Carcinoembryonic antigen (CEA) is a highly-glycosylated, colonic epithelial cell surface glycoprotein which is routinely used as a tumor marker for colon cancer. The sequence and domain structure of CEA, NCA (nonspecific crossreacting antigen), and BGP (biliary glycoprotein) have been determined by us using protein and molecular cloning studies. All three are highly homologous members of the immunoglobulin gene superfamily and have suggested roles in cell-cell and bacterial-cell adhesion. We have shown that they are induced by gamma-interferon and tumor necrosis factor-alpha, further suggesting a role in immunity. As a first step in establishing the role of these proteins, especially CEA and BGP, we will study their gene regulation, including their induction by IFN-gamma and TNFalpha in colonic and noncolonic cell lines. Analyses include DNA mobility shift, footprinting, and methylation interference assays. We will isolate and characterize the transcription factors responsible for the regulation of CEA and BGP expression, and study the mechanism of IFN-gamma and TNFalpha induction relative to other interferon responsive genes. In continuing structural studies, we will determine the oligosaccharide structures at each glycosylation site and the structure of the glycosyl- phosphatidylinositol membrane anchor and relate these structures to their role in bacterial adhesion. We will develop novel, rapid, sensitive methods for the structural analysis of the transcriptional factors and site-specific oligosaccharide units of CEA using laser desorption time of flight mass spectrometry, and fast atom bombardment and electrospray tandem mass spectrometry. These methods will have general utility in the ultrasensitive analysis of rare biological samples. We will characterize the Ig-like structure of CEA using expressed, secreted forms of CEA and its individual Ig-variable and Ig-constant domains using circular dichroism and fluorescence spectroscopy. The structure-function relationships between the ecto- and cytoplasmic domains of CEA, BGP, and E-Cadherin and their role in the vectorial transport of these proteins in cell lines possessing or defective in these properties will be determined using chimeric constructs. Functional assays will also include bacterial binding and treatment with growth factors and growth factor inhibitors. These studies are part of a comprehension approach to the structure-function-regulation of CEA and BGP.
