Identification and Characterization of a Novel Retroviral-Like Aspartic Protease Specifically Expressed in Human Epidermis D. Bernard, B. Méhul, A. Thomas-Collignon,

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Identification and Characterization of a Novel Retroviral-Like Aspartic Protease Specifically Expressed in Human Epidermis D. Bernard, B. Méhul, A. Thomas-Collignon, C. Delattre, M. Donovan, R. Schmidt Journal of Investigative Dermatology Volume 125, Issue 2, Pages 278-287 (August 2005) DOI: 10.1111/j.0022-202X.2005.23816.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Location of Skin ASpartic Protease (SASPase) in a 2D electrophoresis gel of protein extracts of reconstructed human epidermis. Extracts (4 mg protein) from reconstructed epidermis were separated by 2D electrophoresis. Proteins were stained with Amido Black. The newly discovered human protease “SASPase” has a calculated MW of 14 kDa and an isoelectric point (pI) of 5.8 (spots representing known proteins are listed with their respective names). Journal of Investigative Dermatology 2005 125, 278-287DOI: (10.1111/j.0022-202X.2005.23816.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Skin ASpartic Protease (SASPase) sequence and putative 3D structure. (A) Sequence. Nucleotide sequence coding for human SASPase aligned with its deduced amino acid (aa) sequence. Dotted lines underline aa indicating the prosite signature of a retroviral-type aspartyl protease. Active site aspartic acid is in bold. Sequences obtained from Edman sequencing after trypsin digestion of the isolated 2D spot are boxed with double lines. A putative di-leucine domain and transmembrane region are underlined with double lines or a single bold line, respectively. A single nucleotide polymorphism has been detected at aa position 49 replacing threonine (T) by Alanine (A). Two methionines are highlighted with bold letters and correspond to two putative start codons for SASPase. (B–C) Modelization. The program 3D-PSSM was used to calculate the putative 3D structure of SASP14. The equine anemia virus protease structure was calculated as being the best template for the simulation (d1fmb pdb file). The calculated certainty of the structure was 80% and evaluated to be “good” following the software criteria. The structure was represented with the swiss pdb viewer v3.7 software (B) (http://www.exspasy.ch.spdbv) and 3D rendering was improved with the Pov ray software (C). The active site aspartic acid (D24) is highlighted with an arrow. Journal of Investigative Dermatology 2005 125, 278-287DOI: (10.1111/j.0022-202X.2005.23816.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Skin ASpartic Protease (SASPase) sequence and putative 3D structure. (A) Sequence. Nucleotide sequence coding for human SASPase aligned with its deduced amino acid (aa) sequence. Dotted lines underline aa indicating the prosite signature of a retroviral-type aspartyl protease. Active site aspartic acid is in bold. Sequences obtained from Edman sequencing after trypsin digestion of the isolated 2D spot are boxed with double lines. A putative di-leucine domain and transmembrane region are underlined with double lines or a single bold line, respectively. A single nucleotide polymorphism has been detected at aa position 49 replacing threonine (T) by Alanine (A). Two methionines are highlighted with bold letters and correspond to two putative start codons for SASPase. (B–C) Modelization. The program 3D-PSSM was used to calculate the putative 3D structure of SASP14. The equine anemia virus protease structure was calculated as being the best template for the simulation (d1fmb pdb file). The calculated certainty of the structure was 80% and evaluated to be “good” following the software criteria. The structure was represented with the swiss pdb viewer v3.7 software (B) (http://www.exspasy.ch.spdbv) and 3D rendering was improved with the Pov ray software (C). The active site aspartic acid (D24) is highlighted with an arrow. Journal of Investigative Dermatology 2005 125, 278-287DOI: (10.1111/j.0022-202X.2005.23816.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Skin ASpartic Protease (SASPase) Northern blot study in different human tissues. Human multiple tissue mRNA blot (Origene), 2 μg of poly(A) ∼ per lane from tissues indicated above each lane. The calculated mRNA size of SASPase is indicated on the left. The blot was hybridized with a human 1.026 kb radiolabeled SASPase probe. Journal of Investigative Dermatology 2005 125, 278-287DOI: (10.1111/j.0022-202X.2005.23816.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunodetection of Skin ASpartic Protease (SASPase) in normal and diseased skin. Western blot (A): Two repetitions (a, b) were made for each sample. 1, normal human epidermis extract; 2, reconstructed epidermis 13 d; 3, psoriatic stratum corneum extract; and 4, normal non-plantar stratum corneum extract. Immunohistochemistry (B–G) was performed on paraffin sections of normal and diseased skin using the monoclonal antibody SASP7H9 as the primary antibody. The positive staining observed was mostly cytoplasmic and occasionally nuclear in the stratum granulosum of normal skin (B) and in the inner root sheath of hair follicles (C). In psoriasis sample (D, E), increased staining was present in the stratum corneum. In skin with ulceration (F, G), strong positive staining was observed in the stratum granulosum of the intact area (F) but was markedly reduced immediately adjacent to the ulcer (G). SC, stratum corneum; E, epidermis; D, dermis. Journal of Investigative Dermatology 2005 125, 278-287DOI: (10.1111/j.0022-202X.2005.23816.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Recombinant Skin ASpartic Protease (rSASPase) auto-activation of wild-type and mutated forms. The auto-activation reaction was performed in 0.1 M acetate pH 5 at 37°C and monitored by SDS-PAGE. (A) Lanes 1–7 incubation time 0, 5, 10, 20, 30, 80, and 120 min for wild-type (wt) SASPase. Note: In this experiment, rSASPase fusion protein batch is contaminated with an equivalent quantity of free GST possibly due to an uncontrolled auto-activation occurring during the preparation process. (B) Comparison of auto-activation of wt with mutated SASPase (Active site aspartic acid (D) was either replaced by alanine (A: D212A) or glutamic acid (E: D212E)) after 60 min of incubation. The mutated forms have lost the autocatalytic property. Arrow on the left indicates the 14 kDa activated form. (C) A proposed model of SASPase structure. The putative complete protein (SASP37 343 aa, 37.0 kDa, pI 5.3) is represented with the location of its putative transmembrane sequence, the predicted retroviral aspartyl protease signature, and the primary auto-activation sites. The alternative short form is named SASP28 (259 aa, 28.5 kDa, pI 5.22). The major auto-activation protease product is called SASP14 (136 aa, 14.8 kDa, pI 5.2). Journal of Investigative Dermatology 2005 125, 278-287DOI: (10.1111/j.0022-202X.2005.23816.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Indinavir as an inhibitor of Skin ASpartic Protease (SASPase) auto-activation. Recombinant fusion protein (1 mg per mL in Tris buffer saline) was initially acidified to pH 5 with 1 M acetate buffer pH 4.5. Stock solutions of 10 mM inhibitors in DMSO (N, norvir; R, ritonavir; A, amprenavir; P, pepstatine; S, saquinavir) or in water (I, indinavir) were used to bring the test solution to 0.1 or 1 mM final concentrations. A control (C) was run without inhibitors. The final concentration of DMSO was 10% in each case. Samples from the 0 and 30 min timepoints after the initial acidification step were run on Nu-PAGE 12% gels in MES buffer. The black arrow indicates the fusion protein, which is progressively degraded to activated SASPase or SASP14 (dotted arrow). Journal of Investigative Dermatology 2005 125, 278-287DOI: (10.1111/j.0022-202X.2005.23816.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions