Abstract
Retroviral proteases are encoded in the retroviral genome and are responsible for maturation and assembly of infectious virus particles. A number of retroviral protease sequences with retroviral elements are integrated in every eukaryotic genome as endogenous retroviruses. Recently, retroviral-like aspartic proteases that were not embedded within endogenous retroviral elements were identified throughout the eukaryotic and prokaryotic genomes. However, the physiological role of this novel protease family, especially in mammals, is not known. During the high throughput in situ hybridization screening of mouse epidermis, as a granular layer-expressing clone, we identified a mouse homologue of SASPase (Skin ASpartic Protease), a recently identified retroviral-like aspartic protease. We detected and purified the endogenous 32-kDa (mSASP32) and 15-kDa (mSASP15) forms of mSASP from mouse stratum corneum extracts and determined their amino acid sequences. Next, we bacterially produced recombinant mSASP15 via autoprocessing of GST-mSASP32. Purified recombinant mSASP15 cleaved a quenched fluorogenic peptide substrate, designed from the autoprocessing site for mSASP32 maximally at pH 5.77, which is close to the pH of the epidermal surface. Finally, we generated mSASP-deficient mice that at 5 weeks of age showed fine wrinkles that ran parallel on the lateral trunk without apparent epidermal differentiation defects. These results indicate that the retroviral-like aspartic protease, SASPase, is involved in prevention of fine wrinkle formation via activation in a weakly acidic stratum corneum environment. This study provides the first evidence that retroviral-like aspartic protease is functionally important in mammalian tissue organization.
Highlights
21 genes are eukaryotic aspartic proteases, and more than 150 sequences are related to aspartic proteases that are embedded in endogenous retroviral elements as human endogenous retroviruses [5]
We report the purification of processed forms of endogenous mouse SASPase from mouse stratum corneum extracts (SCE) and the determination of their amino acid sequences
We produced rmSASP15 via autoprocessing of GST-mouse SASPase 32-kDa form (mSASP32) expressed in Escherichia coli. 500 ml of E. coli culture expressing GST-mSASP32 was collected by centrifugation, and cell pellets were dissolved in 9 ml of buffer H (50 mM sodium acetate, pH 5.5, 1 mM EDTA, 0.15 M NaCl, 0.1% Triton X-100) containing protease inhibitor mixture (Nakalai Tesque, Japan)
Summary
Materials—Oligonucleotide primers were purchased from Proligo Japan (Kyoto, Japan). N-terminal amino acid sequence analysis and mass spectrometric analysis were performed by Aproscience Co. Purification of mSASP15 from Mouse SCE and C-terminal Amino Acid Analysis—5 mg of mSASP-PR2 pAb was coupled to 0.8 ml of Hitrap NHS-activated HP column (GE Healthcare) according to the manufacturer’s instruction and equilibrated with SCE buffer (mSASP-PR2 pAb column). 500 ml of E. coli culture expressing GST-mSASP32 was collected by centrifugation, and cell pellets were dissolved in 9 ml of buffer H (50 mM sodium acetate, pH 5.5, 1 mM EDTA, 0.15 M NaCl, 0.1% Triton X-100) containing protease inhibitor mixture (Nakalai Tesque, Japan). Separated epidermis was extracted in a buffer containing 62.5 mM Tris-Cl, pH 6.8, 2% glycerol, 1% SDS, 5 mM EDTA, and a protease inhibitor mixture (Nakalai Tesque) followed by sonication on ice 5 times for 3 s and centrifuged at 15,000 ϫ g for 20 min at room temperature. Bound antibodies were visualized with alkaline phosphatase-conjugated goat anti-rabbit IgG and the appropriate substrate as described by the manufacturer (GE Healthcare)
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