ABSTRACT
Pemphigus vulgaris (PV) is an autoimmune blistering disease of skin and mucous membranes caused by autoantibodies to the desmoglein (DSG) family proteins DSG3 and DSG1, leading to loss of keratinocyte cell adhesion. To learn more about pathogenic PV autoantibodies, we isolated 15 IgG antibodies specific for DSG3 from 2 PV patients. Three antibodies disrupted keratinocyte monolayers in vitro, and 2 were pathogenic in a passive transfer model in neonatal mice. The epitopes recognized by the pathogenic antibodies were mapped to the DSG3 extracellular 1 (EC1) and EC2 subdomains, regions involved in cis-adhesive interactions. Using a site-specific serological assay, we found that the cis-adhesive interface on EC1 recognized by the pathogenic antibody PVA224 is the primary target of the autoantibodies present in the serum of PV patients. The autoantibodies isolated used different heavy- and light-chain variable region genes and carried high levels of somatic mutations in complementary-determining regions, consistent with antigenic selection. Remarkably, binding to DSG3 was lost when somatic mutations were reverted to the germline sequence. These findings identify the cis-adhesive interface of DSG3 as the immunodominant region targeted by pathogenic antibodies in PV and indicate that autoreactivity relies on somatic mutations generated in the response to an antigen unrelated to DSG3.
Subject(s)
Autoantibodies/immunology , Autoantigens/immunology , Desmoglein 3/immunology , Immunoglobulin G/immunology , Pemphigus/immunology , Somatic Hypermutation, Immunoglobulin , Amino Acid Sequence , Animals , Animals, Newborn , Antigen-Antibody Reactions , Autoantigens/chemistry , Cells, Cultured , Complementarity Determining Regions/immunology , Desmoglein 3/chemistry , Epitopes/chemistry , Epitopes/immunology , Humans , Immunization, Passive , Immunoglobulin Variable Region/genetics , Keratinocytes/immunology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
During DNA replication and repair, many proteins bind to and dissociate in a highly specific and ordered manner from proliferating cell nuclear antigen (PCNA). We describe a combined approach of in silico searches at the genome level and combinatorial peptide synthesis to investigate the binding properties of hundreds of short PCNA-interacting peptides (PIP-peptides) to archaeal and eukaryal PCNAs. Biological relevance of our combined approach was demonstrated by identification an inactive complex of Pyrococcus abyssi ribonuclease HII with PCNA. Furthermore we show that PIP-peptides interact with PCNA largely in a sequence independent manner. Our experimental approach also identified many so far unidentified PCNA interacting peptides in a number of human proteins.
