Immunocytochemical analysis for intracellular dynamics of C1GalT associated with molecular chaperone, Cosmc |
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Authors: | Narimatsu Yoshiki Ikehara Yuzuru Iwasaki Hiroko Nonomura Chizu Sato Takashi Nakanishi Hayao Narimatsu Hisashi |
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Affiliation: | a Glycogene Function Team of Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan b Molecular Medicine Team of Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan c Department of Function of Biomolecule, Doctoral Program in Medical Sciences for Control of Pathological Processes, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan d Division of Oncological Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 464-8681, Japan |
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Abstract: | The core 1 structure Galβ1-3GalNAcα1-Ser/Thr (T antigen), the major constituent of O-glycan core structure, is synthesized by cooperation of core 1 synthase (C1GalT) and its specific molecular chaperone, Cosmc. The chaperone function of Cosmc has been well investigated biochemically. In this study, we established monoclonal antibodies specifically recognizing either C1GalT or Cosmc, respectively, and investigated the sub-cellular localization of each protein to elucidate how they cooperate to synthesize the core 1 structure.A sequential immunocytochemical analysis of the human colon cancer cell line, LSB, demonstrated different localization of two proteins. C1GalT was localized in Golgi apparatus, while Cosmc was localized in endoplasmic reticulum. In contrast, the LSC cells, which do not have core 1 synthase activity due to a missense mutation in the Cosmc gene, did not express the C1GalT protein. Although the treatment with a proteasome inhibitor, lactacystin, of LSC cells resulted in the increased expression of C1GalT protein, the distribution of C1GalT was not in Golgi apparatus as seen in LSB cells. On the contrary, overexpression of Cosmc but not C1GalT lead to precise localization of C1GalT protein, which distributed in Golgi apparatus and recovered the core 1 synthase activity in LSC cells. These results suggest that the intracellular dynamics of C1GalT is controlled by its specific molecular chaperon, Cosmc, in association with core 1 synthase activity. |
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Keywords: | C1GalT, core 1 β1,3-galactosyltransferase Cosmc, core 1 β1,3-galactosyltransferase-specific molecular chaperone BSA, bovine serum albumin FITC, fluorescein isothiocyanate HEK, human embryonic kidney 293T Ig, immunoglobulin mAb, monoclonal antibody PBS, phosphate-buffered saline TBS, Tris-buffer saline PNA, peanut agglutinin EDTA, ethylenediaminetetraacetic acid ER, endoplasmic reticulum |
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