Characterization of TMEM16E/GDD1 That Causes Gnatho-Diaphyseal Dysplaseia and Limb-Girdle Muscular Dystrophy through Distinct Gene Mutations

Kazumi Kubozono , Department of Oral and Maxillofacial Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Kuniko Mizuta , Department of Oral and Maxillofacial Surgery, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Shinichi Fujimoto , Department of Oral and Maxillofacial Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Masaaki Takechi DDS, PhD, Department of Oral and Maxillofacial Surgery, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Statements of the problem: TMEM16E/GDD1 has been shown to be responsible for the bone-related disease gnathodiaphyseal dysplasia (GDD). GDD is a familial, autosomal dominantly inherited disorder characterized by florid osseous dysplasia in jaw bones, bone fragility, and bowing associated with diaphyseal sclerosis in the long bones.  The mutated dominant allele (i.e., encoding a missense mutation at Cys356) causes GDD, while the recessive alleles (e.g., encoding a nonsense mutation at various positions) result in muscular dystrophy (LGMD). Therefore, the physiological functions of TMEM16E/GDD1 is homeostatic maintenance of muscle have been shown; however, molecular function of TMEM16E/GDD1 is still unclear.  TMEM16E/GDD1 is classified as a member of the TMEM16 family proteins.  Some of the TMEM16 family (e.g., TMEM16A, B, and F) exhibit ion channel activity at cell surface, whereas TMEM16E/GDD1 did not distribute at cell surface when overexpressed in human embryonic kidney 293 (HEK293) cells.  To identify the possible relevance of TMEM16E/GDD1 in muscular homeostasis, we examined subcellular localization of the TMEM16E/GDD in myotubes.

Materials and Method: We analyzed subcellular localization of endogenous TMEM16E/GDD1 by immunofluorescence in myotubes.  To identify molecules that interact with GDD in the gene responsible for muscular dystrophy, we performed Proximity ligation assay that detected an immunofluolecence only when the candidate associated with TMEM16E/GDD1 in situ.

Methods of data analysis: Immunofluorescence was captured using a BZ9000 Fluorescence Microscope at 60×magnification in an oil immersion. Z-stack images were deconvoluted and merged into a full-focus image using BZ-II Image analysis application.

Results: TMEM16E/GDD1 was localized to cytoplasmic vesicles in a myotube.  A molecule that is deficient in patient with LGMD, clearly co-localized to complex with TMEM16E/GDD1 in myotubes.  Surprisingly, another muscle specific surface channel molecule, which showed different cellular distribution from TMEM16E/GDD1, formed a complex with TMEM16E/GDD1.

Conclusion: Our results indicated that TMEM16E/GDD1 cooperates with these associated molecules to protect muscle from dystrophy. 

References:

1. Tran TT, Tobiume K, Hirono C, Fujimoto S, Mizuta K, Kubozono K, Inoue H, Itakura M, Sugita M, Kamata N. TMEM16E (GDD1) exhibits protein instability and distinct characteristics in chloride channel/pore forming ability. J Cell Physiol229: 181-190 (2014)

2. Mizuta K, Tsutsumi S, Inoue H, Sakamoto Y, Miyatake K, Miyawaki K, Noji S, Kamata N, Itakura M. Molecular characterization of GDD1/TMEM16E, the gene product responsible for autosomal dominant gnathodiaphyseal dysplasia. Biochem Biophys Res Commun 357: 126-132 (2007)

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