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A proteomic repertoire of autoantigens identified from the classic autoantibody clinical test substrate HEp-2 cells.

ABSTRACT: Background:Autoantibodies are a hallmark of autoimmune diseases. Autoantibody screening by indirect immunofluorescence staining of HEp-2 cells with patient sera is a current standard in clinical practice. Differential diagnosis of autoimmune disorders is based on commonly recognizable nuclear and cytoplasmic staining patterns. In this study, we attempted to identify as many autoantigens as possible from HEp-2 cells using a unique proteomic DS-affinity enrichment strategy. Methods:HEp-2 cells were cultured and lysed. Total proteins were extracted from cell lysate and fractionated with DS-Sepharose resins. Proteins were eluted with salt gradients, and fractions with low to high affinity were collected and sequenced by mass spectrometry. Literature text mining was conducted to verify the autoantigenicity of each protein. Protein interaction network and pathway analyses were performed on all identified proteins. Results:This study identified 107 proteins from fractions with low to high DS-affinity. Of these, 78 are verified autoantigens with previous reports as targets of autoantibodies, whereas 29 might be potential autoantigens yet to be verified. Among the 107 proteins, 82 can be located to nucleus and 15 to the mitotic cell cycle, which may correspond to the dominance of nuclear and mitotic staining patterns in HEp-2 test. There are 55 vesicle-associated proteins and 12 ribonucleoprotein granule proteins, which may contribute to the diverse speckled patterns in HEp-2 stains. There are also 32 proteins related to the cytoskeleton. Protein network analysis indicates that these proteins have significantly more interactions among themselves than would be expected of a random set, with the top 3 networks being mRNA metabolic process regulation, apoptosis, and DNA conformation change. Conclusions:This study provides a proteomic repertoire of confirmed and potential autoantigens for future studies, and the findings are consistent with a mechanism for autoantigenicity: how self-molecules may form molecular complexes with DS to elicit autoimmunity. Our data contribute to the molecular etiology of autoimmunity and may deepen our understanding of autoimmune diseases.

SUBMITTER: Wang JY

PROVIDER: S-EPMC7507713 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A proteomic repertoire of autoantigens identified from the classic autoantibody clinical test substrate HEp-2 cells.

Wang Julia Y JY Zhang Wei W Rho Jung-Hyun JH Roehrl Michael W MW Roehrl Michael H MH

Clinical proteomics 20200921

<h4>Background</h4>Autoantibodies are a hallmark of autoimmune diseases. Autoantibody screening by indirect immunofluorescence staining of HEp-2 cells with patient sera is a current standard in clinical practice. Differential diagnosis of autoimmune disorders is based on commonly recognizable nuclear and cytoplasmic staining patterns. In this study, we attempted to identify as many autoantigens as possible from HEp-2 cells using a unique proteomic DS-affinity enrichment strategy.<h4>Methods</h4> ...[more]

PMID: 32973414

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Project description:ObjectivesPrimary open-angle glaucoma (POAG) is a neurodegenerative disorder leading to a gradual vision loss caused by progressive damage to the optic nerve. Immunological processes are proposed to be involved in POAG pathogenesis. Altered serological autoantibody levels have been frequently reported, but complete analyses of the natural autoantibodies with respect to disease-related alterations are scarce. Here, we provide an explorative analysis of pathways and biological processes that may involve naturally immunogenic proteins and highlight POAG-specific alterations.MethodsMass spectrometry-based antibody-mediated identification of autoantigens (MS-AMIDA) was carried out in healthy and glaucomatous trabecular meshwork (TM) cell lines, using antibody pools purified from serum samples of 30 POAG patients and 30 non-glaucomatous subjects. Selected antigens were validated by protein microarray (n = 120). Bioinformatic assessment of identified autoantigens, including Gene Ontology (GO) enrichment analysis and protein-protein interaction networks, was applied.ResultsOverall, we identified 106 potential autoantigens [false discovery rate (FDR) < 0.01], from which we considered 66 as physiological targets of natural autoantibodies. Twenty-one autoantigens appeared to be related to POAG. Bioinformatic analysis revealed that the platelet-derived growth factor receptor beta (PDGFRB) pathway involved in TM fibrosis was particularly rich in POAG-related antigens. Antibodies to threonine-tRNA ligase (TARS), component 1 Q subcomponent-binding protein (C1QBP) and paraneoplastic antigen Ma2 (PNMA2) showed significantly (P < 0.05) higher levels in POAG patients as validated by protein microarray.ConclusionThis study provides new insights into autoimmunity in health and glaucoma. Bioinformatic analysis of POAG-related autoantigens showed a strong association with the PDGFRB pathway and also increased levels of PNMA2, TARS, and C1QBP autoantibodies in the serum of POAG patients as potential glaucoma biomarkers.

Project description:BackgroundAutoantibodies against tumor associated antigens are highly related to cancer progression. Autoantibodies could serve as indicators of tumor burden, and have the potential to monitor the response of treatment and tumor recurrence. However, how the autoantibody repertoire changes in response to cancer treatment are largely unknown.MethodsSera of five lung adenocarcinoma patients before and after surgery, were collected longitudinally. These sera were analyzed on a human proteome microarray of 20,240 recombinant proteins to acquire dynamic autoantibody repertoire in response to surgery, as well as to identify the antigens with decreased antibody response after tumor excision or surgery, named as surgery-associated antigens. The identified candidate antigens were then used to construct focused microarray and validated by longitudinal sera collected from a variety of time points of the same patient and a larger cohort of 45 sera from lung adenocarcinoma patients.FindingsThe autoantibody profiles are highly variable among patients. Meanwhile, the autoantibody profiles of the sera from the same patient were surprisingly stable for at least 3 months after surgery. Six surgery-associated antigens were identified and validated. All the five patients have at least one surgery-associated antigen, demonstrating this type of biomarkers is prevalent, while specific antigens are poorly shared among individuals. The prevalence of each antigen is 2%-14% according to the test with a larger cohort.InterpretationTo our knowledge, this is the first study of dynamically profiling of autoantibody repertoires before/after surgery of cancer patients. The high prevalence of surgery-associated antigens implies the possible broad application for monitoring of tumor recurrence in population, while the low prevalence of specific antigens allows personalized medicine. After the accumulation and analysis of more longitudinal samples, the surgery-associated serum biomarkers, combined as a panel, may be applied to alarm the recurrence of tumor in a personalized manner.FundingResearch supported by grants from National Key Research and Development Program of China Grant (No. 2016YFA0500600), National Natural Science Foundation of China (No. 31970130, 31600672, 31670831, and 31370813), Open Foundation of Key Laboratory of Systems Biomedicine (No. KLSB2017QN-01), Science and Technology Commission of Shanghai Municipality Medical Guidance Science &Technology Support Project (16411966100), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20172005), Shanghai Municipal Commission of Health and Family Planning Outstanding Academic Leaders Training Program (2017BR055) and National Natural Science Foundation of China (81871882).

Dataset Information

A proteomic repertoire of autoantigens identified from the classic autoantibody clinical test substrate HEp-2 cells.

Publications

A proteomic repertoire of autoantigens identified from the classic autoantibody clinical test substrate HEp-2 cells.

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