STRUCTURE/FUNCTION OF THE CO17-1A/GA733 ANTIGEN

Wistar Institute, Philadelphia PA

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Abstract

The overall goal of this project is to study the molecular basis of GA733 Ag function in colorectal tumor and normal epithelial cells by investigating structural and functional properties of this protein. The Co17-1A/GA733 antigen (GA733 Ag) is expressed at high levels on most human colorectal carcinomas and on some normal epithelial cells. It is intriguing the monoclonal antibody CO17-1A has shown clinical efficacy without inducing toxicity related to binding of the antibody to normal tissues. This lack of toxicity is probably not due simply to differences in antigen density on tumor and normal cell surfaces; instead it is more likely that there are important structural and/or functional differences of GA733 Ag expressed by tumor versus normal tissues, which will be studied here. The detailed elucidation of structural and functional properties of this putative homotypic cell adhesion protein as described in this project should provide insights into mechanisms for potential anti-tumor effects and potential toxicities in clinical therapy. Molecular biology and protein chemical approaches will be used to identify important structural features of the GA733 Ag and to determine their effects on cell adhesion. One working hypothesis is that cell-cell adhesion mediated by GA733 Ag is signaled across the membrane by a mechanism. involving tyrosine phosphorylation and/or change in cis oligomer state of the cytoplasmic domain, which results in altered interactions with the cytoskeleton. The role of cis dimer site and to engineer secretable, adhesion-active dimers. The cell-cell adhesion binding site will also be mapped using mutagenesis. Standard protein chemical methods, including mass spectroscopy, N-terminal sequencing, HPLC peptide mapping, and chemical crosslinking will be used to identify post- translational modifications, oligomeric state, and other structural features that may affect function. Finally, cell transfetions will be used to evaluate the role of GA733 Ag and its mouse homolog (mEGP) on cell growth, morphology, invasion, and tumorigenicity.