Identification and Characterization of a Novel Retroviral-Like Aspartic Protease Specifically Expressed in Human Epidermis

Abstract

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.

Mouse Homologue of Skin-specific Retroviral-like Aspartic Protease Involved in Wrinkle Formation

“…2). Processing sites of recombinant human SASPase determined from the in vitro autoprocessing product of recombinant protein are also denoted by arrows (13). Note that the processing sites on either side of the protease domain are completely conserved between mouse and human SASPase.…”

ICAM1+ neutrophils promote chronic inflammation via ASPRV1 in B cell–dependent autoimmune encephalomyelitis

“…The substrate and function of ASPRV1 were previously known only in the context of stratified epithelia (e.g., the stratum corneum of the skin), where it processes profilaggrin and enhances skin hydration (8,9,11,12). We have now found that ASPRV1 is also expressed in the immune system solely by neutrophils and promotes chronic inflammation in EAE.…”

“…The results suggest that neutrophils, in EAE, exert macrophage-like functions, as well as a specific effect via the enzyme aspartic peptidase retroviral-like 1 (ASPRV1, also known as SASPase). ASPRV1 is synthesized as a zymogen that contains a putative transmembrane domain and a conserved catalytic domain with a key aspartic acid residue (8)(9)(10). This zymogen can undergo autocleavage under slightly acidic conditions, releasing the catalytic domain that homodimerizes to form an active protease (8,9).…”

“…ASPRV1 is synthesized as a zymogen that contains a putative transmembrane domain and a conserved catalytic domain with a key aspartic acid residue (8)(9)(10). This zymogen can undergo autocleavage under slightly acidic conditions, releasing the catalytic domain that homodimerizes to form an active protease (8,9). ASPRV1 has so far only been detected in stratified epithelia, where it cleaves its only known substrate, profilaggrin (8,9,11,12).…”

“…This zymogen can undergo autocleavage under slightly acidic conditions, releasing the catalytic domain that homodimerizes to form an active protease (8,9). ASPRV1 has so far only been detected in stratified epithelia, where it cleaves its only known substrate, profilaggrin (8,9,11,12). Its knockdown or overexpression causes no major physiological defect (although the skin of adult ASPRV1-deficient mice shows fine wrinkles and reduced hydration) (9,11,12).…”