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Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein.

ABSTRACT: The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte glycoprotein (MOG) by selective unfolding of the antigen domain. The tetramer radioimmunoassay (RIA) was more sensitive for MOG autoantibody detection than other methodologies, including monomer-based RIA, ELISA or fluorescent-activated cell sorting (FACS). Autoantibodies from individuals with acute disseminated encephalomyelitis (ADEM) selectively bound the folded MOG tetramer, whereas sera from mice with experimental autoimmune encephalomyelitis induced with MOG peptide immunoprecipitated only the unfolded tetramer. MOG-specific autoantibodies were identified in a subset of ADEM but only rarely in adult-onset MS cases, indicating that MOG is a more prominent target antigen in ADEM than MS.

SUBMITTER: O'Connor KC

PROVIDER: S-EPMC3429369 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein.

O'Connor Kevin C KC McLaughlin Katherine A KA De Jager Philip L PL Chitnis Tanuja T Bettelli Estelle E Xu Chenqi C Robinson William H WH Cherry Sunil V SV Bar-Or Amit A Banwell Brenda B Fukaura Hikoaki H Fukazawa Toshiyuki T Tenembaum Silvia S Wong Susan J SJ Tavakoli Norma P NP Idrissova Zhannat Z Viglietta Vissia V Rostasy Kevin K Pohl Daniela D Dale Russell C RC Freedman Mark M Steinman Lawrence L Buckle Guy J GJ Kuchroo Vijay K VK Hafler David A DA Wucherpfennig Kai W KW

Nature medicine 20070112 2

The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte ...[more]

PMID: 17237795

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Project description:Cold denaturation is a general property of globular proteins, and the process provides insight into the origins of the cooperativity of protein folding and the nature of partially folded states. Unfortunately, studies of protein cold denaturation have been hindered by the fact that the cold denatured state is normally difficult to access experimentally. Special conditions such as addition of high concentrations of denaturant, encapsulation into reverse micelles, the formation of emulsified solutions, high pressure, or extremes of pH have been applied, but these can perturb the unfolded state of proteins. The cold denatured state of the C-terminal domain of the ribosomal protein L9 can be populated under native-like conditions by taking advantage of a destabilizing point mutation which leads to cold denaturation at temperatures above 0 degrees C. This state is in slow exchange with the native state on the NMR time scale. Virtually complete backbone (15)N, (13)C, and (1)H as well as side-chain (13)C(beta) and (1)H(beta) chemical shift assignments were obtained for the cold denatured state at pH 5.7, 12 degrees C. Chemical shift analysis, backbone N-H residual dipolar couplings, amide proton NOEs, and R(2) relaxation rates all indicate that the cold denatured state of CTL9 (the C-terminal domain of the ribosomal protein L9) not only contains significant native-like secondary structure but also non-native structure. The regions corresponding to the two native alpha-helices show a strong tendency to populate helical Phi and Psi angles. The segment which connects alpha-helix 2 and beta-strand 2 (residues 107-124) in the native state exhibits a significant preference to form non-native helical structure in the cold denatured state. The structure observed in the cold denatured state of the I98A mutant is similar to that observed in the pH 3.8 unfolded state of wild type CTL9 at 25 degrees C, suggesting that it is a robust feature of the denatured state ensemble of this protein. The implications for protein folding and for studies of cold denatured states are discussed.

Dataset Information

Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein.

Publications

Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein.

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