The Intracellular and Extracellular Domains of BP180 Antigen Comprise Novel Epitopes Targeted by Pemphigoid Gestationis Autoantibodies Giovanni Di Zenzo,

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The Intracellular and Extracellular Domains of BP180 Antigen Comprise Novel Epitopes Targeted by Pemphigoid Gestationis Autoantibodies Giovanni Di Zenzo, Valentina Calabresi, Fabiana Grosso, Marzia Caproni, Marina Ruffelli, Giovanna Zambruno Journal of Investigative Dermatology Volume 127, Issue 4, Pages 864-873 (April 2007) DOI: 10.1038/sj.jid.5700594 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The PG sera bind to the selected epitopes of BP180. (a) The diagram represents the BP180 protein aligned with the 15 epitopes tested. The AA residues that define the antigenic sites are indicated under the epitope lines. PG-specific epitopes are shown as black lines, while epitopes recognized with comparable frequency by both PG and control sera are depicted as hatched lines. (b) The reactivity of 15PG sera was assessed by immunoscreening against the selected BP180 epitopes, as shown in some representative filters. Positive signals are indicated (arrows) and correspond to phages exposing BP180 peptides recognized by representative PG sera. The PG2 filter shows the epitope positions in the matrix. (1) E-353; (2) E-508; (3) E-567; (4) corresponds to the phage clone without any peptide exposed on its capsid surface; (5) E-1331; (6) E-282; (7) corresponds to the phage clone with unrelated peptide 1; (8) E-1080; (9) E-915; (10) E-292; (11) E-176; (12) E-773; (13) E-299; (14) E-120; (15), E-151; (16) E-1354; (17), E-266; (18) corresponds to the phage clone with unrelated peptide 2. The control serum (normal healthy individual 1) does not show any positive signal. Journal of Investigative Dermatology 2007 127, 864-873DOI: (10.1038/sj.jid.5700594) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Five PG sera recognize three independent epitopes in the BP180 region encompassing residues 292–399. (a) Competitive inhibition ELISA with PG1, PG9, and PG13 sera against the epitopes E-292, E-299, and E-353. Reactivity of PG sera is almost completely abolished by competition with GST-316 epitope, whereas reactivity against E-353 is not affected. (b) Schematic representation of the overlapping fragments coated an ELISA plate (black lines) and the competitive fusion protein GST-316 (gray line). The assay shows the existence of two independent epitopes (hatched lines) encompassing residues 316–354 and 367–399. (c) Competitive inhibition ELISA with PG3 and PG7 sera against the epitopes E-292 and E-353. Reactivity of PG sera is completely abolished by competition with GST-316 epitope. (d) Schematic representation of the overlapping fragments coated an ELISA plate (black lines) and the competitive fusion protein GST-316 (gray line). The assay shows the existence an epitope (hatched line) encompassing residues 353–367 localized in the overlapping region shared by all the fragments. Journal of Investigative Dermatology 2007 127, 864-873DOI: (10.1038/sj.jid.5700594) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Human and rabbit anti-GST-316 antibodies react with the epidermal side of salt-split human skin and with BP180 antigen. (a) A rabbit polyclonal antibody raised against the fusion protein GST-316 (encompassing residues 316–367) stains the epidermal side of salt-split human skin by IIF and (b) reacts with BP180 antigen by IB on keratinocyte extracts (lane 1, anti-GST-316 polyclonal antibody; lane 2, preimmune rabbit serum). Dilution of polyclonal and preimmune rabbit sera was 1:10,000. (c) In a similar way, immunopurified and concentrated PG3 serum stains the epidermal side of salt-split human skin by IIF and displays reactivity by IB on keratinocyte extracts (d, lane 2PG serum, lane 3PG serum affinity purified against the epitope GST-316). 1A8c is a mAb against BP180 used as positive control (lane 1). Journal of Investigative Dermatology 2007 127, 864-873DOI: (10.1038/sj.jid.5700594) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Journal of Investigative Dermatology 2007 127, 864-873DOI: (10.1038/sj.jid.5700594) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions