Pathogenic Epitopes of Autoantibodies in Pemphigus Reside in the Amino-Terminal Adhesive Region of Desmogleins Which Are Unmasked by Proteolytic Processing.

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Pathogenic Epitopes of Autoantibodies in Pemphigus Reside in the Amino-Terminal Adhesive Region of Desmogleins Which Are Unmasked by Proteolytic Processing of Prosequence Mariko Yokouchi, Marwah Adly Saleh, Keiko Kuroda, Takahisa Hachiya, John R. Stanley, Masayuki Amagai, Ken Ishii Journal of Investigative Dermatology Volume 129, Issue 9, Pages 2156-2166 (September 2009) DOI: 10.1038/jid.2009.61 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Confocal microscopy of anti-desmoglein 1 single-chain variable fragment (scFv) mAbs on human skin. 3-30/3h and 1-18/L12 scFv antibodies stained the cell surface of keratinocytes throughout human epidermis. 3-97/1c and 3-94/O18O8 showed cytoplasmic staining in the superficial layers of the epidermis. Scale bars: 50μm. Journal of Investigative Dermatology 2009 129, 2156-2166DOI: (10.1038/jid.2009.61) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Binding capacity of anti-desmoglein 1 (Dsg1) single-chain variable fragment (scFv) mAbs to the Dsg1 ELISA plate changes after furin treatment. (a) Baculovirally produced recombinant Dsg1 (Dsg1-EHis), visualized by immunoblot using anti-E-tag antibody. Dsg1-EHis was detected as double bands, which were considered to be precursor and mature form of Dsg1-EHis (left lane). After treatment with furin protease, only single band was observed (right lane). White arrowhead indicates the precursor form and black arrowhead indicates the mature form. (b) Comparison of anti-Dsg1 scFv mAbs bindings between furin-treated and Tris-buffered saline-calcium (TBS-Ca)-treated (control) Dsg1 ELISA plates. The ratio was calculated according to the following formula: (optical density (OD) values obtained in furin-treated wells)/(OD values obtained in TBS-Ca-treated wells). Control ratio (ratio=1) is shown by a broken line. The binding of 3-30/3h, a pathogenic mAb, was significantly increased to nearly twofold, whereas that of 3-97/1c, non-pathogenic mAb was decreased. The binding of 1-18/L12 showed no significant change. AM3-13 is an irrelevant scFv and used as a control. Journal of Investigative Dermatology 2009 129, 2156-2166DOI: (10.1038/jid.2009.61) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Binding of anti-desmoglein 1 (Dsg1) single-chain variable fragment (scFv) mAbs to precursor-FacX-Dsg1 and mature Dsg1. (a) Molecular structure of precursor-FacX-Dsg1-EHis and mature-Dsg1-EHis. To obtain precursor form, the recognition sequence in precursor Dsg1 was substituted with that of blood coagulation factor Xa (R-Q-K-R → I-E-G-R) by endogenous proprotein convertases. To obtain mature form of Dsg1, recombinant Dsg1-EHis produced by baculovirus expression system was treated with proprotein convertase furin. (b) Precursor-FacX-Dsg1-EHis and mature-Dsg1-EHis recombinant baculoprotein were immunoprecipitated with representative anti-Dsg1 scFv antibodies or control scFv (AM3-13) and detected on an immunoblot by anti-E-tag antibody. Journal of Investigative Dermatology 2009 129, 2156-2166DOI: (10.1038/jid.2009.61) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Competition ELISA of PF sera binding by anti-desmoglein 1 (Dsg1) single-chain variable fragment (scFv) mAbs. Competition ELISA of 40 PF sera was carried out to see if the binding against standard Dsg1 ELISA plates of PF sera was blocked by adding anti-Dsg1scFv mAbs. 3-30/3h (shown in orange), 3-97/1c, 3-94/O18O8 (shown in green) and 1-18/L12 (shown in blue) were used as competitors. When >20% inhibition was considered as significant (shown in broken line), the bindings were blocked by pathogenic 3-30/3h mAb in 29 PF sera. 3-97/1c and 3-94/O18O8, which recognized the precursor form, blocked the binding in 10 PF sera. Journal of Investigative Dermatology 2009 129, 2156-2166DOI: (10.1038/jid.2009.61) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Epitope mapping of pathogenic 3-30/3h mAb. Wild-type and domain-swapped extracellular domains of human desmoglein 1 (Dsg1) and Dsg3, and of Dsg1 and Dsg2 produced by baculovirus expression system were used for immunoprecipitation with 3-30/3h mAb. The molecular structure of domain-swapped molecules is shown. The results of immunoprecipitation assays with 3-30/3h mAb are listed in the right panel. Journal of Investigative Dermatology 2009 129, 2156-2166DOI: (10.1038/jid.2009.61) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Majority of anti-desmoglein 1 (Dsg1) antibody binding in PF and PV sera targets the mature form more than the precursor form of Dsg1. (a) Coomassie blue staining of purified recombinant baculoproteins used for precursor and mature Dsg1 ELISA plates. White arrowhead indicates the precursor form and black arrowhead indicates the mature form. (b) Binding curves of representative anti-Dsg1 scFv antibodies (3-30/3h, 3-97/1c, 1-18/L12) and control scFv (AM3-13) against mature and precursor Dsg1 ELISA plate. (c) Dispersion of immunoreactivity of 45 PF sera between the mature and precursor Dsg1 ELISA plate. Each closed square represents single patient. Note that most PF sera had stronger reactivity against mature Dsg1 plate than precursor Dsg1 plate. (d) Dispersion of immunoreactivity of 20 mucocutaneous PV sera between the mature and precursor Dsg1 ELISA plate. Each closed triangle represents single patient. Similar to PF patients, most PV sera had stronger reactivity against mature Dsg1 plate than against precursor Dsg1 plate. Journal of Investigative Dermatology 2009 129, 2156-2166DOI: (10.1038/jid.2009.61) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Majority of anti-desmoglein 3 (Dsg3) antibody binding in PV sera targets the mature form more than the precursor form of Dsg1. (a) Coomassie blue staining of purified recombinant baculoproteins used for precursor and mature Dsg1 ELISA plates. White arrowhead indicates the precursor form and black arrow head indicates the mature form. (b) Binding curves of representative anti-Dsg3 mAbs (AK23, AK15) against mature and precursor Dsg3 ELISA plates. (c) Dispersion of immunoreactivity of 47 PV sera between the mature and precursor Dsg3 ELISA plates. Each closed square represents single patient. Most PV sera showed stronger reactivity against mature Dsg3 plate than against precursor Dsg3 plate. Journal of Investigative Dermatology 2009 129, 2156-2166DOI: (10.1038/jid.2009.61) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Time-course analysis of immunoreactivity against precursor and mature form of desmoglein (Dsg) in PF and PV patients. Immunoreactivities against precursor and mature form of Dsg were compared with the disease activity over a time course. four PF patients (a, c, e, and g) and four PV patients (b, d, f, and h) were studied. Each serum was diluted up to 1:100 (a), 1:100 (b), 1:100 (c), 1:200 (d), 1:200 (e), 1:100 (f), 1:6400 (g), and 1:100 (h). Clinical disease activity was subjectively assessed and the disease activity in the first presentation was scored as 10. (0 (normal) to 10 (most severe)). three PF (a, c, and e) and three PV (b, d, and f) were randomly chosen from the PF and PV patients’ sera, which showed stronger reactivity against the mature Dsg plate than against the precursor Dsg plate. As unusual cases, a PF (g) and a PV case (h) who showed stronger reactivity against precursor-Dsg than against the mature-Dsg form were selected. Journal of Investigative Dermatology 2009 129, 2156-2166DOI: (10.1038/jid.2009.61) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Proteolytic processing of the precursor form of desmogleins (Dsgs) and mAb binding to the precursor and mature form of Dsg1. (a) Dsgs are thought to be synthesized as inactive precursor Dsgs with prosequences in the endoplasmic reticulum. The prosequences are then removed by subtillisin-like proprotein convertases before transporting them to the plasma membrane and mature Dsgs are assembled into desmosomes. (b) Binding of anti-Dsg1 mAbs to the mature and the precursor form of Dsg1. The pathogenic mAb, 3-30/3h (shown in orange) binds to the mature Dsg1, but not the precursor Dsg1. Some non-pathogenic mAbs, 3-97/1c and 3-94/O18O8 (shown in green), bind to the precursor Dsg1. A non-pathogenic mAb, 1-18/L12 (shown in blue), which recognizes the middle portion of Dsg1, binds to both mature and precursor Dsg1. The pathogenic mAb bind the region of adhesive interface of Dsg1, which are unmasked by proteolytic processing, perhaps blocking trans-interaction of Dsgs. Journal of Investigative Dermatology 2009 129, 2156-2166DOI: (10.1038/jid.2009.61) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions