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SASPase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing.

Matsui T, Miyamoto K, Kubo A, Kawasaki H, Ebihara T, Hata K, Tanahashi S, Ichinose S, Imoto I, Inazawa J, Kudoh J, Amagai M - EMBO Mol Med (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Medical Top Track (MTT) Program, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan. tmatsui@icems.kyoto-u.ac.jp

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Aberrant expression of filaggrin in SASP−/− hairless miceImmunofluorescence staining of frozen sections of the back skin of SASP+/− and SASP−/− mice stained with anti-filaggrin antibody (red). Nuclei were counterstained with bisbenzimide (blue). The SASP−/− epidermis showed an increased amount of filaggrin-positive stained lower SC. Dashed lines represent the border between the epidermis and dermis. BF, bright field. Scale bar: 10 µm.Enlarged view of A shows an increased amount of lower SC layers in the SASP−/− hairless epidermis.Equivalent amounts of tape-stripped extracts (10 times, 5 µg) from SASP+/+ (n = 2), SASP+/− (n = 2), and SASP−/− (n = 2) mice were immunoblotted with anti-filaggrin antibodies. CBB staining of extracts revealed a reduced expression of filaggrin monomer bands and other major SC proteins in the SASP−/− hairless mice epidermis (left; Coomassie). Immunoblotting with anti-filaggrin demonstrated that an accumulation of aberrant filaggrin degradation products (dimer and trimer sized) was detected (right), whereas mature filaggrin was rarely detected. As equivalent amounts of SC extracts were loaded, the intensity of the profilaggrin band was decreased in SASP−/−, possibly due to an increase in the concentrations of other smear proteins.
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fig04: Aberrant expression of filaggrin in SASP−/− hairless miceImmunofluorescence staining of frozen sections of the back skin of SASP+/− and SASP−/− mice stained with anti-filaggrin antibody (red). Nuclei were counterstained with bisbenzimide (blue). The SASP−/− epidermis showed an increased amount of filaggrin-positive stained lower SC. Dashed lines represent the border between the epidermis and dermis. BF, bright field. Scale bar: 10 µm.Enlarged view of A shows an increased amount of lower SC layers in the SASP−/− hairless epidermis.Equivalent amounts of tape-stripped extracts (10 times, 5 µg) from SASP+/+ (n = 2), SASP+/− (n = 2), and SASP−/− (n = 2) mice were immunoblotted with anti-filaggrin antibodies. CBB staining of extracts revealed a reduced expression of filaggrin monomer bands and other major SC proteins in the SASP−/− hairless mice epidermis (left; Coomassie). Immunoblotting with anti-filaggrin demonstrated that an accumulation of aberrant filaggrin degradation products (dimer and trimer sized) was detected (right), whereas mature filaggrin was rarely detected. As equivalent amounts of SC extracts were loaded, the intensity of the profilaggrin band was decreased in SASP−/−, possibly due to an increase in the concentrations of other smear proteins.

Mentions: 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 regulates stratum corneum hydration through profilaggrin-to-filaggrin processing.

Matsui T, Miyamoto K, Kubo A, Kawasaki H, Ebihara T, Hata K, Tanahashi S, Ichinose S, Imoto I, Inazawa J, Kudoh J, Amagai M - EMBO Mol Med (2011)

Aberrant expression of filaggrin in SASP−/− hairless miceImmunofluorescence staining of frozen sections of the back skin of SASP+/− and SASP−/− mice stained with anti-filaggrin antibody (red). Nuclei were counterstained with bisbenzimide (blue). The SASP−/− epidermis showed an increased amount of filaggrin-positive stained lower SC. Dashed lines represent the border between the epidermis and dermis. BF, bright field. Scale bar: 10 µm.Enlarged view of A shows an increased amount of lower SC layers in the SASP−/− hairless epidermis.Equivalent amounts of tape-stripped extracts (10 times, 5 µg) from SASP+/+ (n = 2), SASP+/− (n = 2), and SASP−/− (n = 2) mice were immunoblotted with anti-filaggrin antibodies. CBB staining of extracts revealed a reduced expression of filaggrin monomer bands and other major SC proteins in the SASP−/− hairless mice epidermis (left; Coomassie). Immunoblotting with anti-filaggrin demonstrated that an accumulation of aberrant filaggrin degradation products (dimer and trimer sized) was detected (right), whereas mature filaggrin was rarely detected. As equivalent amounts of SC extracts were loaded, the intensity of the profilaggrin band was decreased in SASP−/−, possibly due to an increase in the concentrations of other smear proteins.
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Related In: Results  -  Collection

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fig04: Aberrant expression of filaggrin in SASP−/− hairless miceImmunofluorescence staining of frozen sections of the back skin of SASP+/− and SASP−/− mice stained with anti-filaggrin antibody (red). Nuclei were counterstained with bisbenzimide (blue). The SASP−/− epidermis showed an increased amount of filaggrin-positive stained lower SC. Dashed lines represent the border between the epidermis and dermis. BF, bright field. Scale bar: 10 µm.Enlarged view of A shows an increased amount of lower SC layers in the SASP−/− hairless epidermis.Equivalent amounts of tape-stripped extracts (10 times, 5 µg) from SASP+/+ (n = 2), SASP+/− (n = 2), and SASP−/− (n = 2) mice were immunoblotted with anti-filaggrin antibodies. CBB staining of extracts revealed a reduced expression of filaggrin monomer bands and other major SC proteins in the SASP−/− hairless mice epidermis (left; Coomassie). Immunoblotting with anti-filaggrin demonstrated that an accumulation of aberrant filaggrin degradation products (dimer and trimer sized) was detected (right), whereas mature filaggrin was rarely detected. As equivalent amounts of SC extracts were loaded, the intensity of the profilaggrin band was decreased in SASP−/−, possibly due to an increase in the concentrations of other smear proteins.
Mentions: 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.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Medical Top Track (MTT) Program, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan. tmatsui@icems.kyoto-u.ac.jp

Show MeSH
Related in: MedlinePlus