Staphylococcal Exfoliative Toxin B Specifically Cleaves Desmoglein 1

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Staphylococcal Exfoliative Toxin B Specifically Cleaves Desmoglein 1 Masayuki Amagai, Koji Nishifuji Journal of Investigative Dermatology Volume 118, Issue 5, Pages 845-850 (May 2002) DOI: 10.1046/j.1523-1747.2002.01751.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Production of recombinant ETB with exfoliative activity. Coomassie Blue stained SDS-PAGE shows recombinant ETB was produced as a 27 kDa peptide (A). Neonatal mice injected with ETB showed extensive blisters 2 h after injection (B), whereas neonatal mice injected with saline alone did not blister (C). Journal of Investigative Dermatology 2002 118, 845-850DOI: (10.1046/j.1523-1747.2002.01751.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunofluorescence staining of cadherins in the epidermis of mice injected with ETB. Neonatal mice were injected with PBS (A, C, E) or ETB (B, D, F) and stained for Dsg1 (A, B), Dsg3 (C, D), and E-cadherin (E, F). Dsg1 staining showed that the cell surface staining of Dsg1 was removed by injection of ETB. The stainings of Dsg3 and E-cadherin were not affected by injection of ETB. Arrowheads indicate the location of the basement membrane zone. Scale bar: 50 µm Journal of Investigative Dermatology 2002 118, 845-850DOI: (10.1046/j.1523-1747.2002.01751.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunoblot analysis of skin extracts from neonatal mice injected with PBS or ETB. Dsg1 was degraded by ETB injection, whereas Dsg3 and E-cadherin were not affected. Each lane represents an extract from a different mouse. Arrowheads and arrows indicate intact Dsg1 and cleaved Dsg1, respectively. Journal of Investigative Dermatology 2002 118, 845-850DOI: (10.1046/j.1523-1747.2002.01751.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunoblot analysis of extracts from cultured keratinocytes incubated with ETB.(A) HaCaT cells were transduced with recombinant adenovirus with mouse Dsg1 (Dsg1) or mouse Dsg3 (Dsg3) or control adenovirus without any insert (C), and incubated with 50 µg per ml ETB for 1 h. Exogenous Dsg1 and Dsg3 was visualized anti-FLAG tag antibody. Dsg1, but not Dsg3, was degraded by ETB. Arrowheads, asterisk, and arrows indicate intact Dsg1, intact Dsg3, and cleaved Dgs1. (B) HaCaT cells transduced with recombinant adenovirus with Dsg1 was incubated with various amounts of ETB for 10 min or 1 h. Dsg1 was degraded by ETB in a dose- and time-dependent fashion. Journal of Investigative Dermatology 2002 118, 845-850DOI: (10.1046/j.1523-1747.2002.01751.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 In vitro ETB treatment of cryosectioned normal human skin. Non-fixed cryosectioned normal human skin was incubated with ETA (B, E), ETB (C, F, G, H, I)or PBS alone (A, D), and stained for extracellular domain of Dsg1 (A, B, C), extracellular domain of Dsg3 (D, E, F), cytoplasmic domain of Dsg1 (G), cytoplasmic domain of desmocollin (Dsc, H)or desmoplakin (DP, I). The staining for the extracellular domain of Dsg1 was removed by ETB (C)as seen with ETA (B), whereas the staining for Dsg3 was not affected. The staining for the cytoplasmic domain of Dsg1, desmocollins, or desmoplakin was not affected. Scale bar: 50 µm Journal of Investigative Dermatology 2002 118, 845-850DOI: (10.1046/j.1523-1747.2002.01751.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 In vitro ETB treatment of recombinant extracellular domain of Dsg1 and Dsg3. Mouse (A) or human (B) Dsg1 and Dsg3 extracellular domains produced in baculovirus were incubated with various amounts of ETB (A: lane 1, 10 µg per ml; lane 2, 3 µg per ml; lane 3, 1 µg per ml; lane 4, 0.3 µg per ml; lane 5, 0.1 µg per ml; lane 6, 0 µg per ml; B: lane 1, 1 µg per ml; lane 2, 0.3 µg per ml; lane 3, 0.1 µg per ml; lane 4, 0.03 µg per ml; lane 5, 0.01 µg per ml; lane 6, 0 µg per ml), and subjected to immunoblots and visualized with anti-E-tag monoclonal antibody. The extracellular domain of Dsg1, but not that of Dsg3, was cleaved by ETB in a dose-dependent fashion. Arrowheads and arrows indicate the intact recombinant Dsg1 and cleaved product, respectively. Bars on the left side indicate molecular weight standards 250, 100, 50, and 25 kDa, from top to bottom. Journal of Investigative Dermatology 2002 118, 845-850DOI: (10.1046/j.1523-1747.2002.01751.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions