Proteases play a pivotal role in epidermal differentiation and desquamation. Separation of a total protein extract from human reconstructed epidermis by two-dimensional gel electrophoresis and subsequent peptide analysis of a specific protein spot identified a new protein exhibiting similarities with the retroviral aspartic protease family. Cloning of the corresponding full-length cDNA revealed an open reading frame encoding for a new protease of 343 amino acids, containing a putative aspartic protease catalytic domain. We named this protein Skin ASpartic Protease (SASPase). RT-PCR and northern blot analysis of various human tissues revealed that SASPase was specifically expressed within the epidermis. Immunohistochemical analysis showed a particularly intense expression restricted to the granular layers, whereas in diseased skin, its expression was changed. Western blot analysis, using a monoclonal antibody, revealed the expression of two forms of the enzyme: a 28 kDa putative proform and the active 14 kDa form. Recombinant truncated SASPase (SASP28) was generated from a prokaryotic expression system in Escherichia coli as a fusion protein with GST. SASP28 degraded insulin and to a lesser extent casein with a pH optimum of 5. As seen for retroviral proteases, an auto-activation processing was evidenced, generating a 14 kDa protein (SASP14). Site-directed mutagenesis inhibited auto-activation of the enzyme. Indinavir, a potent HIV protease inhibitor used in AIDS therapy, had a significant inhibitory effect on rSASPase auto-activation, which could explain its side effects on skin.
Desquamation is described as a protease-dependent phenomenon where serine proteases with a basic pH optimum play a key role. Recently proteases with an acidic pH optimum were identified in the stratumcorneum and associated with desquamation, e.g., cathepsin D and the stratum corneum thiol protease. The purpose of this study was to investigate if human stratum corneum contains proteases different from the above, exhibiting similar properties. After gel filtration, we identified four distinct proteolytic activities in a human stratum corneum extract, a cathepsin-E-like activity (80 kDa), a cathepsin-D activity (40 kDa), a yet unknown cathepsin-L-like form (28 kDa) exhibiting the highest caseinolytic activity, and a chymotrypsin-like protein (24 kDa) containing the acidic activity of the well described stratum corneum chymotryptic enzyme. We named the new 28 kDa protease stratum corneum cathepsin-L-like enzyme. Characterization of stratum corneum cathepsin-L-like enzyme provided clear evidence that this new protease, despite its membership to the cathepsin-L-like family, is distinct from cathepsin L and from the recently described stratum corneum thiol protease. Its ability to hydrolyze corneodesmosin, a marker of corneocyte cohesion, was in favor of a role of stratum corneum cathepsin-L-like enzyme in the desquamation process. A more detailed analysis did not allow us to identify stratum corneum cathepsin-L-like enzyme at the molecular level but revealed that stratum corneum thiol protease is identical with the recently described cathepsin L2 protease. Reverse transcription polymerase chain reaction studies and the use of a specific antibody revealed that, in contrast to earlier reports, expression of stratum corneum thiol protease in human epidermis is not related to keratinocyte differentiation. Our results indicate that the stratum corneum thiol protease is probably expressed as a pro-enzyme in the lower layers of the epidermis and in part activated by a yet unidentified mechanism in the upper layers during keratinocyte differentiation.
A protein exhibiting endoglycosidase activity was purified from plantar stratum corneum to apparent homogeneity in two sequential column chromatographic steps. Protein sequencing revealed its identity with the recently cloned human heparanase 1, an enzyme, the expression of which is reported to be related to the metastasic potential of tumor cells. By using a heparanase 1 specific antibody we were able to demonstrate that, in the plantar stratum corneum, heparanase 1 exists in two forms, the active 50 kDa protein and the inactive 63 kDa form, probably a proform of the enzyme. The antibody also decorated numerous degradation fragments. Reverse transcription polymerase chain reaction studies as well as immunohistochemical analysis using reconstructed and normal human epidermis demonstrated clearly a keratinocyte differentiation related expression of heparanase 1. Interestingly, the antibody also strongly decorated dendritic cells, which after double labeling could be identified to be a subpopulation of the epidermal Langerhans cells. Based on our findings and the known history of this enzyme, we advanced the hypothesis that heparanase 1 has multiple physiologic functions in the epidermis: (i) it plays an important role in epidermal differentiation, possibly by modulating the liberation of heparan sulfate bound (growth) factors; (ii) in the stratum corneum, the endoglycosidase activity of heparanase 1 might be indispensable and represent the first step in the desquamation process; and (iii) in Langerhans cells, its catalytic activity is required for the trans-tissue migration of these cells.
In atopic dermatitis (AD), the skin barrier is disturbed, and the expression of calcium-dependent S100 proteins and the calcium gradient is also altered in the epidermis. The calmodulinlike skin protein (CLSP), which is expressed in the differentiated epidermis, is believed to modulate the function of calciumdependent proteins involved in barrier formation and is significantly increased in the epidermis of psoriatic patients. We, therefore, investigated the CLSP level in skin biopsies taken from patients with acute exacerbated and non-exacerbated AD as well as from healthy control subjects. Immunohistochemical, Western blot and ELISA analyses showed significant increases (P < 0.03) in CLSP level in the epidermis from patients with acute exacerbated AD as compared to that from patients with non-exacerbated AD and from control subjects. Such increased expression of CLSP may help re-establish a functional epidermal barrier in acute AD.Abbreviations: AD, atopic dermatitis; CLSP, calmodulin-like skin protein; AEAD, acute exacerbated AD; NEAD, non-exacerbated AD.
Skin aspartic acid protease (SASPase) is believed to be a key enzyme involved in filaggrin processing during epidermal terminal differentiation. Since little is known about the regulation of SASPase function, the aim of this study was to identify involved protein partners in the process. Yeast two hybrid analyses using SASPase as bait against a human reconstructed skin library identified that the N-terminal domain of filaggrin 2 binds to the N-terminal fragment of SASPase. This interaction was confirmed in reciprocal yeast two hybrid screens and by Surface Plasmon Resonance analyses. Immunohistochemical studies in human skin, using specific antibodies to SASPase and the N-terminal domain of filaggrin 2, showed that the two proteins partially co-localized to the stratum granulosum. In vitro enzymatic assays showed that the N-terminal domain of filaggrin 2 enhanced the autoactivation of SASPase to its 14 kDa active form. Taken together, the data suggest that the N-terminal domain of filaggrin 2 regulates the activation of SASPase that may be a key event upstream of filaggrin processing to natural moisturizing factors in the human epidermis.
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