Immunocytochemical analysis for intracellular dynamics of ClGaIT associated with molecular chaperone, Cosmc

The core 1 structure Gal beta 1-3GalNAc alpha 1-Ser/Thr (T antigen), the major constituent of O-glycan core structure, is synthesized by cooperation of core 1 synthase (ClGaIT) 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 ClGaIT 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. ClGaIT 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 ClGaIT protein. Although the treatment with a proteasome inhibitor, lactacystin, of LSC cells resulted in the increased expression of ClGaIT protein, the distribution of ClGaIT was not in Golgi apparatus as seen in LSB cells. On the contrary, overexpression of Cosmc but not ClGAIT lead to precise localization of ClGaIT protein, which distributed in Golgi apparatus and recovered the core 1 synthase activity in LSC cells. These results suggest that the intracellular dynamics of ClGaIT is controlled by its specific molecular chaperon, Cosmc, in association with core 1 synthase activity. (c) 2007 Elsevier Inc. All rights reserved.