Degradation of Corneodesmosome Proteins by Two Serine Proteases of the Kallikrein Family, SCTE/KLK5/Hk5 and SCCE/KLK7/Hk7

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Degradation of Corneodesmosome Proteins by Two Serine Proteases of the Kallikrein Family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7

Ce´ cile Caubet,Ã Nathalie Jonca,Ã Maria Brattsand,w Marina Guerrin,Ã Dominique Bernard,z Rainer Schmidt,z Torbjo¨ rn Egelrud,w Michel Simon,Ã and Guy SerreÃ ÃUMR5165 ‘‘Epidermis Differentiation and Rheumatoid Autoimmunity’’, CNRS-P. Sabatier University (Institut Fe´ de´ ratif de Recherche 30 and INSERM-CNRS- Universite´ P. Sabatier-Centre Hospitalier Universitaire), Toulouse, France; wDepartment of Dermatology, University Hospital, Umea˚ ,Sweden;zL’OREAL, Life Science Research, Centre Charles Zviak, Clichy, France

Corneodesmosin (CDSN), desmoglein 1 (DSG1), and desmocollin 1 (DSC1) are adhesive proteins of the extracellular part of the corneodesmosomes, the junctional structures that mediate corneocyte cohesion. The degradation of these proteins at the epidermis surface is necessary for desquamation. Two serine proteases of the kallikrein family synthesized as inactive precursors have been implicated in this process: the stratum corneum chymotryptic enzyme (SCCE/KLK7/hK7) and the stratum corneum tryptic enzyme (SCTE/KLK5/hK5). Here, we analyzed the capacity of these enzymes to cleave DSG1, DSC1, and epidermal or recombinant forms of CDSN, at an acidic pH close to that of the stratum corneum. SCCE directly cleaved CDSN and DSC1 but was unable to degrade DSG1. But incubation with SCTE induced degradation of the three corneodesmosomal components. Using the recombinant form of CDSN, either with its N-glycan chain or enzymatically deglycosylated, we also demonstrated that oligosaccharide residues do not protect CDSN against proteolysis by SCCE. Moreover, our results suggest that SCTE is able to activate the proform of SCCE. These results strongly suggest that the two kalikreins are involved in desquamation. A model is proposed for desquamation that could be regulated by a precisely controlled protease–protease inhibitor balance. Key words: cadherin/desmosomes/epidermis/kallikreins/proteinases/stratum corneum J Invest Dermatol 122:1235 –1244, 2004

Within the stratum corneum, strong inter-corneocyte cohe- proteolysed in the stratum corneum, to yield a fragment, sion is provided by corneodesmosomes, cellular junctions probably devoid of any adhesive properties, detected only derived from desmosomes (Skerrow et al, 1989; Chapman at the skin surface (Lundstrom et al, 1994; Simon et al, et al, 1991) and named corneodesmosomes (Serre et al, 2001a). Previous data evidenced a role for CDSN in 1991). Ultrastructural studies demonstrated that the degra- corneocyte cohesion and strongly suggested that its dation of corneodesmosomes is concomitant to desqua- degradation is necessary for desquamation (Lundstrom mation (Chapman and Walsh, 1990; Fartasch et al, 1993). et al, 1994; Haftek et al, 1997; Simon et al, 2001b; Jonca Moreover, proteolytic cleavage of the extracellular part of et al, 2002). Fragments of DSC1 were observed to these cell–cell adhesive structures was shown to be a key accumulate in the stratum corneum of normal epidermis event in this process, in plantar (Lundstrom and Egelrud, (King et al, 1987) and DSC1 proteolysis is reduced in the 1988) as well as non-palmoplantar stratum corneum stratum corneum of dry skin.1 The cleavage of DSG1 is also (Egelrud and Lundstrom, 1990; Suzuki et al, 1994). Three concomitant with scale shedding, and its persistence is a proteins have been described as components of the characteristic of hyperkeratosis (Lundstrom and Egelrud, extracellular part of corneodesmosomes: the two desmo- 1990; Suzuki et al, 1996). somal cadherins, desmoglein 1 (DSG1) and desmocollin 1 Molecules responsible for the cleavage of corneodes- (DSC1) that associate in a calcium-dependent manner mosomal proteins and their mode of regulation are not well (Kowalczyk et al, 1999), and corneodesmosin (CDSN), a understood. A number of different proteases of the serine, glycoprotein secreted by granular keratinocytes and then cysteine, or aspartic protease families have been identiﬁed incorporated into the desmosomes (Serre et al, 1991). in the stratum corneum and might play a role in desquama- Synthesized as a 52–56 kDa protein, CDSN is progressively tion (Suzuki et al, 1993; Horikoshi et al, 1999; Watkinson, 1999; Bernard et al, 2003, for a review see Rawlings, 2003).

Abbreviations: CDSN, corneodesmosin; DSC, desmocollin; DSG, desmoglein; MoAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; SCCE, stratum corneum chymotryptic en- 1Long S, Banks J, Watkinson A, Harding C, Rawlings AV: J zyme; SCTE, stratum corneum tryptic enzyme Invest Dermatol 106: 872 (abstract), 1996

Copyright r 2004 by The Society for Investigative Dermatology, Inc. 1235 1236 CAUBET ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Their direct involvement in degradation of the corneodes- Protogold staining of total proteins and immunoblotting mosome components, however, has not been demon- analysis with a serum directed against involucrin, another strated. Among these proteases, the stratum corneum epidermal protein, indicated that the incubation with SCTE chymotryptic enzyme (SCCE) and the stratum corneum in an acidic environment did not induce degradation of the tryptic enzyme (SCTE) (Hansson et al, 1994; Suzuki et al, major proteins of the extract, and that involucrin is not a 1994; Brattsand and Egelrud, 1999; Ekholm et al, 2000) are substrate of the enzyme (Fig 1). CDSN, however, was serine proteases of the kallikrein family named KLK7/hK7 degraded during the incubation. Among the produced and KLK5/hK5, respectively (Diamandis et al, 2000). They fragments, some were only detected by F28-27 (around are both highly expressed in granular keratinocytes and 48 and 33 kDa) and others were detected by both anti- present in the intercellular spaces of the stratum corneum CDSN MoAb (around 36 kDa). The degradation of DSC1 (Sondell et al, 1995; Ekholm et al, 2000). Synthesized as was time dependent but the protein was still detected after inactive proforms, they are both activated by cleavage of a 2 h of incubation. Surprisingly, as opposed to the results short amino-terminal domain performed by an enzyme with obtained at pH 7.2 (Simon et al, 2001a), acidic pH allowed the characteristics of a trypsin-like protease (Hansson et al, degradation of DSG during incubation with SCTE. No 1994; Brattsand and Egelrud, 1999). Moreover, glycosi- degradation was observed for the proteins tested when dases may also be involved in corneodesmosome degra- incubations were done in the absence of the enzyme (only dation at the skin surface since pre-treatment of stratum shown for CDSN). corneum biopsies with endoglycosydases allowed more Recombinant SCCE also induced complete degradation complete cell dissociation following the action of exogen- of CDSN at pH 5.6, generating fragments of 48 and 31 kDa ous proteases (Walsh and Chapman, 1991). The importance (Fig 2). The CDSN fragment of 48 kDa was stained by both of pH for stratum corneum cohesion and desquamation was MoAb G36-19 and F28-27, whereas the 31 kDa one was also recently evidenced using superbases topically applied only recognized by G36-19. Control experiments in the on hairless mouse skin (Hachem et al, 2003). absence of the active SCCE showed that CDSN remained For a better understanding of the desquamation me- intact throughout the incubation. SCCE treatment at pH 5.6 chanisms and to test the direct implication of SCCE and also induced the proteolysis of DSC1, but not of DSG and SCTE in corneodesmosome degradation, we analyzed in involucrin. vitro, the capacity of the two enzymes to cleave the To evaluate the importance of pH on the degradation of corneodesmosome extracellular proteins. In particular, we the corneodesmosome extracellular components, the im- asked whether the serine proteases, which both have an in munoreactive bands were quantified by scanning, and rates vitro neutral pH optimum when tested with synthetic of proteolysis at pH 5.6 and 7.2 were compared (Fig 3).The substrates, conserve sufficient activity at pH 5.6, close to results clearly indicated that both SCCE and SCTE induced the physiological pH of the stratum corneum (Ohman and degradations of CDSN and DSC1 with similar efficiency at Vahlquist, 1994), to be involved in normal desquamation. the acidic and neutral pH. We next investigated the influence of the CDSN carbohydrates on its cleavage by SCCE. We report that the Production of glycosylated recombinant CDSN The combined action of SCCE and SCTE allowed degradation results described above strongly suggested that CDSN, at pH 5.6 of CDSN, DSG1, and DSC1, and that sugars did DSC1 and DSG1 are substrates of SCCE and/or SCTE. An not protect CDSN against proteolysis by SCCE. A model of indirect effect of one of the enzymes mediated by activation proteolytic events necessary for desquamation is proposed of another protease already present in the epidermal involving a tight balance between proteases and protease extract, however, cannot be excluded. To test the direct inhibitors, in a particular microenvironment. action of either SCCE or SCTE without the presence of other epidermal proteases, the use of recombinant proteins becomes a necessary tool. We produced a recombinant Results form of CDSN using the EBNA-293 human cell line transfected with CDSN cDNA (Fig 4A). To compare the SCTE- and SCCE-induced proteolysis of corneodesmo- carbohydrate moiety of the recombinant and epidermal somal components at pH 5.6 Since SCCE and SCTE have CDSN forms, treatments with O- and N-glycosidase both been implicated in desquamation, we analyzed (glycopeptidases specific for serine/threonine- or aspara- whether proteins of the extracellular part of corneodesmo- gine-linked sugars, respectively) were performed. Incuba- somes are substrates for these proteases in vitro. Although tion with O-glycanase did not induce any modifications in the proteolysis of CDSN has been demonstrated during the apparent molecular mass of either form (data not incubation with SCCE and SCTE at pH 7.2 (Simon et al, shown). The epidermal, as well as the recombinant CDSN, 2001a), so far no studies had been carried out to cleave was shown to contain mainly N-linked oligosaccharides. corneodesmosome components at acidic pH, the physio- Indeed, treatment with N-glycosidase F induced a decrease logical pH of the horny layer. Epidermal proteins were of 4–5 kDa in the apparent molecular mass of the therefore treated for 2 h with these enzymes at pH 7.2 or recombinant CDSN (Fig 4B), as observed with the epider- 5.6, and the incubated extracts were analyzed by immuno- mal form. For more detailed information on the oligosac- blotting with anti-CDSN (G36-19 and F28-27 monoclonal charide composition, the recombinant CDSN was purified antibodies (MoAb)), anti-DSG (DG3.10 MoAb), and anti- by affinity chromatography and analyzed by affinodetection DSC antibodies (a MoAb specifically recognizing DSC1, and with biotinylated lectins, as previously described (Simon a serum directed against all three DSC isoforms). et al, 1997). Wheat germ and Pisum sativum agglutinins 122 : 5 MAY 2004 KALLIKREIN INVOLVEMENT IN DESQUAMATION 1237

Figure 1 SCTE-induced degradation of epidermal proteins at pH 5.6. Proteins extracted from human epidermis in pH 5.6 sodium-acetate buffer were incubated with ( þ ) or without () SCTE at pH 5.6 for increasing periods of time as indicated above the gels. After addition of Laemmli’s sample buffer to stop the reaction, proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Proteins were either stained with Protogold or immunodetected with DG3.10, G36-19, F28-27, or anti-DSC1 MoAb or with sera directed against involucrin or DSC1-3, as indicated. Arrows indicate the 52–56 kDa corneodesmosin (CDSN). Stars and open arrowheads show the immunodetected CDSN fragments, already present in the non-incubated extract or produced during incubation, respectively. The estimated molecular mass of CDSN-derived peptides (kDa) is shown on the right. The purity of the highly enriched fraction of SCTE used, separated by SDS-PAGE and stained with protogold, is shown in the lower-left inset. The position of molecular mass standards (kDa) is indicated on the left. SCTE, stratum corneum tryptic enzyme; DSC, desmocollin; MoAb, monoclonal antibodies; CDSN, corneodesmosin; DSG, desmoglein.

(specific for b-N-acetylglucosamine groups and for a-D- may be due to SCTE-mediated activation of another mannose and a-D-glucose, respectively), that were shown protease already present in the extract (see Discussion). to bind purified epidermal CDSN (Simon et al, 1997), were also found to bind the recombinant CDSN form (not shown). Absence of influence of CDSN carbohydrates on its proteolysis by SCCE Oligosaccharides can protect cor- Cleavage of the recombinant CDSN by SCCE and neodesmosomal proteins from protease degradation and influence of CDSN carbohydrates on its proteolysis The may prevent premature desquamation as suggested by recombinant CDSN was incubated at pH 5.6 with either Walsh and Chapman (1991). To assess the importance of the active SCCE or the inactive pro-SCCE for 2 h at 371C. carbohydrates in CDSN proteolysis, degradation of the The resulting proteolytic fragments were then analyzed by recombinant CDSN by SCCE at pH 5.6 was compared immunodetection with G36-19 (Fig 5A) and F28-27 (data not before and after N-glycosidase treatment. Samples were shown). Two of the fragments generated with an apparent analyzed by immunoblotting with G36-19 and F28-27 and molecular mass of about 48 and 31 kDa were detected by with 4 sera directed to various domains of CDSN (A4055, G36-19. As observed on proteolysis of native epidermal B102115,C409423,D472486). Whatever the anti-CDSN anti- CDSN, the 31 kDa CDSN fragment was only recognized by bodies used, results showed that removal of oligosacchar- G36-19, whereas the 48 kDa fragment was also stained by ides from CDSN did not modify its cleavage by SCCE (Fig 5 F28-27. Incubation with the recombinant inactive pro-SCCE and data not shown). All the fragments generated in each was without effect on the recombinant CDSN. These results case were the same after subtracting the mass of the clearly demonstrated that SCCE is able to directly cleave carbohydrate moiety (compare Fig 5A with B and Fig 5C with CDSN. D). Incubation with the recombinant inactive pro-SCCE was In opposition to the epidermal protein, the recombinant without effect on the recombinant deglycosylated CDSN, as it CDSN was not cleaved during incubation with the active was on the glycosylated CDSN either native or recombinant. SCTE at pH 5.6, even when the SCTE/protein ratio was increased by a factor of 20 (data not shown). This suggests SCTE-induced cleavage of pro-SCCE Activation of SCCE that the SCTE-induced degradation of the epidermal CDSN requires the proteolytic cleavage of its propeptide by a 1238 CAUBET ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 2 SCCE-induced degradation of epidermal proteins at pH 5.6. Proteins extracted from human epidermis in pH 5.6 sodium-acetate buffer were incubated with ( þ ) or without () SCCE at pH 5.6 for increasing periods of time as indicated above the gels. After addition of Laemmli’s sample buffer to stop the reaction, proteins were immunoblotted with G36-19, F28-27, DG3.10, anti-DSC1 MoAb and with sera directed against DSC1-3 or involucrin, as indicated. Arrows show the 52–56 kDa CDSN. Stars and open arrowheads show the immunodetected CDSN fragments, already present in the non-incubated extract or produced during incubation, respectively. The estimated molecular mass of CDSN-derived peptides (kDa) is shown on the right. The puriﬁed fraction of SCCE used, separated by SDS-PAGE and stained with protogold, is shown in the lower- right inset. The position of molecular mass standards (kDa) is indicated on the left. SCCE, stratum corneum chymotryptic enzyme; MoAb, monoclonal antibodies; DSC, desmocollin; CDSN, corneodesmosin; DSE, desmoglein.

hitherto unknown trypsin-like protease. To test whether this serum speciﬁc for the inactive enzyme, and with an anti- could be achieved by SCTE, the TENP-40 epidermal extract SCCE serum recognizing both the inactive and active forms was incubated with SCTE or with proteolysis buffer alone (Fig 6). As expected, the 31 kDa SCCE precursor was de- for 2 h, then immunodetected with the anti-pro-SCCE23–31 tected by both sera in the epidermal extract (lanes 1 and 3).

Figure 3 Rates of SCCE- and SCTE-induced degradation of CDSN and Figure 4 DSC1 at pH 5.6 or 7.2 are similar. Epidermal extracts were incubated Production of a glycosylated recombinant CDSN. (A) EBNA-293- at 371C for 5–120 min with SCCE or SCTE either at pH 7.2 or 5.6. CDSN cells were grown 3 d in medium without fetal calf serum. Samples were separated by SDS-PAGE and analyzed by immunoblot- Proteins of conditioned medium or epidermal extract (TENP-40 buffer ting with monoclonal antibodies anti-DSC1 and anti-CDSN (F28-27). extract) were separated by SDS-PAGE and immunodetected with G36- Immunoreactivities of both antibodies were scanned and quantiﬁed by 19. (B) Proteins of either the conditioned medium dialyzed against densitometry. Rates of DSC1 and CDSN degradation were expressed phosphate-buffered saline or the epidermal extract were treated ( þ )or as percentages of remaining proteins as a function of time. SCCE, not () with N-glycosidase (PNGase). Then, the samples were stratum corneum chymotryptic enzyme; SCTE, stratum corneum immunoblotted with G36-19. The position of molecular mass standards tryptic enzyme; CDSN, corneodesmosin; DSC1, desmocollin 1. (kDa) is indicated on the left. CDSN, corneodesmosin. 122 : 5 MAY 2004 KALLIKREIN INVOLVEMENT IN DESQUAMATION 1239

Figure 5 CDSN proteolysis by SCCE is not inﬂuenced by the carbohydrate moiety of the protein. (A, C) Conditioned medium of EBNA-293-CDSN cells was dialyzed against phosphate-buffered saline and incubated at pH 5.6 with the active SCCE or with its inactive precursor pro-SCCE for increasing periods of time. After addition of Laemmli’s sample buffer to stop the reaction, proteins were immunoblotted with G36-19 or with the serum C409423. (B, D) The dialyzed conditioned medium (NT) was treated with N-glycosidase (PNGase) for 3 h, then incubated with the active SCCE or with the inactive pro-SCCE for increasing periods of time as indicated above the gels. Samples were immunoblotted with G36-19 or with the serum C409423 (B, D). Open arrowheads on the left show the immunodetected CDSN fragments. The position of molecular mass standards (kDa) is indicated on the left. CDSN, corneodesmosin; SCCE, stratum corneum chymotryptic enzyme.

The anti-SCCE serum indicated that incubation with SCTE led to the appearance of a 28 kDa form of SCCE (lane 4). Since this form was not immunodetected by the anti-pro-

SCCE2131 (lane 2), it very likely represents the active form of the enzyme.

Discussion

In vitro and in vivo studies have demonstrated that the cleavage of DSG1 is concomitant with scale shedding and that its persistence at the surface of the stratum corneum is a characteristic of hyperkeratosis (Lundstrom and Egelrud, 1990; Suzuki et al, 1996). Moreover, another protein of the Figure 6 Cleavage of pro-SCCE using SCTE. Proteins extracted from human corneodesmosomes, CDSN, is progressively processed by epidermis in pH 5.6 sodium-acetate buffer were incubated with ( þ )or proteolysis from the stratum granulosum up to the stratum without () SCTE at pH 5.6 for 2 h. Samples were immunoblotted with corneum surface (Simon et al, 2001a). In the last step, most anti-pro-SCCE2331 and anti-SCCE sera. Arrows indicate the pro- of the protein is degraded and the fragment remaining is a SCCE and its proteolysed form, probably corresponding to SCCE (31 and 28 kDa, respectively). The position of molecular mass standards part of its central domain, probably devoid of adhesive (kDa) is indicated on the left. SCCE, stratum corneum chymotryptic properties. Reduced proteolysis of CDSN is also observed enzyme; SCTE, stratum corneum tryptic enzyme. in xerosis and hyperkeratotic diseases (Haftek et al, 1997; Simon et al, 2001b). This study strongly suggests that the proteases SCCE and SCTE are the proteases involved in 1240 CAUBET ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY these degradation processes, supporting the hypothesis already present in the TENP-40 epidermal extract in its that they are among the actors responsible for corneocyte proform. Regarding this result, we cannot totally exclude dissociation and desquamation. Indeed, their combined that degradation of the two desmosomal cadherins DSG1 action could degrade all the molecules essential for and DSC1 during the incubations with SCTE is also due to a corneocyte cohesion. In agreement, both trypsin-like and second SCTE-activated protease. chymotrypsin-like proteases have been suggested to be Walsh and Chapman (1991) reported that in vitro, pre- involved in the proteolysis of DSG1 (Suzuki et al, 1994, 1996). treatments of stratum corneum with glycosidases render In our assays, we used 50 ng of protease for 70 mgof corneodesmosomes more sensitive to protease degrada- epidermal proteins, which is a rather low protease/substrate tion probably by making corneodesmosomal proteins more ratio, as compared to a 1:10 (w/w) ratio of hK6:extracellular accessible to proteases. The recombinant CDSN also matrix protein used by others (Bernett et al, 2002). We allowed us to test the effect of its N-glycan chain on its cannot claim, however, that the ratio is actually physiolo- proteolysis. Like the epidermal CDSN, the recombinant gically relevant. Particularly because, to our knowledge, the form is mainly N-glycosylated, detected by the same concentration of proteases in the microenvironment close lectins, and its sugar moiety also presents a molecular to the corneodesmosomes is unknown. mass of 4–5 kDa. To assess the importance of the glycan We have shown that CDSN and DSC1 are good chain, the susceptibility to SCCE degradation, at pH 5.6, of substrates for both SCCE and SCTE in vitro at pH 7.2, the glycosylated and the enzymatically deglycosylated unlike DSG (Simon et al, 2001a; Fig 3). We showed in this recombinant CDSN were compared. We demonstrated that paper that degradation of the corneodesmosome proteins the removal of the sugars does not modify SCCE cleavage by SCCE and SCTE is effective at pH 5.6, the pH of the sites on CDSN and has no incidence on the proteolysis stratum corneum. CDSN was a good substrate for SCCE at efficacy in vitro. These results are in agreement with our both pH values tested, the immunoreactive fragments previous study, showing that the larger fragments of human produced being distinct, whereas cleavage of DSC1 by this CDSN extracted from the stratum corneum are still protease seemed to be more prominent at the acidic pH. glycosylated (Simon et al, 2001a). Although carbohydrate CDSN and DSC1 were also proteolysed during incubation moieties do not protect CDSN against proteolysis by SCCE with SCTE at the two pH values, but degradation of DSG1 in vitro, they could nevertheless protect it against degrada- occurred only at the acidic pH. These results suggest that tion by other epidermal proteases. More likely, they could SCCE and SCTE have become adapted to retain activity modulate its adhesive properties. within the acidic stratum corneum intercellular space. Cleavage of its propeptide by an unknown trypsin-like Likewise, SCCE activity is assumed to have become protease is necessary for SCCE activation. Hansson et al tolerant to the dry conditions of the stratum corneum (1994) demonstrated that in vitro, tryptic digestion of a (Watkinson et al, 2001). The hydratation and pH of the recombinant form of pro-SCCE removed the propeptide stratum corneum probably constitute important elements in and yielded a proteolytically active protein with the same regulation of its enzyme activities including proteolysis, and NH2-terminal amino acid sequence as the native SCCE. therefore contribute to epidermis homeostasis (Rippke et al, SCTE has already been proposed to participate in this 2002; Hachem et al, 2003). activation but our results provide the first experimental In a recent study using in situ zymographic assays, P. evidence for its actual involvement. Indeed, we observed Elias and his team did not detect serine protease activity in that the SCCE precursor, present in the epidermal extracts the stratum corneum of hairless mouse skin. Increasing the used, is cleaved and probably activated by SCTE. In stratum corneum pH, however, induced the detection of the agreement, we observed that native as well as recombinant serine protease activity and the enhancement of cor- SCTE activate recombinant pro-SCCE but at low rates (T. neodesmosome degradation (Hachem et al, 2003). This Egelrud and M. Brattsand, unpublished data). Such a apparent discrepancy with our in vitro observation of a clear cascade of protease activation is well known in other activity of SCCE and SCTE at pH 5.6 could be explained by physiological processes, e.g. coagulation. a difference either in the sensitivity of the assays or between Considering our results and other published data, we species. Alternatively, it may suggest that, in vivo, the pH suggest that desquamation is the result of a tightly controls the activation rate of serine protease proforms regulated multi-step proteolytic event and we propose a rather than protease activity. model involving proteases and protease inhibitors in the Production of a recombinant form of CDSN using desquamation process (Fig 7). In addition to SCCE and transfected 293-EBNA cells allowed us to prove the action SCTE, it has been proposed that other proteases take part of SCCE on CDSN. Incubation at pH 5.6 of an epidermal in desquamation according to data from zymographic extract containing CDSN and the recombinant form of analyses and assays with specific protease inhibitors. The CDSN generated the same fragments. Therefore, we enzymes are two cysteine proteases, cathepsin L2/stratum conclude that SCCE acts directly without an intermediate corneum thiol protease (SCTP) stratum corneum cathepsin step. SCTE, however, was unable to degrade the recombi- L-like enzyme (Watkinson, 1996; Bernard et al, 2003), and nant CDSN in the same conditions. This could indicate that two aspartic proteases, cathepsin D-like and cathepsin E- an inhibitor of SCTE was present in the conditioned medium like (Horikoshi et al, 1998, 1999; Bernard et al, 2003). In containing the recombinant protein and was not eliminated agreement, DSC1 is degraded by partially purified SCTP during dialysis. Alternatively, the degradation of the native (Watkinson, 1996) and CDSN was recently shown to be a epidermal CDSN during incubation with SCTE may be due substrate of the stratum corneum cathepsin L-like enzyme to an indirect effect, SCTE activating another protease (Bernard et al, 2003). The importance of another cathepsin 122 : 5 MAY 2004 KALLIKREIN INVOLVEMENT IN DESQUAMATION 1241

Figure 7 Regulation of desquamation: a model. Both SCCE/KLK7 and SCTE/KLK5, which we propose to be involved in the degradation of the extracellular components of corneodesmosomes (CDSN, DSG1, and DSC1) and in desquamation, are produced as inactive proforms. They are activated by tryptic cleavage of a short amino-terminal domain. Whereas the SCTE activator is unknown – it may be an autoactivation – pro-SCCE seems to be activated by SCTE. Other proteases, mainly cathepsins, are detected in the stratum corneum and have been proposed to participate in degradation of the corneodesmosome components. At the acidic pH of the stratum corneum, the active SCCE cleaves CDSN and DSC1. The active SCTE also induces proteolysis of CDSN, DSC1, and DSG1, either directly or indirectly activating another protease. Some cathepsins (see details in Discussion) have also been shown to cleave CDSN or DSC1. Proteolytic activities of SCCE and SCTE are regulated by their chemical and biochemical microenvironment in the stratum corneum. Among these regulatory elements, elafin, secretory leukocyte protease inhibitor (SLPI), lymphoepithelial Kazal-type 5 serine protease inhibitor (LEKTI), and cystatins, inhibitors of serine and/or cysteine proteases, are probably involved in regulating desquamation. When the direct effect of a protease has not been demonstrated, the corresponding proteolytic activity is shown by an open arrow. The question marks indicate unknown protease activators. SCCE, stratum corneum chymotryptic enzyme; SCTE, stratum corneum tryptic enzyme; CDSN, corneodesmosin; DSG1, desmoglein 1; DSC1, desmocollin 1. in desquamation was also evidenced by the recent ber of the trappin protein family, has also been shown to be identification of cathepsin C missense and nonsense a weak but efficient inhibitor of SCCE and to significantly mutations, resulting in undetectable activity, in Papillon– reduce the shedding of corneocytes in vitro (Franzke et al, Lefe` vre syndrome, a periodontal disease associated with 1996). Although principally detected during inflammation palmoplantar hyperkeratosis (Toomes et al, 1999). Synthe- processes, elafin was found covalently bound to the sized as proenzymes, cathepsins also undergo proteolytic corneocyte cornified envelopes in the normal stratum maturation but the proteases involved have not been corneum (Steinert and Marekov, 1995; Schalkwijk et al, definitely identified. These cathepsins are present in the 1999). Also expressed in the upper layers of the epidermis, stratum corneum and are active in an acidic environment the lymphoepithelial Kazal-type 5 serine protease inhibitor but their real role in desquamation remains to be elucidated. (LEKTI), encoded by the SPINK-5 gene may also target Their in vitro actions on corneodesmosome proteins will be SCCE and SCTE. Mutations of SPINK-5 cause Netherthon tested in a future work. syndrome, a congenital ichthyosis characterized by a A variety of serine protease inhibitors is also present in defective inhibitory regulation of desquamation leading to the extracellular spaces of the stratum corneum and they a severe skin permeability barrier dysfunction (Chavanas et probably participate in the regulation of desquamation- al, 2000; Komatsu et al, 2002). Finally, human epidermis associated proteolysis. Among them, antileukoproteinase contains specific cysteine protease inhibitors: cystatin a (ALP) also called secretory leukocyte protease inhibitor and cystatin M/E. Interestingly, cystatin M/E expression is (SLPI) has been found, in vitro, to prevent the detachment of restricted to the epidermis (Zeeuwen et al, 2001) and a null corneocytes from human plantar epidermis and to strongly mutation in its gene causes hyperkeratotic epidermis and inhibit SCCE activity on synthetic substrates (Franzke et al, abnormal hair follicles in mice (Zeeuwen et al, 2002). This 1996). Thus, it may be a relevant human inhibitor for SCCE strongly supports the participation of cystatins in the in vivo. Interestingly, ALP can be cleaved and inactivated by physiological regulatory events of desquamation. members of the cathepsin family in vitro (Taggart et al, As a whole, our data strongly suggest that in the stratum 2001). In addition to their potential role in degradation of corneum protease activation and levels of protease in- corneodesmosomal components, cathepsins may thus take hibitors are in fine balance which is essential for skin part in the control of SCCE activity. Elafin, formerly known homeostasis. A better understanding of the proteolytic as the skin-derived-antileukoprotease (SKALP) and mem- cascade and of the regulatory mechanisms involved in 1242 CAUBET ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table I. Antibodies used for immunoblotting

Antibodies Speciﬁcity Source, reference Monoclonal antibodies G36-19 Human CDSN (amino acids 306–309) Serre et al (1991); Guerrin et al (1998) F28-27 Human CDSN (amino acids 349–354) Serre et al (1991); Guerrin et al (1998)

DG3.10 Human, mouse, and bovine DSG1 and DSG2 Progen Biotechnik GmbH

Anti-DSC1 Human DSC1 Research Diagnostic Inc. Sera

Serum A40–55 Human CDSN (amino acids 40–55) Simon et al (2001)

Serum B112–115 Human CDSN (amino acids 112–115) Simon et al (2001)

Serum C409–423 Human CDSN (amino acids 409–423) Simon et al (2001)

Serum D472–486 Human CDSN (amino acids 472–486) Simon et al (2001) Serum anti-DSC1-3 Human DSC1, DSC2, and DSC3 Serotec Ltd Serum anti-involucrin Human involucrin Biomedical Technologies Inc. Serum anti-pro-SCCE Human pro-SCCE (amino acids 23–31) Simon et al (2002) Serum anti-SCCE Human SCCE (amino acids 71–84, 87–96 and 169–183) Simon et al (2002)

desquamation should enable the design of new treatments Extraction of proteins from human epidermis Dermo-epidermal for skin disorders associated with disturbance in stratum cleavage of breast skin obtained from female undergoing plastic corneum turnover. surgery (after informed consent and in accordance with Helsinki principles) and without any history of skin diseases was performed by heat treatment. The epidermis was extracted in a Tris-EDTA Materials and Methods buffer containing Nonidet P-40 at pH 7.2 (TENP-40 buffer extract), as previously described (Simon et al, 1997). To obtain protein Antibodies Characteristics of the antibodies used in this study extracts at pH 5.6, the epidermis was extracted on ice in the are summarized on Table I. Affinity-purified polyclonal antibodies following buffer: 100 mM sodium acetate pH 5.6, 0.15 M NaCl, 0.5% Nonidet P-40. The results reported in this paper were A4055,B102115,C409423, and D472486 directed against various domains of CDSN (amino acids 40–55, 102–115, 409–423, 472–486, reproducibly obtained using at least two separate extracts respectively), and the anti-CDSN MoAb G36-19 and F28-27 were prepared from two different individuals. produced and characterized in our laboratory (Serre et al, 1991; Recombinant CDSN Guerrin et al, 1998; Simon et al, 2001a). DG3.10, a MoAb directed Sub-cloning experiments The p14.9 plasmid (Guerrin et al, 1998) to human DSG1 and DSG2, was purchased from Progen was digested with the restriction enzymes HindIII and NotI and the Biotechnik GmbH (Heidelberg, Germany). Rabbit sera directed resulting fragment containing human CDSN cDNA sequence against the three human DSC (anti-DSC1-3) and against involucrin (GenBank accession number AF030130, CDSN allele 1.71) was were purchased from Serotec (Oxford, UK) and from Biomedical sub-cloned into the pCEP4 vector (Invitrogen Corporation, Technologies Inc. (Stoughton, California), respectively. A MoAb Carlsbad, CA) giving rise to the pCEP4-CDSN plasmid. directed to human DSC1 was from Research Diagnostic Inc. (Flanders, New Jersey). Affinity-purified polyclonal antibodies Expression in mammalian cells The human embryonic kidney cell specific for the pro-SCCE or directed to both pro-SCCE and line EBNA-293 (Invitrogen) was grown in Dulbecco’s modified active SCCE were elicited in rabbits by injection of synthetic Eagle’s medium supplemented with 10% fetal calf serum (Bio peptides conjugated via a C-terminal cysteine residue to keyhole media, Toulouse, France) and 250 mg per mL Geneticin (G418 limpet hemocyanine, as reported before (Simon et al, 2001a). sulfate). Cells (2 106) were transfected using 6 mL of a non- According to the predicted amino acid sequence of the enzyme liposomal formulation (FuGENE 6 Transfection Reagent, Roche (Genbank accession number L33404) the peptide EEAQGDKIIC Molecular Biomedicals Roche Diagnostics Corporation, Indiana- (amino acids 23–31) with an added C-terminal cysteine residue polis, IN) and 2 mg of pCEP4-CDSN. Stable transfected cells was used to produce the serum anti-pro-SCCE2331, directed to (EBNA-293-CDSN) were selected and maintained in the presence the region specific for the precursor. A mixture of three synthe- of 0.2 mg per mL hygromycin. They were rinsed three times with tic peptides, with an added N-terminal cysteine residue, serum-free medium and grown in the absence of serum for 3 d. CKMNEYTVHLGSDT (amino acids 71–84), CDRRAQRIKASKSF The resulting conditioned medium was then centrifuged for 10 min (amino acids 87–99), and CKLISPQDCTKVYKDL (amino acids 169– at 3000 g to eliminate cell debris and stored at 801C. 183), was used to produce the serum anti-SCCE recognizing both inactive and active forms of SCCE. The anti-sera were affinity Recombinant SCCE and pro-SCCE Recombinant pro-SCCE was purified on their corresponding peptide(s), as already described produced using murine C127 cells, purified and activated with (Simon et al, 2001a). The specificity of the affinity-purified agarose-bound trypsin as previously described (Hanson et al, 1994). antibodies was evidenced using recombinant pro-SCCE and SCCE, and a fraction of plantar stratum corneum highly enriched Epidermal SCTE A fraction highly enriched in native SCTE and in pro-SCCE and SCCE (Simon et al, 2002). None of the sera devoid of other protease activities as detected by zymography was reacted with SCTE (data not shown). prepared as described (Brattsand and Egelrud, 1999). Briefly, 122 : 5 MAY 2004 KALLIKREIN INVOLVEMENT IN DESQUAMATION 1243 plantar stratum corneum was ground in a mortar, homogenized in Address correspondence to: Michel Simon, CNRS-UPS UMR5165 Tris-HCl buffer, pH 7 and incubated for 1 h at room temperature. Faculte´ de Me´ decine, 37 a´ llees J Guesde, 31073 Toulouse, France. The recovered pellet was extracted with 1 M acetic acid. The Email: [email protected] extract was cleared by centrifugation and filtration and subjected to a reversed phase chromatography. Fractions containing SCTE, as detected by zymography and immunoblotting, were purified by References gel exclusion in 0.2 M acetic acid and 0.3 M NaCl buffer. Bernard D, Mehul B, Thomas-Collignon A, Simonetti L, Remy V, Bernard MA, Protein electrophoresis and immunoblotting Proteins were Schmidt R: Analysis of proteins with caseinolytic activity in a human separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) stratum corneum extract revealed a yet unidentified cysteine protease and electro-transferred to reinforced nitrocellulose membranes. and identified the so-called ‘‘stratum corneum thiol protease’’ as Membranes were stained with either Ponceau Red or Protogold cathepsin l2. J Invest Dermatol 120:592–600, 2003 (British BioCell International, Cardiff, UK) and probed with Bernett MJ, Blaber SI, Scarisbrick IA, Dhanarajan P, Thompson SM, Blaber M: antibodies as previously reported (Simon et al, 1997). G36-19 Crystal structure and biochemical characterization of human kallikrein 6 reveals that a trypsin-like kallikrein is expressed in the central nervous and F28-27 were diluted to 0.1 mg per mL, DG3-10 and anti-DSC1 system. J Biol Chem 277:24562–24570, 2002 to 0.5 mg per mL. Affinity-purified anti-peptide antibodies to CDSN Brattsand M, Egelrud T: Purification, molecular cloning, and expression of a were diluted to 1/100, anti-pro-SCCE23–31 and anti-SCCE to 1/ human stratum corneum trypsin-like serine protease with possible 1000, anti-DSC1-3 to 1/2000 and anti-involucrin to 1/200. function in desquamation. J Biol Chem 274:30033–30040, 1999 Immunoreactivities were revealed with the ECL Western blotting Chapman SJ, Walsh A: Desmosomes, corneosomes and desquamation. An kit as described by the manufacturer (Amersham Pharmacia ultrastructural study of adult pig epidermis. Arch Dermatol Res 282:304– Biotech, Uppsala, Sweden). 310, 1990 Chapman SJ, Walsh A, Jackson SM, Friedmann PS: Lipids, proteins and Deglycosylation experiments Medium conditioned by EBNA- corneocyte adhesion. Arch Dermatol Res 283:167–173, 1991 293-CDSN cells was dialyzed against phosphate-buffered saline Chavanas S, Bodemer C, Rochat A, et al: Mutations in SPINK5, encoding a serine using dialysis tubing WMCO 10,000 (Pierce, Tattenhall, Cheshire, protease inhibitor, cause Netherton syndrome. Nat Genet 25:141–142, UK). Ten microgram of protein and either 2 U of N-glycosidase F 2000 (EC 3.5.1.52; Roche Molecular Biochemicals) or an equal volume Diamandis EP, Yousef GM, Luo LY, Magklara A, Obiezu CV: The new human of buffer were incubated at 371C for 3 h. The reactions were kallikrein gene family: Implications in carcinogenesis. Trends Endocrinol stopped by boiling for 2 min in sample buffer. Treated and mock Metab 11:54–60, 2000 samples were separated by SDS-PAGE and analyzed by immuno- Egelrud T, Lundstrom A: The dependence of detergent-induced cell dissociation blotting. in non-palmo-plantar stratum corneum on endogenous proteolysis. J Invest Dermatol 95:456–459, 1990 Proteolysis experiments Treatments were performed either at pH Ekholm IE, Brattsand M, Egelrud T: Stratum corneum tryptic enzyme in normal 7.2 or 5.6 as described previously (Simon et al, 2001a) with the epidermi: A missing link in the desquamation process? J Invest Dermatol 114:56–63, 2000 following modifications. When treatments were performed with Fartasch M, Bassukas ID, Diepgen TL: Structural relationship between epidermal SCTE, a solution of 0.5 M Tris was used to adjust the pH of the lipid lamellae, lamellar bodies and desmosomes in human epidermis: An protease fraction (initial pH of 2). At the neutral pH, 50 ng of ultrastructural study. Br J Dermatol 128:1–9, 1993 recombinant pro-SCCE or SCCE, or 100 ng of enriched epidermal Franzke CW, Baici A, Bartels J, Christophers E, Wiedow O: Antileukoprotease SCTE (buffered at pH 7.2) or an equivalent volume of the inhibits stratum corneum chymotryptic enzyme. Evidence for a regulative proteolysis buffer (10 mM sodium phosphate buffer, pH 7.2, 0.15 function in desquamation. J Biol Chem 271:21886–21890, 1996 M NaCl), were added to 70 mg of proteins from the TENP-40 buffer Guerrin M, Simon M, Montezin M, Haftek M, Vincent C, Serre G: Expression extract. At the acidic pH, the same amount of proteases (buffered cloning of human corneodesmosin proves its identity with the product of at pH 5.6) or an equivalent volume of the acidic proteolysis buffer the S gene and allows improved characterization of its processing during (50 mM sodium acetate buffer, pH 5.6, 0.15 M NaCl), were added keratinocyte differentiation. J Biol Chem 273:22640–22647, 1998 to 70 mg of epidermal proteins extracted at pH 5.6. To cleave the Hachem JP, Crumrine D, Fluhr J, Brown BE, Feingold KR, Elias PM: pH directly recombinant CDSN at acidic pH, conditioned medium of EBNA- regulates epidermal permeability barrier homeostasis, and stratum 293-CDSN cells, was dialyzed against acidic proteolysis buffer and corneum integrity/cohesion. J Invest Dermatol 121:345–353, 2003 70 mg of total proteins were mixed with 500 ng of SCCE or pro- Haftek M, Simon M, Kanitakis J, Marechal S, Claudy A, Serre G, Schmitt D: Expression of corneodesmosin in the granular layer and stratum corneum SCCE or with various amounts (100 ng–2 mg) of the fraction of normal and diseased epidermis. Br J Dermatol 137:864–873, 1997 enriched in epidermal SCTE. In all proteolysis experiments, Hanson L, Stromqvist M, Backman A, Wallbrandt P, Carlstein A, Egelrud T: 1 mixtures were incubated at 37 C for 2 h and reactions were Cloning, expression, and characterization of stratum corneum chymo- stopped by boiling for 2 min in sample buffer. Samples were tryptic enzyme. A skin-specific human serine proteinase. J Biol Chem separated by SDS-PAGE and analyzed by immunoblotting as 269:19420–19426, 1994 described above. The immunoblotting reactivities were scanned Horikoshi T, Arany I, Rajaraman S, et al: Isoforms of cathepsin D and human and quantified by densitometry using TotalLab v2003.2 software epidermal differentiation. Biochimie 80:605–612, 1998 (Nonlinear Dynamics, Durham, North Carolina). Horikoshi T, Igarashi S, Uchiwa H, Brysk H, Brysk MM: Role of endogenous cathepsin D-like and chymotrypsin-like proteolysis in human epidermal desquamation. Br J Dermatol 141:453–459, 1999 Jonca N, Guerrin M, Hadjiolova K, Caubet C, Gallinaro H, Simon M, Serre G: We thank Pr J.-P. Chavoin (Plastic Surgery Department, Rangueil Corneodesmosin, a component of epidermal corneocyte desmosomes, Teaching Hospital, Toulouse, France) for providing us with human skin. The technical assistance of C. Pons is gratefully acknowledged. This displays homophilic adhesive properties. J Biol Chem 277:5024–5029, 2002 King IA, Tabiowo A, Fryer PR: Evidence that major 78-44 kDa concanavalin A- study was supported in part by grants from the Universite´ Paul Sabatier-Toulouse III, from INSERM (U563 and CJF 96-02) and from binding glycopolypeptides in pig epidermis arise from the degradation of L’Ore´ al (Paris, France). C. Caubet was a recipient of a grant from the desmosomal glycoproteins during terminal differentiation. J Cell Biol French Ministry of Research and Technology. N. Jonca was supported 105:3053–3063, 1987 by a postdoctoral fellowship from the Socie´ te´ de Secours des Amis Komatsu N, Takata M, Otsuki N, Ohka R, Amano O, Takehara K, Saijoh K: des Sciences and from the Fondation pour la Recherche Me´ dicale. Elevated stratum corneum hydrolytic activity in Netherton syndrome suggests an inhibitory regulation of desquamation by SPINK5-derived DOI: 10.1111/j.0022-202X.2004.22512.x peptides. J Invest Dermatol 118:436–443, 2002 Kowalczyk AP, Bornslaeger EA, Norvell SM, Palka HL, Green KJ: Desmosomes: Manuscript received September 23, 2003; revised November 25, 2003; Intercellular adhesive junctions specialized for attachment of intermediate accepted for publication December 4, 2003 filaments. Int Rev Cytol 185:237–302, 1999 1244 CAUBET ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Lundstrom A, Egelrud T: Cell shedding from human plantar skin in vitro: Evidence Sondell B, Thornell LE, Egelrud T: Evidence that stratum corneum chymotryptic of its dependence on endogenous proteolysis. J Invest Dermatol 91:340– enzyme is transported to the stratum corneum extracellular space via 343, 1988 lamellar bodies. J Invest Dermatol 104:819–823, 1995 Lundstrom A, Egelrud T: Evidence that cell shedding from plantar stratum Steinert PM, Marekov LN: The proteins elafin, filaggrin, keratin intermediate corneum in vitro involves endogenous proteolysis of the desmosomal filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide protein desmoglein I. J Invest Dermatol 94:216–220, 1990 cross-linked components of the human epidermal cornified cell envelope. Lundstrom A, Serre G, Haftek M, Egelrud T: Evidence for a role of J Biol Chem 270:17702–17711, 1995 corneodesmosin, a protein which may serve to modify desmosomes Suzuki Y, Koyama J, Moro O, Horii I, Kikuchi K, Tanida M, Tagami H: The role of during cornification, in stratum corneum cell cohesion and desquamation. two endogenous proteases of the stratum corneum in degradation of Arch Dermatol Res 286:369–375, 1994 desmoglein-1 and their reduced activity in the skin of ichthyotic patients. Ohman H, Vahlquist A: In vivo studies concerning a pH gradient in human stratum Br J Dermatol 134:460–464, 1996 corneum and upper epidermis. Acta Derm Venereol 74:375–379, 1994 Suzuki Y, Nomura J, Hori J, Koyama J, Takahashi M, Horii I: Detection and Rawlings A: Trends in stratum corneum research and the management of dry skin conditions. Int J Cosmetic Sci 25:63–95, 2003 characterization of endogenous protease associated with desquamation Rippke F, Schreiner V, Schwanitz HJ: The acidic milieu of the horny layer: New of stratum corneum. Arch Dermatol Res 285:372–377, 1993 findings on the physiology and pathophysiology of skin pH. Am J Clin Suzuki Y, Nomura J, Koyama J, Horii I: The role of proteases in stratum corneum: Dermatol 3:261–272, 2002 Involvement in stratum corneum desquamation. Arch Dermatol Res Schalkwijk J, Wiedow O, Hirose S: The trappin gene family: Proteins defined by 286:249–253, 1994 an N-terminal transglutaminase substrate domain and a C-terminal four- Taggart CC, Lowe GJ, Greene CM, Mulgrew AT, O’Neill SJ, Levine RL, McElvaney disulphide core. Biochem J 340 (Part 3):569–577, 1999 NG: Cathepsin B, L, and S cleave and inactivate secretory leucoprotease Serre G, Mils V, Haftek M, et al: Identification of late differentiation antigens of inhibitor. J Biol Chem 276:33345–33352, 2001 human cornified epithelia, expressed in re-organized desmosomes and Toomes C, James J, Wood AJ, et al: Loss-of-function mutations in the cathepsin bound to cross-linked envelope. J Invest Dermatol 97:1061–1072, 1991 C gene result in periodontal disease and palmoplantar keratosis. Nat Simon M, Bernard D, Guerrin M, Egelrud T, Schmidt R, Serre G: Expression Genet 23:421–424, 1999 analysis of stratum corneum chymotryptic enzyme and its precursor at Walsh A, Chapman SJ: Sugars protect desmosome and corneosome glycopro- the surface of human epidermis, using affinity-purified anti-peptide teins from proteolysis. Arch Dermatol Res 283:174–179, 1991 antibodies. In: Marks R, Le´ veˆ que J-L, Voegeli R (eds). Stratum Corneum. Watkinson A: US patent 5545402, Elizabeth Arden Co., Division of Conopco Inc., London: Martin Dunitz Limited Edition, 2002; p 81–83 1996. Simon M, Bernard D, Minondo AM, et al: Persistence of both peripheral and non- Watkinson A: Stratum corneum thiol protease (SCTP): A novel cysteine protease peripheral corneodesmosomes in the upper stratum corneum of winter of late epidermal differentiation. Arch Dermatol Res 291:260–268, 1999 xerosis skin versus only peripheral in normal skin. J Invest Dermatol Watkinson A, Harding C, Moore A, Coan P: Water modulation of stratum corneum 116:23–30, 2001b chymotryptic enzyme activity and desquamation. Arch Dermatol Res Simon M, Jonca N, Guerrin M, et al: Refined characterization of corneodesmosin 293:470–476, 2001 proteolysis during terminal differentiation of human epidermis and its Zeeuwen PL, van Vlijmen-Willems IM, Hendriks W, Merkx GF, Schalkwijk J: A null relationship to desquamation. J Biol Chem 276:20292–20299, 2001a Simon M, Montezin M, Guerrin M, Durieux JJ, Serre G: Characterization and mutation in the cystatin M/E gene of ichq mice causes juvenile lethality purification of human corneodesmosin, an epidermal basic glycoprotein and defects in epidermal cornification. Hum Mol Genet 11:2867–2875, associated with corneocyte-specific modified desmosomes. J Biol Chem 2002 272:31770–31776, 1997 Zeeuwen PL, van Vlijmen-Willems IM, Jansen BJ, et al: Cystatin M/E expression Skerrow CJ, Clelland DG, Skerrow D: Changes to desmosomal antigens and is restricted to differentiated epidermal keratinocytes and sweat glands: A lectin-binding sites during differentiation in normal human epidermis: A new skin-specific proteinase inhibitor that is a target for cross-linking by quantitative ultrastructural study. J Cell Sci 92 (Part 4):667–677, 1989 transglutaminase. J Invest Dermatol 116:693–701, 2001