The stratum corneum (SC), the outermost layer of the epidermis, acts as a barrier against the external environment. It is hydrated by endogenous humectants to avoid desiccation. However, the molecular mechanisms of SC hydration remain unclear. We report that skin‐specific retroviral‐like aspartic protease (SASPase) deficiency in hairless mice resulted in dry skin and a thicker and less hydrated SC with an accumulation of aberrantly processed profilaggrin, a marked decrease of filaggrin, but no alteration in free amino acid composition, compared with control hairless mice. We demonstrated that recombinant SASPase directly cleaved a linker peptide of recombinant profilaggrin. Furthermore, missense mutations were detected in 5 of 196 atopic dermatitis (AD) patients and 2 of 28 normal individuals. Among these, the V243A mutation induced complete absence of protease activity in vitro, while the V187I mutation induced a marked decrease in its activity. These findings indicate that SASPase activity is indispensable for processing profilaggrin and maintaining the texture and hydration of the SC. This provides a novel approach for elucidating the complex pathophysiology of atopic dry skin.
The paper explained
The SC is the outermost layer of the skin in terrestrial animals and thus acts as a barrier against the external environment. It is hydrated by endogenous substances to avoid desiccation; however, the mechanisms responsible for maintaining hydration of the SC remain unclear at the molecular level. Dry skin is a common phenotype in patients with atopic dermatitis (AD). Recent reports have indicated that the protein filaggrin is mutated in ichthyosis vulgaris patients and is a major predisposing factor for development of atopic eczema, asthma, and allergies. Approximately 50 and 80% of European and Japanese AD patients, respectively, have normal filaggrin alleles, suggesting the presence of previously unidentified predisposing factors.
We produced ‘hairless’ mice that were deficient in the enzyme skin‐specific retroviral‐like aspartic protease (SASPase). The decreased activity of this enzyme in the mice resulted in dry skin with an accumulation of incorrectly processed profilaggrin, a precursor of the filaggrin protein. This incorrectly processed and accumulated profilaggrin subsequently results in a marked decrease of filaggrin production. We also demonstrated that SASPase directly cleaved a profilaggrin linker peptide in vitro. Several missense mutations were detected in 5 of 196 AD patients and 2 of 28 normal individuals. Among these, the V243A mutation resulted in complete ablation of protease activity in vitro, while the V187I mutation induced a marked decrease in SASPase activity.
This is the first report demonstrating that a deficiency of the protease SASPase is a likely cause of dry skin in vivo. We clarified that the activity of SASPase plays a key role in determining the texture of the SC by modulating SC hydration. This molecular mechanism will provide clues in revealing the role of the SC in terrestrial animals and how they adapted to life on land. Our results also provide novel concepts to assist in determining the complex pathophysiology of atopic dry skin.
Skin is the outermost tissue of terrestrial animals. It forms an effective barrier between the organism and the environment, which is indispensable for the prevention of the invasion of microorganisms, chemical compounds and allergens, and the maintenance of moisture levels of the skin. Skin is composed of three layers: the epidermis, dermis, and hypodermis. The epidermis is a stratified squamous, keratinized epithelium composed of the stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG), and stratum corneum (SC) (Watt, 1989). Epidermal cells (keratinocytes) divide and differentiate as they move upward from the SB to the SC. At the SG‐to‐SC transition, the keratinocytes dramatically transform themselves from three‐dimensional living cells to two‐dimensional, flattened and dead cells without intracellular organelles containing keratin bundles and lipids to constitute the SC (Candi et al, 2005). During this abrupt and dynamic terminal differentiation, keratinocytes express various proteins such as keratins, profilaggrin/filaggrin, involucrin, small proline‐rich proteins, loricrin, cystatin A, and elafin, which form the cornified envelope of mature corneocytes (Candi et al, 2005).
It has long been recognized that there is a heritable component for the development of atopic dermatitis (AD) (Barnes, 2010). Recent reports indicate that ‘filaggrin’ has nonsense mutations in ichthyosis vulgaris (IV) patients and is the major predisposing factor for atopic eczema, asthma, and allergies (Smith et al, 2006; Irvine, 2007; Palmer et al, 2006; Sandilands et al, 2009). These reports further indicate that early onset of AD is caused by outside‐to‐inside paradigms, namely a primary barrier abnormality (Elias & Steinhoff, 2008), and filaggrin nonsense mutations are carried by nearly 10% of Europeans. However, such filaggrin mutations are found in ∼50 and 20% of European and Japanese AD patients, respectively (Barker et al, 2007; Nomura et al, 2008; Sandilands et al, 2007, 2009), indicating that patients with AD who have normal filaggrin alleles are likely affected by other previously unidentified predisposing factors.
Filaggrin is expressed as a profilaggrin of >400 kDa in humans, which is a major component of keratohyalin granules in the SG of the epidermis (Dale et al, 1985; Presland et al, 2006). Profilaggrin is an insoluble phosphoprotein that consists of an amino terminal Ca2+‐binding protein of the S‐100 family, linked to 10–12 tandem filaggrin monomer repeats (Dale et al, 1985; Harding & Scott, 1983; McGrath & Uitto, 2008). At the SG‐to‐SC transition, each filaggrin repeat is processed by certain protease(s) to generate the filaggrin monomer (37 and 28 kDa in human and mouse, respectively). The resulting monomer filaggrin has an unusual cationic charge, strongly binds to and bundles the keratin cytoskeleton to form microfibrils, and is believed to contribute to the production of flattened cells in the lower SC (Dale et al, 1978). Additionally, part of the filaggrin–keratin complex is cross‐linked by transglutaminase to build the skin barrier (Candi et al, 1998, 2005; Harding & Scott, 1983; Steinert & Marekov, 1995). Keratin‐bound filaggrin is citrullinated and further degraded into amino acids, which constitute a part of the natural moisturizing factor (NMF) in the upper SC (Tarcsa et al, 1996; Mechin et al, 2005; Nachat et al, 2005; Ishida‐Yamamoto et al, 2002; Denecker et al, 2007; Kamata et al, 2009). Therefore, at the transition of the SG‐to‐SC, processing from profilaggrin to filaggrin is the rate‐limiting, critical step for the profilaggrin processing cascade leading to SC moisturizing.
Several proteases are involved in profilaggrin to filaggrin processing. Calpain I and profilaggrin endopeptidase I (PEP‐I) are important for the processing of the linker peptides between the filaggrin monomer repeats to generate the monomeric filaggrin in vitro (Resing et al, 1989, 1993a,b, 1995; Yamazaki et al, 1997); furin or convertase are involved in cleavage of the N‐terminus from profilaggrin (Pearton et al, 2001); and knockout mice of matriptase/MT‐SP1 and prostasin (CAP1/Prss8) show a defect in the conversion of profilaggrin to filaggrin (Leyvraz et al, 2005; List et al, 2003). Although the N‐ and C‐terminal sequences of mouse, rat and human filaggrin have been determined (Resing et al, 1989, 1993b; Thulin & Walsh, 1995; Thulin et al, 1996), whether the linker sequences of profilaggrin are cleaved by matriptase or prostasin remains unclear.
Human and mouse retroviral‐like aspartic protease, SASPase (skin aspartic protease; Asprv1), was the first identified stratified epithelia‐specific protease expressed exclusively in the SG (Bernard et al, 2005; Matsui et al, 2006). This protease has been cloned as a 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced gene (Taps) from mouse back skin epidermis (Rhiemeier et al, 2006). Recently, aberrant SASPase expression in transgenic mice was reported to cause impaired skin regeneration and skin remodelling after cutaneous injury and chemically induced hyperplasia (Hildenbrand et al, 2010). We have previously reported that SASPase‐deficient mice showed increased fine wrinkles on the side of the adult body, suggesting that SASPase plays a certain physiological role in the SG and SC, although precise physiological and biochemical analysis was difficult due to the presence of hair. In this report, we produced SASPase‐deficient ‘hairless’ mice. Through physiological and biochemical analyses of the mice, we demonstrate that SASPase activity plays a key role in determining the texture of SC by modulating SC hydration as well as profilaggrin processing. Moreover, we also report the identification of loss‐of‐function mutation of SASPase in the human genome.
Generation of SASPase‐deficient hairless mice
Using high‐throughput in situ hybridization screening, we previously identified a mouse homologue of SASPase and showed that SASPase‐deficient mice displayed fine wrinkles on the side of their bodies (Matsui et al, 2004, 2006). Because SASPase is expressed exclusively in the SG and SC, these wrinkles were hypothesized to be derived from aberrant functions of the SG and SC. To further analyse the effect of a deficiency of SASPase on the epidermal surface, we transferred the ablated allele to a Hos/HR‐1 hairless background. Hos:HR‐1 mice (BALB/c background) were crossed with SASP−/− mice (C57BL/6J background) using a speed congenic method (Wakeland et al, 1997). Immunoblotting of the skin in SASP+/+, SASP+/−, and SASP−/− hairless mice showed no expression of SASPase in the SASP−/− epidermis (Supporting information Fig 1A). Frozen sections of SASP+/− and SASP−/− hairless mouse ears were stained with anti‐SASP pAb and revealed a loss of the SASPase signal in the SG of the SASP−/− epidermis (Supporting information Fig 1B). Therefore, immunoblotting and immunofluorescence staining of frozen sections did not detect SASPase in SASPase deficient mice, indicating that the epidermis of SASP−/− mice was successively deficient in SASPase protein in the hairless background.
Appearance of SASP−/− hairless mice
Like normal mice, hairless mice (Hos:HR‐1) grow hair after birth; however, at 17–23 days, they lose this hair due to hair root atrophy in 3–4 days, starting with the head, and become completely hairless at 3.5 weeks of age. Thereafter, we observed that SASP−/− hairless mice had more fine wrinkles and drier, rougher skin than their SASP+/+ and SASP+/− counterparts (Fig 1A left). Female mice tended to show a more marked phenotype than male mice (data not shown). Stereomicroscopic examination revealed that SASP+/+ and SASP+/− mice had smooth, moist‐looking skin with fine grooves, whereas SASP−/− hairless mice had skin with raised scales composed of many horny cells (Fig 1A right). When hung by their tails, more apparent differences were observed (Fig 1B). Specimens were fixed by standard techniques, dehydrated, dried at a critical point and examined employing scanning electron microcopy (SEM). After drying, epithelial scales of the skin of the SASP+/− hairless mice had fallen off, exposing the surface structure and resulting in many fine grooves. In contrast, coarse grooves were observed in the SASP−/− hairless mice (Fig 1C). Similarly, SEM revealed that the individual horny cells were easily identified in SASP+/− hairless mice, whereas the intercellular spaces between the horny cells could not be readily discerned in their SASP−/− counterparts (Fig 1D).
Morphology of SASP−/− mice epidermis
Next, we examined the histological analysis of SASP−/− hairless mice. The epidermis of the SASP−/− mice showed a normal layer organization of keratinocytes, and keratohyalin‐granules were observed in both SASP+/+ and SASP−/− mice at the level of hematoxylin–eosin (HE)‐stained paraffin section images (Fig 2A and B). However, the cornified cells of the SASP−/− SC were compacted more tightly, and the number of layers was increased compared with SASP +/+ mice (Fig 2A and B). Ultrathin section electron microscopy confirmed these findings at a higher resolution (Fig 2C–G). The appearance of individual cells from the SB to SS was indistinguishable between the SASP+/+ and SASP−/− skin, and the SG contained both F‐ and L‐granules in the SASP−/− SC (Fig 2C and D). Furthermore, the SC of SASP−/− mice showed 7–10 layers of electron dense and tightly compacted cell layers compared with that of SASP+/+, which usually exhibited 5–7 layers (Fig 2C–H). These results suggest that a deficiency of SASPase in hairless background mice induced an abnormality in the SC.
Physiological features of SASP−/− epidermis
To physiologically characterize the epidermal surface of the SASP−/− hairless mice, we measured the trans‐epidermal water loss (TEWL) and the SC hydration of SASP+/+ (n = 7) and SASP−/− (n = 11) mice (Fig 3A and B). Although the TEWL was not significantly changed between SASP+/+ and SASP−/− mice (Fig 3A), a clear difference was observed in SC hydration. As such, SC hydration of SASP−/− mice was significantly lower than in SASP+/+ mice (Fig 3B). These results indicate that SASP−/− hairless mice showed markedly decreased SC hydration without alteration of barrier function.
Aberrant profilaggrin processing in SASP−/− hairless mice
Next, to analyse the epidermal differentiation of SASP−/− hairless mice, the expression of various epidermal differentiation markers were examined. Immunofluorescence staining of frozen sections of back skin epidermis with anti‐keratin 14, keratin 1, involucrin and loricrin pAbs revealed normal expressions and localization of epidermal differentiation markers (Supporting information Fig 2A). Immunoblotting of back skin epidermal urea extracts with anti‐keratin 14, keratin 1, involucrin and loricrin pAbs also revealed that the corresponding expression levels were not altered (Supporting information Fig 2B). On the other hand, immunofluorescence staining with anti‐filaggrin pAb revealed that filaggrin‐positive layers of the SC (lower SC) were increased in the back skin epidermis of the SASP−/− hairless mice (Fig 4A and B). To carefully compare the filaggrin in the lower SC, we tape‐stripped the SC of the SASP+/− and SASP−/− epidermis. Coomassie brilliant blue (CBB) staining of equivalent amounts of the extracts revealed that all the major bands were decreased, suggesting there were increased concentrations of certain smear proteins (Fig 4C, left). Immunoblotting of the same samples with anti‐filaggrin pAb revealed the accumulation of primarily two smear bands below the size of dimeric and trimeric filaggrin and that a mature filaggrin band was rarely detected (Fig 4C, right). These results suggest that a deficiency of SASPase resulted in the accumulation of premature processed dimeric and trimeric filaggrin and that mouse SASPase cleaves the linker sequence of mouse profilaggrin in vivo. The processing of mouse profilaggrin in the C57BL/6J mouse was reported to occur in a two‐step process via two types of profilaggrin linker sequences with or without FYPV, respectively (Resing et al, 1989). First, a profilaggrin linker sequence containing FYPV may be cleaved, resulting in the accumulation of a two‐domain intermediate (2DI) and a three‐domain intermediate (3DI). Second, the linker type without FYPV, which connects 2DI and 3DI of monomeric filaggrin, is potentially cleaved by a Ca2+ dependent protease (Resing et al, 1989, 1993a). Some amino acid residues are then removed from the exposed sites by further exoprotease activity (Resing et al, 1989). Therefore, accumulation of dimeric and trimeric‐like profilaggrin in the SASP−/− epidermis in Hos:HR‐1 background suggests that SASPase may be involved in either the first or second processing steps.
SASPase directly cleaves the profilaggrin linker sequence in vitro
Accumulation of aberrant dimeric and trimeric profilaggrin in the lower SC of the SASP−/− hairless mice implies that the profilaggrin linker peptide is a direct substrate for SASPase. To confirm this hypothesis, we examined whether SASPase directly cleaves the profilaggrin linker peptide in vitro. Because it was difficult to produce recombinant mouse filaggrin with a linker peptide in Escherichia coli because of excess degradation, we produced human filaggrin (hFilaggrin) as a fusion protein of maltose binding protein (MBP) connected with the human profilaggrin linker peptide in E. coli. Purified MBP‐hFilaggrin showed a triple banding pattern. The N‐terminal amino acid sequence of the major upper two bands revealed that both proteins had an intact N‐terminus of MBP indicating that C‐terminally processed filaggrin (MBP‐hFilaggrin‐ΔC) was copurified with MBP‐hFilaggrin. To prepare the active form of SASPase (14 kDa; hSASP14; 191‐326aa), we expressed and purified a 28 kDa form of human SASPase (hSASP28; 85‐343aa) as a fusion protein with GST (GST‐hSASP28) in E. coli (Bernard et al, 2005). After complete autoprocessing under weakly acidic conditions (pH 6.0), which is the optimum pH of SASPase, the produced hSASP14 was purified (Fig 5A; Bernard et al, 2005; Matsui et al, 2006). Next, hSASP14 was incubated with MBP‐hFilaggrin/‐hFilaggrin‐ΔC under weakly acidic conditions (pH 6.0). As shown in Fig 5B, MBP‐hFilaggrin/hFilaggrin‐ΔC was cleaved into mainly 42, 37 and 23 kDa proteins (Fig 5B and C). The 42 kDa protein was recognized by anti‐MBP pAb (data not shown), and the N‐terminal amino acid sequencing confirmed that the protein at 42 kDa was the MBP protein itself. The N‐terminal amino acid sequencing of the 37 and 23 kDa identified the same peptide, ‘QVSTH’. This N‐terminal amino acid sequence corresponded to the previously reported N‐terminal amino acid sequence of native monomeric human filaggrin, in which Q is modified into pyrrolidone carboxylic acid (PCA) (Thulin & Walsh, 1995; Thulin et al, 1996). These results indicate that hSASP14 cleaved MBP‐hFilaggrin and produced MBP (42 kDa) and hFilaggrin (37 kDa), of which the N‐terminus was QVSTH. Furthermore, MBP‐hFilaggrin‐ΔC was cleaved at the same site and produced MBP (42 kDa) and hFilaggrin‐ΔC (23 kDa), of which the N‐terminus was QVSTH (Fig 5B and C). Thus, we conclude that hSASP14 cleaved the linker sequence of human profilaggrin between ‘GSFLY’ and ‘QVSTH’ in vitro (Fig 5C and D).
Normal free amino acid composition in SC of SASP−/− mice
Previous reports suggest that NMFs serve as natural humectants of SC (reviewed in Rawlings & Matts, 2005). To investigate whether aberrant processing of filaggrin had a detrimental effect on the composition of NMFs, we performed free amino acid analysis of tape stripped SC of SASP+/+ (n = 7) and SASP−/− (n = 11) mice. There were no alterations in the total amount or the composition of free amino acids (Fig 6A and B). These results suggest the NMFs were normal in SASP−/− SC, despite the marked decrease in SC hydration.
Mutations of human SASPase affect autoprocessing and profilaggrin linker cleavage activity
Analysis of our SASP−/− hairless mice indicates that loss of SASPase activity may result in dry skin accompanied by an accumulation of unprocessed profilaggrin. Thus, we investigated whether the human genome possesses SASPase mutations, which consequently affect protease activity, i.e. profilaggrin to filaggrin processing activity. We investigated mutations in the SASPase gene in 28 control subjects and 196 AD‐patients (Sasaki et al, 2008). As a result of the mutation search on the human SASPase gene, two types of missense mutations (D232Y and V243A: 3.5% [1/28]) in the control subjects, four types of missense mutations (A54S: 0.5% [1/196], I186T: 0.5% [1/196], V187I: 1.5% [3/196], R311C: 0.5% [1/196]), and three types of silent mutations (F101F: 0.5% [1/196], P206P: 3.1% [6/196], N276N: 1.5% [3/196]) in the AD patients were identified (Fig 7A, Supporting information Tables I and II). All mutations were heterozygous. V187I was most frequently identified (identified in three AD patients). A54S and R311C were found in the same patient and in the same allele. The number of patients and mutation types are shown in Supporting information Tables I and II.
To examine whether these mutations affect the protease activity of SASPase, we bacterially expressed and purified GST‐hSASP28 bearing the mutations I186T, V187I, R311C, D232Y, or V243A, and performed an in vitro autoprocessing assay as previously described (Bernard et al, 2005; Matsui et al, 2006). To compare the autoprocessing activity in vitro, we purified GST‐hSASP28 mutants (Fig 7B). We could not examine the autoprocessing activity of GST‐hSASP28(I186T)/(R311C), because full‐length GST‐hSASP28(I186T)/(R311C) was barely purified, possibly due to the higher autoprocessing activity in E. coli. The rest of the purified GST‐hSASP28(WT)/(D232Y)/(V243A) was incubated under weakly acidic conditions (pH 6.0), which is the optimal pH of SASPase (Bernard et al, 2005; Matsui et al, 2006). As shown in Fig 7C, GST‐hSASP28(D232Y) showed autoprocessing activity similar to that of the WT, and V243A showed no autoprocessing activity.
We next examined whether the V187I mutation, which was identified most frequently in AD patients (three patients), affected autoprocessing activity in vitro. The purified GST‐hSASP28(WT)/(V187I) was incubated under weakly acidic conditions (pH 6.0). As shown in Fig 7D, GST‐hSASP28(V187I) showed decreased autoprocessing activity in both 300 and 400 mM NaCl. Fig 7E shows the time course by semi‐quantification of produced hSASP14. Because V187I is located outside of the cleaved hSASP14, autoprocessed hSASP14 from hSASP28(V187I) should be the same as the WT. Therefore, the initial autoprocessing reaction (1–30 min in 300 mM NaCl) should reflect the difference between WT and V187I. The reaction rate of GST‐hSASP28(V187I) at 300 mM NaCl was estimated at 5.6‐fold over 15–30 min, which is lower than that of GST‐hSASP28(WT). These results indicate that hSASP28(V187I) has substantially reduced activity at pH6.0 in vitro compared to WT. Finally, we examined whether the V187I mutation had an effect on the filaggrin linker cleavage activity of GST‐hSASP28 in vitro. To examine whether the mutation of V187I affected the profilaggrin cleavage activity, we incubated GST‐hSASP28(WT) or GST‐hSASP28(V187I) with MBP‐hFilaggrin/MBP‐hFilaggrin‐ΔC under weakly acidic conditions (pH 6.0). As expected, the MBP‐hFilaggrin cleavage activity by GST‐hSASP28 was suppressed in GST‐hSASP28(V187I) compared with GST‐hSASP28(WT), relative to the production of hSASP14 (Fig 7E). These results indicate that GST‐hSASP28(V187I) showed decreased autoprocessing activity at pH 6.0, resulting in decreased profilaggrin linker cleavage activity in vitro.
It is important to elucidate whether these heterogenic loss‐of‐function mutations of SASPase affect human skin physiology. And it is possible there was some dry skin in our non‐AD cohort as criteria of this cohort did not discriminate against dry skin (Sasaki et al, 2008). We measured TEWL and SC hydration of a non‐AD individual with a mutation (V243A/+) as well as three normal individuals without mutations. The appearance of the skin surface, TEWL and SC hydration of V243A/+ were similar to the three normal individuals (Supporting information Fig 3). These results did not provide conclusive evidence due to the small numbers tested. In the case of complex disorders like AD, it is difficult to prove a direct role of the sequence variant in the pathogenesis. In the future, it is necessary to perform a large‐scale cohort analysis to clarify the clinicopathological significance of SASPase mutation.
Physiological role of SASPase
Figure 8 summarizes our findings. In normal epidermis, non‐phosphorylated profilaggrin is orderly processed into filaggrin and bundle keratin filaments at the ‘lower SC’, then degraded into free amino acids which constitute most of the NMFs in the ‘upper SC’. SASPase deficiency causes incomplete linker cleavage of profilaggrin resulting in an accumulation of trimeric and dimeric profilaggrins slightly degraded from either N‐ or C‐terminal ends in the ‘lower SC’. Such aberrant profilaggrin may bind to keratin filaments, finally degrade, and produce a normal composition of free amino acids in the ‘upper SC’. Finally, the SC of SASP−/− epidermis has an increased number of layers and produces a wrinkled, dry, rough skin.
In our previous study, we reported increased fine wrinkles at the side of the body in SASP−/− mice raised on a C57BL/6J background. However, we were not able to distinguish any morphological changes between SASP+/− and SASP−/− mice, possibly due to the thin epidermis of the back skin (Matsui et al, 2006). In this paper, we generated SASP−/− mice with Hos:HR‐1 background. The increased thickness of the epidermis of these mice enabled us to distinguish a clear difference in SC structure and profilaggrin processing pattern in the SC. SASP−/− hairless mice showed a marked decrease in SC hydration and an increased number of electron dense layers in the SC. Such aberrant layers in the SC consisted of an upper SC without filaggrin staining and a lower SC stained by anti‐filaggrin Ab with the accumulation of aberrant dimeric and trimeric filaggrin, i.e. premature profilaggrin processing (Figs 5B and 8). Interestingly, the composition and quantity of major free amino acids were not altered in the dry skin‐like SC of SASP−/− hairless mice (Fig 6).
Hydration of the SC plays an important role in maintaining metabolic activity, enzyme activity, mechanical properties, appearance and barrier function of the skin and is dependent on (i) organization of the tight and semi‐permeable barrier of intercellular lamellar lipids, (ii) the diffusion path length created by the SC layers and corneocyte envelopes, and (iii) the presence of NMFs (Rawlings & Harding, 2004; Rawlings & Matts, 2005). NMFs comprise up to 10% of the SC and are reported to be an important natural humectants of the SC because of their hygroscopic features (Rawlings & Harding, 2004; Rawlings & Matts, 2005). Most NMFs are composed of free amino acids derived from the degradation products of filaggrin and its derivatives, PCA and urocanic acid (UCA). There are also non‐amino‐acid‐derived (non‐filaggrin‐derived) NMFs, such as sugars, hyaluronic acid, urea, citrate, lactate, and glycerol (Fluhr et al, 2008; Rawlings & Harding, 2004; Rawlings & Matts, 2005). Although we did not analyse the PCA or UCA of SASP−/− hairless mice, there were no changes in free amino acids, which suggests that filaggrin‐derived NMFs were not altered. The accumulation of aberrantly processed profilaggrin without monomer filaggrin in the ‘lower SC’ accompanied by a normal free amino acid content in the ‘upper SC’ in SASP−/− SC implies disruption of several mechanisms for maintenance of SC hydration other than the contribution of NMFs.
As described in the Introduction section, filaggrin is thought to have two major functions: the formation of keratin microfibrils in the lower SC and the production of NMFs in the upper SC (Rawlings & Harding, 2004; Fig 8). It is possible that dry skin of SASP−/− hairless mice is derived from the lower SC where aberrantly processed filaggrin accumulates, but not from the upper SC where the end‐products of filaggrin (NMFs) are located. In the normal epidermis of humans, rats and mice, intermediate processing products of mouse filaggrin, 2DI and 3DI, have been reported, and they possess keratin binding activity similar to monomeric filaggrin (Harding & Scott, 1983). Thus, it is suggested that aberrant dimeric and trimeric filaggrins in the SASP−/− SC also possess keratin binding activity. It is widely believed that the SC hydration and physiological and physical mechanics are closely linked and depend on keratin structural organization. Accumulation of premature processed profilaggrin, even if there is keratin binding activity, may alter the cubic‐like, rod‐packing symmetry of keratin filaments at the SG‐to‐SC transition and/or at the lower SC, and this may cause alteration of the SC hydration level in the SASP−/− epidermis (Norlen & Al‐Amoudi, 2004). The SC of flaky tail mice, in which filaggrin is absent in the cornified cell layers of the epidermis, did not show decreased SC hydration nor an increased number of layers in the SC (Fallon et al, 2009; Presland et al, 2000; Scharschmidt et al, 2009). This suggests that aberrantly processed profilaggrin and a marked decrease of mature filaggrin affect the texture and hydration of the SC. The importance of the amount of mature filaggrin for SC hydration was reported by Ginger et al, who found an inverse relationship between the profilaggrin‐12‐repeat allele and the occurrence of self‐perceived dry skin (Ginger et al, 2005).
SASPase may cleave other as yet unidentified substrates, in addition to profilaggrin, resulting in decreased SC hydration via abnormalities in the intercellular lamellar lipids barrier, the diffusion path length and/or the composition of non‐filaggrin‐derived NMFs. These possibilities can be examined by crossing SASP−/− mice with filaggrin‐deficient mice to examine whether dry skin is derived from aberrant profilaggrin processing.
In normal, healthy controls and AD patients, we identified several missense mutations, which may have affected the activity of SASPase. Among them, we identified a loss‐of‐function mutation of SASPase (V243A) in non‐AD controls. This mutation was located inside the protease domain and no longer showed any activity at pH 6.0. Another missense mutation, V187I, was the most frequently identified in the AD patients (three AD patients). It was located outside of the protease domain at the autoprocessing site, the ‘P4’ position of the substrate recognition site. A change of the amino acid at P4 resulted in decreased autoprocessing activity in vitro. GST‐hSASP28(V187I) did not show any autoprocessing activity in E. coli (neutral pH), suggesting that this mutation had no activity in the cytoplasm of the SG, resulting in the same loss‐of‐function effect as SASPase(V243A). SASPase, as a retroviral aspartic protease, must undergo homodimeric formation for its protease activity (Bernard et al, 2005; Matsui et al, 2006). In the HIV protease, a subunit exchange reaction with a catalytically defective protease results in 50% inhibition of enzymatic activity (Darke, 1994). In the case of a heterozygous loss‐of‐function mutation of bi‐allelic expression, half of the molecules are catalytically inactive and induce ‘heterodimeric inhibition’ of SASPase activity, resulting in a quarter decrease of total activity. The newly found, rare mutations of SASPase (V187I)/(V243A) possibly behave in a dominant negative manner in the person who has the heterozygous mutation.
Of note, the SASP−/− mice showed decreased SC hydration without alteration of TEWL, suggesting a decreased ability of water retention in the SC under normal barrier function. Although the TEWL is an important hallmark for skin barrier function, TEWL is not necessarily correlated with dry skin (Berry et al, 1999; Engelke et al, 1997; Wilhelm et al, 1991). Decreased SC hydration is found in a number of diseases, such as AD, eczema or psoriasis (Harding et al, 2000). There are a number of human epidermal diseases that include the aberrant expression and processing of profilaggrin to filaggrin (reviewed in Dale et al, 1990). Therefore, it is possible that patients who do not have a nonsense mutation of filaggrin, but who exhibit xerosis or AD, might have an aberrant profilaggrin processing pattern. Involvement of SASPase in progression of these diseases from the aspect of the profilaggrin processing pathway should be examined. The profilaggrin degradation pattern could be useful for the diagnosis of xerosis and the early onset of AD.
Collectively, these results indicate that activity of SASPase plays a key role in determining the texture of the SC by modulating SC hydration as well as profilaggrin‐to‐filaggrin processing. Moreover, these results, in combination with clinicopathological investigations of epidermal diseases derived from the aberrant processing of profilaggrin by SASPase mutation, will provide a novel concept to dissect the complex mechanisms of percutaneous antigen priming in atopic diseases.
MATERIALS AND METHODS
Oligonucleotide primers were purchased from Sigma–Aldrich Japan (Kyoto, Japan). N‐terminal amino acid sequence analysis was performed by Shimadzu Techno Research (Kyoto, Japan).
For immunofluorescence and immunoblotting, antibodies against keratin 14, keratin 1, involucrin, loricrin and filaggrin were used (Covance, Berkeley, CA). A polyclonal antibody against SASPase, anti‐SASP‐C, which recognizes the C‐terminus of both 28 kDa (human) and 32 kDa (mouse) SASPase, and anti‐SASP‐PR1 polyclonal antibody, which recognizes both 14 kDa (human) and 15 kDa (mouse) SASPase, have been described previously (Matsui et al, 2006).
Hairless mice (Hos/HR‐1) were obtained from Hoshino Experimental Animal Supply (Ibaragi, Japan). SASP+/− mice of a C57BL/6J background were bred from six generations to a pure Hos/HR‐1 background through the speed congenic services of the Central Institute for Experimental Animals (Kawasaki, Japan). Mice heterozygous for a SASPase deletion were interbred, and SASP+/+, SASP+/− and SASP−/− littermates were used for experiments. Wild‐type (WT, SASP+/+), SASPase heterogenic (SASP+/−) and SASPase knockout (SASP−/−) mice on a Hos/HR‐1 backgrounds were used in the study. All mice were maintained under specific pathogen‐free (SPF) conditions that are required for maintaining mouse colonies. All animal procedures were approved by the Animal Studies Subcommittee (Institutional Animal Care and Use Committee) of the Tokyo Medical and Dental University and performed in accordance with their guidelines. Basal SC hydration was measured with ASA‐M1 (Asahi Biomed, Tokyo, Japan) on the back skin of SASP+/+ (n = 7) and SASP−/− (n = 11) mice. From the same mice, TEWL measurements were taken under basal conditions with a VAVO SCAN (Asahi Biomed). Experiments were performed with adult female mice only (2–5 months old).
Tape‐stripped epidermal extract
The dorsal and back skin of SASP+/+, SASP+/− and SASP−/− hairless mice were sequentially stripped with Scotch Book Tape 10 times (3M, St. Paul, MN). Corneocytes adherent to the tape surface were eluted by 5 ml of urea‐buffer and concentrated by an Amicon‐ultra 10 kDa (Millipore) into 50 µl. Protein concentrations were estimated by the Bradford method. Samples were mixed with 25 µl of 3 × SDS sample buffer.
Free amino acid analysis
Surface samples of SC were obtained by adhesive tape striping (Scotch Book Tape) performed five times. Each tape corresponded to a skin area of about 3 × 10 cm2 derived from anesthetized SASP+/+ (n = 7) and SASP−/− (n = 11) hairless mice. Water‐soluble amino acids on the adhesive tapes were extracted with 5 ml 0.1% Triton X‐100. After sonication for 30 min at 37 °C, amino acids were quantified using an amino acid analyser (Hitachi model L‐8500).
Fifty microlitres of GST‐hSASP28 mutants (1.4 mg/ml or 0.9 mg/ml) in buffer D (50 mM phosphate buffer, pH 6.0, 0.7 M NaCl) containing 1 mM EDTA and protease inhibitor cocktail) was incubated at 37°C. Five‐microlitres aliquots were recovered at different times during the incubation step, and the reaction was stopped by the addition of 20 µl Laemmli buffer. All the aliquots (5 µl) were analysed by SDS‐PAGE on 15% acrylamide gels followed by staining with CBB R‐250. Semi‐quantification of hSASP14 was analysed densitometrically using Adobe Photoshop™ CS3.
Purification of recombinant hSASP14
All procedures were performed at 4°C. Purified GST‐hSASP28 was dialysed against buffer D using NAP‐10 (GE Healthcare, Japan) and concentrated and frozen at −80 °C. Next, 300 µl of concentrated GST‐SASP28 (5 mg/ml) was incubated for 60 min at 37°C to commence autoprocessing. Each sample was diluted in 1 ml of buffer D and passed over 200 µl of Glutathione Sepharose 4B beads (GE Healthcare) twice. The flow‐through fraction containing hSASP14 was collected and subjected to the protease assay.
Human filaggrin cleavage assay
Purified hSASP14 (419 pmol) was incubated with 3.6 µM MBP‐hFilaggrin in 100 µl of buffer D in the presence of 1 mM EDTA and protease inhibitor cocktail for 60 min at 37°C. After incubation, the reactions were stopped by adding 50 µl of 3 × SDS sample buffer, and 10 µl was subjected to SDS‐PAGE. Cleaved fragments were subjected to N‐terminal amino acid sequencing.
For more information
OMIM: SKIN ASPARTIC PROTEASE: http://www.ncbi.nlm.nih.gov/omim/611765
TM conceived of the study, participated in its design and coordination, carried out the analysis of knockout mice and the biochemical studies and drafted the manuscript; KM and JK carried out the mutation search; AK participated in the design of the study and helped to draft the manuscript; HK and TE conducted the human study; KH and ST helped to analyse the knockout mice; SI carried out the electron microscopic analysis; II and JI participated in the design and coordination of the study and helped to draft the manuscript; MA conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer‐reviewed, but not copy‐edited or typeset. They are made available as submitted by the authors.
We thank Sayaka Katahira‐Tayama and Itsumi Ohmori for technical assistance. We thank Dr. Tsuyohi Hata (KOSÉ Corporation) for helpful discussions. We thank KAN Research Institute Inc. for providing materials. This work was supported by a Grant‐in‐Aid for Scientific Research to TM to AK, and to HK, ‘Program for Improvement of Research Environment for Young Researchers’ from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to TM and to AK, research grants from the Nakatomi Foundation, the Cosmetology Research Foundation and the Naito Foundation to TM, the Keio University Global Center of Excellence Program for In vivo Human Metabolomic Systems Biology from MEXT and Health and Labour Sciences Research Grants for Research on Allergic Diseases and Immunology from the Ministry of Health, Labour and Welfare.
Supporting information is available at EMBO Molecular Medicine online.
The authors declare that they have no conflicts of interest.
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