Flor Evangelista, David A. Dasher, Luis A. Diaz, Phillip S

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E-cadherin Is an Additional Immunological Target for Pemphigus Autoantibodies Flor Evangelista, David A. Dasher, Luis A. Diaz, Phillip S. Prisayanh, Ning Li Journal of Investigative Dermatology Volume 128, Issue 7, Pages 1710-1718 (July 2008) DOI: 10.1038/sj.jid.5701260 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Baculovirus expression of the ectodomain of human E-cadherin, Dsg1, and Dsg3. IB analysis of the High Five culture supernatants containing the secreted recombinant proteins E-cadherin (lane 1), Dsg1 (lane 2), and Dsg3 (lane 3). Proteins were electrophoresed on 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies to the (a) His-tag or (b) human E-cadherin. Journal of Investigative Dermatology 2008 128, 1710-1718DOI: (10.1038/sj.jid.5701260) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Anti-E-cadherin IgG autoantibodies detected in PF and FS patients. Representative IP–IB results from six PF (lanes 1–6) and six FS (lanes 7–12) sera are shown. The E-cadherin- and Dsg1-recombinant proteins were immunoprecipitated by all of the PF and FS sera, but none immunoprecipitated Dsg3. Well-characterized serum samples from a PF or PV patient were used as positive controls (+); normal human serum was used as a negative control (−). One PF serum (lane 3) and another FS (lane 7) produced a weak E-cadherin band. Journal of Investigative Dermatology 2008 128, 1710-1718DOI: (10.1038/sj.jid.5701260) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Anti-E-cadherin IgG autoantibodies detected in PV patients. Representative IP–IB results from (a) mPV and (b) mcPV sera are shown. All PV sera reacted with Dsg3. E-cadherin was not recognized by mPV sera but was bound by the majority of mcPV sera. A clear correlation between the reaction with E-cadherin and Dsg1 was observed. The reaction varies from strong reaction (lanes 1–3), moderate reaction (lanes 4, 8, and 9), weak reaction (lane 5) to no reaction (lanes 6, 7, 10). (+) Positive control; (−), negative control. Journal of Investigative Dermatology 2008 128, 1710-1718DOI: (10.1038/sj.jid.5701260) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 IP–IB analysis of sera from patients with other diseases. Representative results from sera of other diseases are shown: systemic lupus erythematosus (lanes 1–3), epidermolysis bullosa acquisita (lane 4), Sezary syndrome (lane 5), and BP (lanes 6–12). A weak positive reaction with two BP sera to Dsg1 was detected (lanes 6 and 10). None of these sera reacted with E-cadherin or Dsg3. (+) Positive control; (−) negative control. Journal of Investigative Dermatology 2008 128, 1710-1718DOI: (10.1038/sj.jid.5701260) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Elimination of E-cadherin IP reactivity in pemphigus serum by preincubation with rDsg1. Sera samples from (a) FS and PF patients or (b) mcPV patients were preincubated with a culture supernatant containing rDsg1, rDsg3, or no recombinant protein and then IP with E-cadherin. The precipitations were subjected to IB probed with a goat antiserum against E-cadherin. As shown, preincubation with a culture supernatant containing no recombinant protein did not affect the reactivity of E-cadherin (a, lanes 1 and 3; b, lane 1). However, preincubation with a culture supernatant containing rDsg1 eliminated or greatly reduced the E-cadherin reactivity (a, lanes 2 and 4; b, lane 2). In contrast, preincubation with a culture supernatant containing rDsg3 did not affect the reactivity of E-cadherin (b, lane 3). Journal of Investigative Dermatology 2008 128, 1710-1718DOI: (10.1038/sj.jid.5701260) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Competitive inhibition of the ELISA. A set of eight pemphigus sera were (a) preincubated with purified recombinant E-cadherin and tested for Dsg1 binding or (b) preincubated with purified rDsg1 and tested for E-cadherin binding by ELISA. The black section of each bar shows the % inhibition. Journal of Investigative Dermatology 2008 128, 1710-1718DOI: (10.1038/sj.jid.5701260) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 IP of normal human epidermal extracts with PF/FS sera. Epidermal extracts from neonatal foreskins were immunoprecipitated with a normal human serum (lane 1), PF sera (lanes 2–5), or an FS serum (lane 6) and subjected to IB using a mouse mAb to human E-cadherin intracellular domain. As shown, all the five patients' sera precipitated the 120-kDa E-cadherin (lanes 2–6). The normal human serum did not precipitate E-cadherin (lane 1). Journal of Investigative Dermatology 2008 128, 1710-1718DOI: (10.1038/sj.jid.5701260) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions