Identification and characterization of a novel retroviral-like aspartic protease specifically expressed in human epidermis.
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.
Bernard
D
D
L'Oréal Recherche, Clichy, France. dbernard@rd.loreal
Méhul
B
B
Thomas-Collignon
A
A
Delattre
C
C
Donovan
M
M
Schmidt
R
R
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004817:Epidermis / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D006801:Humans; D019469:Indinavir / Q000494:pharmacology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000032:analysis; D012190:Retroviridae / Q000201:enzymology*
D017320:HIV Protease Inhibitors; D012333:RNA, Messenger; D019469:Indinavir; D016282:Aspartic Acid Endopeptidases; C503513:skin aspartic protease, human