. Mitsudo K. Henderson Y. Frederick M.J. Wang M. El Naggar A.K. Marx U.C. Briggs K. Clayman G.L. Protein Expr. Purif. 2004; 35: 93-101Crossref PubMed Scopus (39) Google Scholar, 50Raghunath M. Tontsidou L. Oji V. Aufenvenne K. Schurmeyer-Horst F. Jayakumar A. Stander H. Smolle J. Clayman G.L. Traupe H. J. Invest. Dermatol. 2004; 123: 474-483Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Recombinant neutrophil elastase (NE) and elafin were purchased from Calbiochem and Sigma-Aldrich, respectively, diluted in water to a final concentration of 0.5 g/liter, and stored at –80 °C. Recombinant SLPI and a recombinant DSG1/Fc chimera were obtained from R&D Systems Inc. (Minneapolis, MN), reconstituted in phosphate-buffered saline (pH 7.4) to final concentrations of 0.5 g/liter and 0.25 g/liter, respectively, and stored at –80 °C. Anti-LEKTI monoclonal antibodies 1C11G6 and 1D6G8 were produced as previously described (50Raghunath M. Tontsidou L. Oji V. Aufenvenne K. Schurmeyer-Horst F. Jayakumar A. Stander H. Smolle J. Clayman G.L. Traupe H. J. Invest. Dermatol. 2004; 123: 474-483Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Kinetic Inhibition Assays—The putative inhibitory effect of LEKTI domains 1–6, 6–9′, 9–12, and 12–15, SLPI, and elafin on multiple KLKs was assessed by measuring residual protease activity against AMC peptide substrates after incubation with individual inhibitors. The inhibition of NE by SLPI and elafin was used as a positive control. Assays were performed using optimal substrates (PFR-AMC for KLK1; VPR-AMC for KLK5, KLK6, and KLK13; QAR-AMC for KLK14; and AAPV-AMC for NE) and buffer conditions (KLK1: 0.1 m Tris-HCl, 0.1 m NaCl, 0.01% Tween-20, pH 8.0; KLK5, KLK13, and KLK14: 0.1 m sodium phosphate, 0.01% Tween 20, pH 8.0; KLK6: 50 mm Tris, 0.1 m NaCl, 0.2% bovine serum albumin, pH 7.3; and neutrophil elastase: 0.2 m Tris-HCl, 0.01% Tween-20, pH 7.5) (45Michael I.P. Sotiropoulou G. Pampalakis G. Magklara A. Ghosh M. Wasney G. Diamandis E.P. J. Biol. Chem. 2005; 280: 14628-14635Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 51Chao J. Barrett A.D. Rawlings N.D. Woessner J.F. 2nd Ed. Handbook of Proteolytic Enzymes. 2. Elsevier Academic Press, London2004: 1577-1580Google Scholar, 52Magklara A. Mellati A.A. Wasney G.A. Little S.P. Sotiropoulou G. Becker G.W. Diamandis E.P. Biochem. Biophys. Res. Commun. 2003; 307: 948-955Crossref PubMed Scopus (133) Google Scholar, 53Kapadia C. Yousef G.M. Mellati A.A. Magklara A. Wasney G.A. Diamandis E.P. Clin. Chim. Acta. 2004; 339: 157-167Crossref PubMed Scopus (24) Google Scholar, 54Borgono C.A. Michael I.P. Shaw J.L. Luo L.Y. Ghosh M.C. Soosaipillai A. Grass L. Katsaros D. Diamandis E.P. J. Biol. Chem. 2006; (November 16, 2006)10.1074/jbc.M608348200Google Scholar). Proteases (final concentration of 12 nm for KLK1, KLK5, KLK13, KLK14, and NE or 30 nm for KLK6) were preincubated with LEKTI fragments (0–60 nm), SLPI (0–1.2 μm), and/or elafin (0–1.2 μm) in 10 μl of optimal buffer at 25 °C (for LEKTI reactions) or 37 °C (for SLPI and elafin reactions) with gentle agitation for different time points (10 min for KLK1 and KLK6, 5 min for KLK6, 1 min for KLK5, and 10 s for KLK14 with LEKTI fragments; 30 min for KLK1, KLK6, and KLK13, 5 min for KLK6 and KLK14, and 1 min for NE with SLPI and elafin). The mixtures were subsequently added to 90 μl of optimal buffer containing several fixed AMC peptide concentrations ranging from 4 to 3000 μm within polystyrene microtiter plate wells. Initial rates of protease-mediated peptide hydrolysis were monitored by measuring free AMC fluorescence on the Wallac 1420 Victor2TM fluorometer (PerkinElmer Life Sciences) with excitation and emission filters of 380 and 480 nm, respectively, at 1-min intervals for 20 min at 37 °C. Protease-free reactions, for each substrate concentration, were used as negative controls, and the background counts obtained were subtracted from each value. A standard curve was constructed using known concentrations of AMC to convert rates of reaction from AMC fluorescence counts/min to free AMC produced/min. The slope of the resultant AMC standard curve was 19.184 AMC fluorescence counts/nm AMC. The rate changes (nanomolar AMC/min) of inhibited and control reactions were determined from the velocity plots. Activities were expressed relative to control incubations from which inhibitors were excluded. Average IC50 values, i.e. the inhibitor concentration required for 50% inhibition of protease activity, were determined by non-linear regression analysis using Prism (Version 4.0, GraphPad, San Diego, CA). Michaelis-Menten parameters (Km and Vmax), the equilibrium inhibition constant (Ki), and inhibitory mechanisms were determined by linear and non-linear regression analysis using the Enzyme Kinetics Module 1.1 (Sigma Plot, SSPS, Chicago, IL). All experiments were performed in triplicate and repeated at least twice. In Vitro Digestion of LEKTI and SLPI by KLKs—LEKTI (10 ng) or SLPI (500 ng) were incubated separately with KLK1, KLK5, KLK6, KLK13, and KLK14 (1 ng for LEKTI reactions; 50 ng for SLPI reactions) in a final optimal buffer volume of 20 μl for different time points ranging from 0 to 24 h at 37 °C with shaking. Control reactions, i.e. KLKs, LEKTI, and SLPI incubated alone, were also performed. Reactions were terminated by freezing in liquid nitrogen and were subsequently resolved by SDS-PAGE using the NuPAGE Bis-Tris electrophoresis system and 4–12% gradient pre-cast polyacrylamide gels under reducing conditions at 200 V for 45 min (Invitrogen). For KLK-SLPI reactions, protein mixtures were visualized by silver staining with the Silver Xpress kit (Invitrogen), according to the manufacturer's instructions. For KLK-LEKTI reactions, proteins were transferred to Hybond-C Extra nitrocellulose membrane (Amersham Biosciences) at 30 V for 1 h, blocked in Tris-buffered saline-Tween (TBS-T; 0.1 mol/liter Tris-HCl buffer (pH 7.5) containing 0.15 mol/liter NaCl and 0.1% Tween 20) supplemented with 5% nonfat dry milk overnight at 4 °C and probed with anti-LEKTI monoclonal antibody 1C11G6 (for rLEKTI(1–6), -(6–9′), and -(9–12)) or 1D6G8 (for rLEKTI(12–15)), both diluted 1:1,000 in TBS-T, for 1 h at room temperature. Membranes were washed three times for 15 min with Tris-buffered saline-Tween and treated with alkaline phosphatase-conjugated goat anti-mouse antibody (1:10,000 in Tris-buffered saline-Tween; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature. Finally, the membranes were washed again as above, and fluorescence was detected on x-ray film using a chemiluminescent substrate (Diagnostic Products Corp., Los Angeles, CA). Effect of KLKs on SLPI Activity—Prior to measuring the inhibitory activity of SLPI against NE, individual KLKs were incubated with SLPI at several molar ratios (i