Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies

There is increasing interest in the role of antibodies targeting specific membrane proteins in neurological and other diseases. The target(s) of these pathogenic antibodies is known in a few diseases, usually when candidate cell surface proteins have been tested. Approaches for identifying new antigens have mainly resulted in the identification of antibodies to intracellular proteins, which are often very useful as diagnostic markers for disease but unlikely to be directly involved in disease pathogenesis because they are not accessible to circulating antibodies. To identify cell surface antigens, we developed a “conformational membrane antigen isolation and identification” strategy. First, a cell line is identified that reacts with patient sera but not with control sera. Second, intact cells are exposed to sera to allow the binding of presumptive autoantibodies to their cell surface targets. After washing off non-bound serum components, the cells are lysed, and immune complexes are precipitated. Third, the bound surface antigen is identified by mass spectrometry. As a model system we used a muscle cell line, TE671, that endogenously expresses muscle-specific tyrosine receptor kinase (MuSK) and sera or plasmas from patients with a subtype of the autoimmune disease myasthenia gravis in which patients have autoantibodies against MuSK. MuSK was robustly detected as the only membrane protein in immunoprecipitates from all three patient samples tested and not from the three MuSK antibody-negative control samples processed in parallel. Of note, however, there were many intracellular proteins found in the immunoprecipitates from both patients and controls, suggesting that these were nonspecifically immunoprecipitated from cell extracts. The conformational membrane antigen isolation and identification technique should be of value for the detection of highly relevant antigenic targets in the growing number of suspected antibody-mediated autoimmune disorders. The approach would also be very suitable for the analysis of human or experimental antitumor responses.Autoimmune diseases are conditions in which aberrant immune responses cause damage to and dysfunction of the body''s own tissue. They range from prevalent conditions, such as type 1 diabetes mellitus and rheumatoid arthritis, to various types of autoimmune thyroiditis (1), inflammatory bowel diseases (2), skin conditions such as bullous pemphigoid (3), and rarer neurological disorders such as myasthenia gravis (4).Understanding of most of these diseases is still highly incomplete. Fundamental knowledge includes the identity of the antigenic target of the immune response and whether the response is predominantly T cell- or antibody-mediated. In some of the above examples, “candidate” antigens have been proposed as a result of study of the pathophysiology of the disease (e.g. see Ref. 5). The detection of a disease-specific autoantibody allows the development of diagnostic tests, and if the target is a cell surface protein it usually implies that the disease will respond clinically to treatments that reduce the levels of the pathogenic antibodies.In recent years, there has also been increasing interest in natural (or experimental) immune responses to tumor cells that may slow the growth or spread of a tumor. In some cases, however, this immune response may result in pathogenic autoimmunity. For example, antibodies directed to voltage-gated calcium channels expressed on the surface of small cell lung cancer cells can cause neurological dysfunction by binding to similar calcium channels on the motor nerve endings (see Ref. 4). In other cancer-associated (paraneoplastic) disorders, however, there are antibodies to intracellular antigens, which are also shared between the tumor and neuronal tissue, that are highly useful as diagnostic markers for the disorders. In these patients, T cell immunity is thought to be responsible for the neurological disease (see Ref. 6), which generally does not improve with immunosuppressive treatments.Attempts to identify autoantigens and tumor antigens in many autoimmune and cancer-related syndromes have generally used techniques involving screening of mRNA expression libraries or, more recently, separation of soluble extracts of tissue or cell lines by one- or two-dimensional electrophoresis and blotting of the separated proteins onto membranes where they are probed with patient sera. Typically in any one experiment, a large number of protein bands or spots are bound by serum antibodies, and some of the corresponding bands or spots on the gel are then excised, digested, and analyzed by mass spectrometry (e.g. Refs. 7 and 8). The identified proteins have been claimed as novel antigens associated with the condition with sometimes a whole array of proteins identified from a single experiment and claimed to represent a disease-associated “autoimmune profile.” However, the identified proteins are often common intracellular proteins with the same or closely related proteins repeatedly implicated in seemingly unrelated autoimmune, allergic, and malignant diseases (see “Discussion”). The intracellular location of these proteins where they would be inaccessible to circulating antibodies and their lack of disease specificity cast doubt upon their relevance.The best understood example of an antibody-mediated disease is myasthenia gravis with acetylcholine receptor antibodies (for a review, see Ref. 9). Another subgroup of myasthenia gravis patients has antibodies to a muscle-specific tyrosine kinase (MuSK).¹ These antibodies are known to bind to the cell surface and to inhibit the clustering function of MuSK (10). Although the mechanisms of disease are not fully understood, the patients respond to immunotherapies, and the identification of this antigen by a candidate approach has revolutionized the diagnosis and treatment of this subtype of myasthenia (11). In many other conditions, however, no suitable candidate antigens have yet been proposed, limiting the diagnosis and treatment of the disorders.To develop a novel proteomics strategy for identifying cell membrane autoantigens, we used a model system involving antibodies from MuSK antibody-positive patients and from MuSK antibody-negative subjects. We first allowed the antibodies to bind to their target(s) on the intact cell surface, rather than after extraction and denaturation in detergents, so that the antibodies could recognize fully conformational epitopes. The cells, with antibodies already bound, were then solubilized, and the ready formed immune complexes were isolated and either visualized by SDS-PAGE and immunoblotting or identified by mass spectrometry. Although we show the current results as a “proof of principle,” the “conformational membrane antigen isolation and identification” (CMAII) technique could easily be adapted for use in studies of other diseases.