Departmental Bulletin Paper A modified application of the luciferase immunoprecipitation systems for detecting antibodies to the G protein-coupled receptors

Mukaino, Akihiro  ,  Higuchi, Osamu  ,  Nakane, Shunya  ,  Maeda, Yasuhiro  ,  Sakai, Waka  ,  Nakamura, Hideki  ,  Matsuo, Hidenori  ,  Kawakami, Atsushi

60 ( 2 )  , pp.69 - 76 , 2016-01 , Nagasaki University School of Medicine
Background: When multipass transmembrane molecules are located on the cell surface, there may be interaction with not only bioactive molecules but also pathogenic molecules in areas protruding outside the cell. In antibody-mediated autoimmune disorders, it has been found that the autoantibodies occasionally attack membrane molecules on the cell surface, thus causing the disease such as myasthenia gravis. In such cases, highly sensitive autoantibody detection technology is required for early diagnosis. However, autoantibody analysis technology that is specialized for membrane molecules is still under development. Here we demonstrate a novel method for detecting of antibodies against the extracellular portions of multipass transmembrane molecules. Methods: Antibodies for muscarinic acetylcholine receptor type3 (M3R) were detected with two kinds of luciferase immunoprecipitaion systems (LIPS), conventional LIPS (cLIPS) and its modified application, termed modified LIPS (mLIPS). In mLIPS, antibodies against extracellular portions of membrane molecules could be preferentially detected. Results: An antibody to the amino-terminal portion of human M3R was detected with modified LIPS with a high sensitivity. In contrast, an antibody to the carboxyl-terminal portion was not detected with mLIPS, because it did not interact with intracellular portions of M3R in living cells. We also found antibodies for M3R in a patient serum with Sjögren’s syndrome. Conclusion: Our technology has a promising future, and we hope that it will be applied in the analysis of antibodies against a diverse range of multipass transmembrane molecules, including GPCRs.

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