Antigen Selection of Anti-DSG1 Autoantibodies During and Before the Onset of Endemic Pemphigus Foliaceus Ye Qian, Stephen H. Clarke, Valeria Aoki, Gunter.

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Antigen Selection of Anti-DSG1 Autoantibodies During and Before the Onset of Endemic Pemphigus Foliaceus Ye Qian, Stephen H. Clarke, Valeria Aoki, Gunter Hans-Filhio, Evandro A. Rivitti, Luis A. Diaz Journal of Investigative Dermatology Volume 129, Issue 12, Pages 2823-2834 (December 2009) DOI: 10.1038/jid.2009.184 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Anti-desmoglein 1 (DSG1) hybridoma antibodies are specific for DSG1. (a) Representative anti-DSG1 MAbs (all are IgG1, except FGS-4E4 and GCDS-2D11 (IgG4)) from different fogo selvagem (FS) patients were used to immunoprecipitate DSG1. Hybridoma antibodies bound to protein G agarose beads were used to immunoprecipitate His-tagged DSG1 ectodomain. The immunoprecipitated samples were used in western blots using an anti-His horseradish peroxidase conjugate to detect DSG1 protein. (b) Similar to serum from an FS patient, anti-DSG1 MAbs (FGS-4E4) show typical indirect immunofluorescence staining patterns using monkey esophagus as the substrate. Bar in (b)=50μm. Journal of Investigative Dermatology 2009 129, 2823-2834DOI: (10.1038/jid.2009.184) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 V gene use by fogo selvagem (FS) and pre-FS clonally independent anti-desmoglein 1 (DSG1) hybridomas. (a) VH gene use. (b) VL gene use. IgM and IgG are summarized separately for FS (upper panels) and pre-FS (lower panels). The number of hybridomas identified by sequence similarity to use each gene is indicated. Genes used by both FS and pre-FS hybridomas are marked by an asterisk. Journal of Investigative Dermatology 2009 129, 2823-2834DOI: (10.1038/jid.2009.184) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The expressed VH genes from fogo selvagem (FS) and pre-FS hybridomas share replacement mutations. Sequence comparisons were conducted using Vector NTI (Invitrogen Life Technologies, Carlsbad, CA). All the sequences shown are from independent clones. Complementary determining regions (CDRs) (1 and 2) are boxed and replacement mutations are highlighted. Positions in which no mutations occurred are in yellow, whereas conservative replacement mutations are highlighted in blue and non-conservative replacement mutations highlighted in white. Positions where shared mutations occurred are indicated with arrows. Germline genes are shown at the bottom of each VH gene set. The amino acid differences at positions 33 (G or A) and 98 (K or R) of IGHV3-30 may be polymorphisms and are not considered in this analysis. Journal of Investigative Dermatology 2009 129, 2823-2834DOI: (10.1038/jid.2009.184) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions