Role of EBAG9 protein in coat protein complex I-dependent glycoprotein maturation and secretion processes in tumor cells.

Many proteins mature within the secretory pathway by the acquisition of glycans. Failure to maintain the proper distribution of the glycosylation machinery might lead to disease. High expression levels of the ubiquitous Golgi protein estrogen receptor-binding fragment-associated gene 9 (EBAG9) in human tumors correlate with poor clinical prognosis, and EBAG9 overexpression in epithelial cell lines induces truncated glycans, typical of many carcinomas. Here, we addressed the pathogenetic link between EBAG9 expression and the alteration of the cellular glycome. We applied confocal microscopy, live imaging, pulse-chase labeling in conjunction with immunoprecipitation, and enzymatic activity assays in a variety of EBAG9-overexpressing or depleted epithelial tumor cell lines. EBAG9 shuttles between the ER-Golgi intermediate compartment and the cis-Golgi, and we demonstrate association of EBAG9 with coat protein complex I (COPI)-coated transport vesicles. EBAG9 overexpression imposes delay of endoplasmic reticulum-to-Golgi transport and mislocalizes components of the ER quality control and glycosylation machinery. Conversely, EBAG9 down-regulation accelerates glycoprotein transport through the Golgi and enhances mannosidase activity. Thus, EBAG9 acts as a negative regulator of a COPI-dependent ER-to-Golgi transport pathway in epithelial cells and represents a novel pathogenetic principle in which interference with intracellular membrane trafficking results in the emergence of a tumor-associated glycome.

Reevaluation of the 22-1-1 antibody and its putative antigen, EBAG9/RCAS1, as a tumor marker.

BACKGROUND: Tumor-associated antigens are appreciated as diagnostic markers, but they have also prompted tremendous efforts to develop tumor-specific immunotherapy. A previously cloned tumor-associated antigen, EBAG9, was initially defined by reactivity with the monoclonal antibody 22-1-1. Functionally, the EBAG9-encoded gene-product was believed to induce apoptosis in activated immune cells. However, using a cell-biological approach we identified EBAG9 as a Golgi-resident modulator of O-linked glycan expression, the latter product was then recognized by the 22-1-1 antibody. Secondly, EBAG9 expression was found physiologically in all murine tissues examined. This raised the question if EBAG9 is tumor-specific and mediates apoptosis itself or through O-linked glycans generated, among them the cognate 22-1-1 antigen Tn. METHODS: We have used immunohistochemistry to detect the expression of 22-1-1 and EBAG9 in various tissues. Correlation between expression of both antigens in cell lines was analysed by immunoblot and flow cytometry. Apoptosis was studied by using flow cytometry and Caspase-Glo 3/7 assay kit. Cellular distribution of EBAG9 was analysed by electron and confocal microscopy. RESULTS: Here, we compared expression of the 22-1-1 and EBAG9-defined antigens in normal and neoplastic tissues in situ. In contrast to 22-1-1 staining, EBAG9 is a ubiquitously expressed antigen in all normal and cancerous tissues. Functional studies on the role of 22-1-1 reactive material did not support any evidence for apoptosis induction. Employing electron and confocal microscopy, a refined subcellular localization of EBAG9 at the Golgi was obtained. CONCLUSION: We suggest that the estrogen-inducible EBAG9 gene-product and the 22-1-1 defined antigen are structurally and functionally separate antigens.