A synthetic non-histone substrate to study substrate targeting by the Gcn5 HAT and sirtuin HDACs.

Gcn5 and sirtuins are highly conserved histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes that were first characterized as regulators of gene expression. Although histone tails are important substrates of these enzymes, they also target many nonhistone proteins that function in diverse biological processes. However, the mechanisms used by these enzymes to choose their nonhistone substrates are unknown. Previously, we used SILAC-based MS to identify novel nonhistone substrates of Gcn5 and sirtuins in yeast and found a shared target consensus sequence. Here, we use a synthetic biology approach to demonstrate that this consensus sequence can direct acetylation and deacetylation targeting by these enzymes in vivo Remarkably, fusion of the sequence to a nonsubstrate confers de novo acetylation that is regulated by both Gcn5 and sirtuins. We exploit this synthetic fusion substrate as a tool to define subunits of the Gcn5-containing SAGA and ADA complexes required for nonhistone protein acetylation. In particular, we find a key role for the Ada2 and Ada3 subunits in regulating acetylations on our fusion substrate. In contrast, other subunits tested were largely dispensable, including those required for SAGA stability. In an extended analysis, defects in proteome-wide acetylation observed in ada3Δ mutants mirror those in ada2Δ mutants. Altogether, our work argues that nonhistone protein acetylation by Gcn5 is determined in part by specific amino acids surrounding target lysines but that even optimal sequences require both Ada2 and Ada3 for robust acetylation. The synthetic fusion substrate we describe can serve as a tool to further dissect the regulation of both Gcn5 and sirtuin activities in vivo.

Overview of the generation, validation, and application of phosphosite-specific antibodies.

Protein phosphorylation is a universal key posttranslational modification that affects the activity and other properties of intracellular proteins. Phosphosite-specific antibodies can be produced as polyclonals or monoclonals in different animal species, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is key for their use in proteomics and profiling of disease.

Pathogenic human monoclonal antibody against desmoglein 3.

Pemphigus vulgaris (PV) is a potentially fatal autoimmune mucocutaneous disease associated with production of IgG autoantibodies to desmoglein 3 (Dsg3), a 130-kDa epidermal cadherin protein. The binding of pathogenic antibody to Dsg3 on epidermal keratinocytes leads to loss of intercellular adhesion and results in intraepithelial blister formation. Here, we describe a human monoclonal antibody, PVMAB786, a Dsg3-specific IgG4 antibody, from an untreated patient with active PV. The antibody reacts with a 130-kDa protein on keratinocyte cell surfaces and recombinant Dsg3 protein, but not desmoglein 1 protein. PVMAB786 induces acantholysis in normal human skin and mucous membranes and induces a clinical and histological profile similar to human PV when injected into neonatal mice. PVMAB786 will be a valuable tool in identifying the role of Dsg3 in epithelial cell adherence and acantholysis, mechanisms of Dsg3 processing/presentation and V gene and isotype usage in PV pathogenesis.

Role of intramolecular epitope spreading in pemphigus vulgaris.

Pemphigus vulgaris (PV) is an acquired immunobullous disorder. At the early stage of the disease (mucosal PV), patients display only autoimmunity to desmoglein (Dsg) 3 and develop mucosal blisters; while at the later stage of the disease (mucocutaneous PV), patients exhibit non-cross-reactive autoimmunity to both Dsg3 and Dsg1 and acquire cutaneous as well as mucosal blisters. At these two disease stages, Dsg3 autoantibodies exhibit different tissue-binding patterns and pathogenic activities, suggesting that they may recognize distinct epitopes. To test this hypothesis and to investigate the mechanism underlying the disease transition, we studied Dsg3 autoantibody epitopes from mucosal PV patients and patients exhibiting disease transition to mucocutaneous PV. We demonstrated that autoantibodies from the majority of mucosal PV patients target epitopes at the COOH-terminal portion of the Dsg3 ectodomain. Interestingly, only autoantibodies against the Dsg3 NH2-terminal epitope(s) are able to bind human skin. Moreover, we discovered that the intramolecular epitope spreading from Dsg3(87-566) to Dsg3(1-88) is a critical step that precedes the intermolecular epitope spreading from Dsg3 to Dsg1. During disease transition, this mechanism dictates the development of Dsg3 autoantibodies that recognize human skin and lead to expression of cutaneous PV lesions.

Characterization of polyclonal antibodies raised against a linear peptide determinant of desmoglein-3.

Our labs are pursueing the goal of exactly defining the qualities of peptide antigenicity, immunogenicity and pathogenicity in order to develop safe specific immunotherapies by utilizing specific portions of disease-associated-proteins (DAPs). Using as a model the Pemphigus vulgaris antigen (PVA) desmoglein 3 (Dsg3), we have studied the murine humoral response against the Dsg3 amino acid 49-60 peptide sequence, previously characterized as sequence having low similarity to the mouse proteome. The results show that the low-similarity Dsg3(49-60)REWVKFAKPCRE peptide does not elicit pathogenic antibodies.

Epitope definition by proteomic similarity analysis: identification of the linear determinant of the anti-Dsg3 MAb 5H10.

BACKGROUND: Walking along disease-associated protein sequences in the search for specific segments able to induce cellular immune response may direct clinical research towards effective peptide-based vaccines. To this aim, we are studying the targets of the immune response in autoimmune diseases by applying the principle of non-self-discrimination as a driving concept in the identification of the autoimmunogenic peptide sequences. METHODS: Computer-assisted proteomic analysis of the autoantigen protein sequence and dot-blot/NMR immunoassays are applied to the prediction and subsequent validation of the epitopic sequences. RESULTS: Using the experimental model Pemphigus vulgaris/desmoglein 3, we have identified the antigenic linear determinant recognized by MAb 5H10, a monoclonal antibody raised against the extracellular domain of human desmoglein-3. The computer-assisted search for the Dsg3 epitope was conducted by analyzing the similarity level to the mouse proteome of the human desmoglein protein sequence. Dot-blot immunoassay analyses mapped the epitope within the sequence Dsg349-60 REWVKFAKPCRE, which shows low similarity to the mouse proteome. NMR spectroscopy analyses confirmed the specificity of MAb 5H10 for the predicted epitope. CONCLUSIONS: This report promotes the concept that low level of sequence similarity to the host's proteome may modulate peptide epitopicity.

T cell receptor gene usage of BP180-specific T lymphocytes from patients with bullous pemphigoid and pemphigoid gestationis.

BP180 is the autoantigen of different immunobullous diseases, including bullous pemphigoid (BP) and pemphigoid gestationis (PG). Previously, we demonstrated that the NC16A domain of this autoantigen harbors key epitopes of autoantibodies and T cells, indicating that it plays an essential role in the pathogenesis of diseases. Moreover, NC16A-specific T cell clones derived from these patients were shown to express a CD4+ memory T cell phenotype and secrete cytokines that may promote autoantibody production. In this study, we further characterize the properties of these T cells by analyzing their epitope specificity and T cell receptor (TCR) gene usage. We discovered that 83% of T cell clones obtained from BP patients preferentially express TCRBV13, while clones derived from a PG patient express the TCRBV3 gene. However, no preferential TCRBJ gene usage was identified. In conclusion, our results provide an advanced understanding of the characteristics of autoimmune T cells in immunobullous diseases.

A patient with both bullous pemphigoid and epidermolysis bullosa acquisita: an example of intermolecular epitope spreading.

Bullous pemphigoid (BP) and epidermolysis bullosa acquisita are distinct autoimmune blistering disorders. BP is characterized by autoantibodies directed against the NC16A domain of collagen XVII, whereas patients with epidermolysis bullosa acquisita have autoantibodies against the NC1 domain of type VII collagen. We followed up a patient with BP for 9 years. During that time his clinical disease took on several features suggestive of epidermolysis bullosa acquisita. The objective of this study was to determine if the patient's autoantibody profile reflected the change in his clinical picture. Enzyme-linked immunosorbent assay and immunoblotting for detection and subclass determination of autoantibodies to type XVII and type VII collagen were performed on banked patient sera from the 9-year period. The patient's initial autoantibodies were exclusively IgG1 directed against collagen XVII. During the course of his illness, the subclass specificity of the patient's type XVII collagen autoantibodies shifted to the IgG4 subclass and during the same time interval the patient developed IgG2 autoantibodies directed against type VII collagen. This patient with BP exhibited both subclass shifting and development of a second autoantibody system that correlated with a change in the clinical appearance of the disease. The analysis of the patient's autoantibodies provides strong evidence for the involvement of epitope spreading in the evolution of his autoimmune disease.

T cell receptor gene usage in desmoglein-3-specific T lymphocytes from patients with pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune disease mediated by autoantibodies against desmoglein-3 (Dsg3). It has been documented that both humoral and cellular autoimmunity play essential roles in the development of PV. Recently, we identified that T cells from PV patients respond to three antigenic fragments on the ectodomain of Dsg3. These T cells are CD4 alpha/beta cells secreting a Th2-like cytokine profile, and responding of Dsg3 in a restriction to HLA-DRBI*0402 or 1401 alleles. Other characteristics of these cells, such as detailed epitope(s) and T cell receptors (TCRs) usage, however, have not been investigated. The purpose of this study is to determine detailed T cell epitope(s) and TCR genes utilized by Dsg3-specific T cells. Here, we found that Dsg3(AA145-192)-specific cells preferentially utilize the TCRVbeta13 gene, while Dsg3(AA240-303)- and Dsg3 (AA570-614)-specific cells utilize Vbeta7 and Vbeta17 genes, respectively. Analysis of TCRValpha gene expression, it appears that Valpha22 gene is expressed by Dsg3(AA145-192)-specific cells, whereas the Valpha10 gene is predominantly utilized by Dsg3(AA240-303)-specific T cells. There are no specific utilization of Valpha gene in the group of cells proliferate to Dsg3 (AA570-614). We believe that this information will further our understanding of the properties of autoimmune T cells in patients with PV.

Pathogenicity and epitope characteristics of anti-desmoglein-1 from pemphigus foliaceus patients expressing only IgG1 autoantibodies.

Pemphigus foliaceus (PF) is an antibody-mediated autoimmune disorder with IgG1 and IgG4 as the predominant subclasses of autoantibodies against a desmosomal glycoprotein, desmoglein-1 (Dsg1). Previously, we found that the IgG4 anti-Dsg1 autoantibodies only recognize a conformational epitope(s), whereas the IgG1 autoantibodies recognize both conformational and linear epitopes but do not display pathogenicity in the passive transfer animal model. The purpose of this study was to analyze the epitopes recognized by autoanti-bodies from a subset of PF patients who only express anti-Dsg1 of the IgG1 isotype throughout the course of their diseases and to further characterize the pathogenicity of their IgG1 anti-Dsg1. We found that IgG1 auto-antibodies in this subset of PF patients, similar to IgG4 autoantibodies from other PF patients, are able to bind both human and mouse skin and induce the experimental PF in mice. Moreover, a detailed epitope mapping reveals that the conformational epitopes recognized by IgG1 autoantibodies from these PF patients are restricted to the first 161 amino acids of Dsg1, whereas the linear epitopes are spread throughout the entire ectodomain. In conclusion, our study reveals that the isotype of IgG does not necessarily determine the epitopes and pathogenicity of pemphigus autoantibodies.

Isotypes and antigenic profiles of pemphigus foliaceus and pemphigus vulgaris autoantibodies.

In this study we systematically characterized isotype profiles and antigenic and tissue specificity of antidesmoglein autoantibodies from patients with pemphigus foliaceus (PF) and pemphigus vulgaris (PV) using enzyme-linked immunoabsorbent assays (ELISA), indirect immunofluorescence (IIF) staining, and immunoblotting (IB). In PF, we found that IgG1 antidesmoglein-1 (Dsg1) reacts with a linear epitope(s) on the ectodomain of Dsg1, while its IgG4 counterpart recognizes a conformational epitope(s). These two subclasses of anti-Dsg1 are both capable of recognizing tissues from monkey esophagus and adult human skin, but IgG1 is not able to react with mouse skin, which may explain why this isotype of anti-Dsg1 failed to induce PF-like lesions in the passive transfer animal model. In mucosal PV patients, we found that both IgG1 and IgG4 only recognized monkey esophagus tissue by IIF, except in one patient, indicating that these antibodies react with a unique conformational epitope(s) that is present in mucosal but not skin tissue. In generalized PV, IgG1 anti-Dsg3 autoantibodies appeared to recognize a linear epitope(s) on the Dsg3 ectodomain. In contrast, IgG4 anti-Dsg3 antibodies recognized both linear and conformational epitopes on the Dsg3 molecule. Interestingly, the IgG1 anti-Dsg3 antibodies failed to react with human and mouse skin tissues, suggesting that this subclass of autoantibodies may not play an essential role in the development of PV suprabasilar lesions. In summary, we conclude that this study further elucidates the pathological mechanisms of PF and PV autoantibodies by revealing their distinct isotype and antigenic profiles. This information may help us to better understand the autoimmune mechanisms underlying the development of pemphigus.

T cell receptor Beta chain gene usage in endemic pemphigus foliaceus (fogo selvagem).

The trimolecular complex comprised of the major histocompatibility complex, peptide antigen, and the T cell receptor is a requisite for T cell activation in normal and autoimmune responses. T cell receptor analysis is critical to further our understanding regarding mechanisms of T cell epitope selection and autoimmune initiation and progression and may help to identify targets for immunotherapy. Pemphigus foliaceus is an autoimmune blistering skin disease characterized by intraepidermal blisters and circulating autoantibodies directed against desmoglein 1, a 160 kDa transmembrane desmosomal molecule expressed in keratinocytes. As tissue damage is mediated by anti-desmoglein 1 antibodies, an initial T cell response is a likely requirement for autoantibody generation in this disease. To elucidate the role of pathogenic T cells in autoimmunity further, we have directly characterized the T cell receptor of T cells derived from pemphigus foliaceus patients. Complementary DNA was isolated from 17 desmoglein 1 specific T cell clones generated from pemphigus foliaceus patients by clonal expansion in vitro. To analyze the T cell repertoire, a panel of primers, collectively specific for the known human T cell receptor beta variable region (TCRBV) families were paired with a constant region primer to polymerase chain reaction to amplify one distinct T cell receptor beta variable region allele for each T cell clone studied. Polymerase chain reaction products were sequenced to determine exact beta chain gene usage. In the 17 clones tested, 10 distinct T cell receptor beta variable region usages and nine T cell receptor beta joining gene segment usages were identified. Furthermore, T cell receptor beta variable region and beta joining usage did not appear to be random, but oligoclonal in nature, with some preference shown for T cell receptor beta variable region 5S1 and T cell receptor BJ2S5.

A subset of pemphigus foliaceus patients exhibits pathogenic autoantibodies against both desmoglein-1 and desmoglein-3.

In pemphigus vulgaris the major pathogenic antibody binds desmoglein-3, and mediates mucosal disease. Development of cutaneous disease is associated with acquisition of antibodies to desmoglein-1. In pemphigus foliaceus, and its endemic form, fogo selvagem by contrast, the major pathogenic antibody recognizes desmoglein-1 and mediates cutaneous disease only. In this study, we sought to determine the prevalence of antibodies to desmoglein-3 in patients with pemphigus foliaceus and fogo selvagem. We produced recombinant desmoglein-1 and desmoglein-3, and used them in highly sensitive and specific enzyme-linked immunosorbent assays, as well as immunoprecipitation assays. We detected antibodies to desmoglein-3 in 19 of 276 patients with pemphigus foliaceus and fogo selvagem, who had cutaneous disease only. We showed that these antibodies to desmoglein-3 could be absorbed in a concentration-dependent manner by desmoglein-3 but not by desmoglein-1. Also antibodies to desmoglein-1 could be absorbed in a concentration-dependent manner by desmoglein-1 but not desmoglein-3. This suggests that two separate species of antibody are present rather than one antibody capable of cross-reacting with both desmoglein-1 and desmoglein-3. Finally, it was shown that affinity-purified antibodies to desmoglein-3 from patients with pemphigus foliaceus and fogo selvagem induced a pemphigus vulgaris-like skin disease in mice by passive transfer. These results suggest that a subset of patients with pemphigus foliaceus and fogo selvagem have antibodies to desmoglein-3 that may be involved in the pathogenesis of their cutaneous disease.

Autoimmune responses in patients with linear IgA bullous dermatosis: both autoantibodies and T lymphocytes recognize the NC16A domain of the BP180 molecule.

Linear IgA bullous disease (LABD) is an autoimmune skin disease characterized by subepidermal blisters and IgA autoantibodies directed against the epidermal basement membrane zone (BMZ) of the skin. Various antigens have been identified as targets of IgA autoantibodies including BP180, a type II glycoprotein that spans the BMZ and lamina lucida. Previously, we have identified a subset of LABD patients whose sera contained IgA antibodies against the 16th noncollagenous (NC16A) domain of BP180. NC16A was previously shown to harbor epitopes that are recognized by both autoantibodies and T cells from patients with bullous pemphigoid and herpes gestationis and is thought to be associated with the development of these immunobullous diseases. The aim of this study was to determine whether T lymphocytes from LABD patients with anti-NC16A IgA autoantibodies respond to epitopes in the same region of the BP180 protein. Indeed, of the four LABD patients in our study, all had T cells that specifically proliferated in response to NC16A. Moreover, two subfragments of NC16A were identified as the predominant targets of LABD T cells. Further analysis of T cell lines and clones derived from these patients revealed that these cells express a CD4 memory T cell phenotype and secrete a Th1/Th2 mixed-cytokine profile, characteristics similar to those of T cells in bullous pemphigoid patients. Our data suggest that the BP180 protein, typically the NC16A region, is the common target of both cellular and humoral immune responses in some LABD patients. This information helps to further elucidate the autoimmune mechanisms in this disease.