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.
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.
A proteomic analysis of stratum corneum (SC) samples of normal healthy skin revealed the presence of more than 70 proteins by 2D electrophoresis. The majority of these proteins to our knowledge have not yet been described in normal SC. We analysed by Western blot the levels of 25 proteins in the SC taken from postmenopausal and dry skin compared with young and normal skin, respectively. In postmenopausal skin, there was a significantly increased amount of heat shock protein 27, plakoglobin and desmoglein 1, whereas transglutaminase 3, apolipoprotein D and acid ceramidase levels were significantly reduced compared with the SC of young skin. We confirmed corneodesmosin as a marker of dry skin. In addition, we showed for the first time that the levels of both phosphatidylethanolamine-binding protein 1 and annexin A2 were significantly increased in the SC of dry skin compared with the SC of normal skin. These results suggest that a proteomic analysis of the SC obtained using a non-invasive varnish stripping method is an attractive alternative to invasive methods to better characterize changes in the physiology of ageing and dry skin.
The study aimed at detecting differentially expressed proteins in the stratum corneum of dandruff versus non-dandruff scalps to better understand dandruff aetiology. iTRAQ-based quantitative proteomic analysis revealed a total of 68 differentially expressed biomarkers. A detailed analysis of their known physiological functions provided new insights into the affected metabolic pathways of a dandruff scalp. Dandruff scalp showed (1) profound changes in the expression and maturation of structural and epidermal differentiation related proteins, that are responsible for the integrity of the skin, (2) altered relevant factors that regulate skin hydration, and (3) an imbalanced physiological protease-protease inhibitor ratio. Stratum corneum proteins with antimicrobial activity, mainly those derived from sweat and sebaceous glands were also found modified. Comparing our data with those reported for atopic dermatitis revealed that about 50 % of the differentially expressed proteins in the superficial layers of the stratum corneum from dandruff and atopic dermatitis are identical.
The calmodulin-like skin protein (CLSP) or so-called calmodulin-like protein 5, a recently discovered skin-specific calcium-binding protein, is closely related to keratinocyte differentiation. The 16-kDa protein is proteolytically degraded in the upper layers of the stratum corneum (SC) of healthy skin. With the use of specific new monoclonal antibodies to CLSP, we were able to demonstrate that the abnormal elevated levels of CLSP, characteristic of psoriatic epidermis, were probably not due to an overexpression of the protein, but most likely the result of its non-degradation. Further in vitro experiments using recombinant CLSP and in situ data clearly showed that calcium protected and chelator accelerated CLSP degradation. These data indicate that CLSP degradation in the SC of psoriatic skin might be hindered by the abnormally elevated calcium concentration. No degradation of CLSP in psoriatic epidermis keeping its ability to bind protein as transglutaminase 3 may have a physiological role in skin diseases such as psoriasis.
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