RESEARCH STRATEGY Abstract of parent grant: GM033225 Mammalian cells must respond to signals in their environment in complex ways in order to properly develop, form tissues, and marshal a defense against invading organisms. Much of the cell-cell communication needed is provided by a class of cell adhesion and signaling proteins that share a topology that includes an extracellular variable immunoglobulin-like (IgV) domain, one or more constant immunoglobulin-like (IgC) domains, a membrane anchor, and sometimes a cytoplasmic domain that carries signaling elements. The extra cellular domains of these molecules are heavily glycosylated and the glycans (carbohydrates) on these domains mediate or modify interactions with similar molecules on other cells and thus modulate biologically important signals. CEACAMs (carcinoembryonic antigen related cell adhesion molecules) are members of this class. Our work in the previous funding segment focused on CEACAM1, the product of one of twelve genes encoding CEACAMs in humans. We were able to produce a dimer structure of the IgV domain of CEACAM1, but more importantly, show that dimerization was affected by even minimal glycosylation. There are relatively few systematic structural studies of the effects of glycosylation in the CEACAMs or any of the related cell- surface signaling molecules, despite clear evidence that glycosylation modulates function. This arises because of the heterogeneity of native glycosylation, the challenges in preparing samples that are homogeneously glycosylated, and the limitations glycosylation places on common structure determination methods. Hence, to undertake this study we have assembled a team of investigators from three laboratories with complementary expertise in mammalian cell expression and glycan engineering, mass spectrometry-based protein and carbohydrate analysis, and NMR-based structure determination of glycosylated proteins.
Aims i nclude the production of specific glycoforms of IgV-IgC constructs of CEACAM1 and CEACAM5 with isotopic labeling suitable for NMR structure determination, mass spectrometry based screening of glycoforms to identify those displaying altered conformation or dimerization tendencies, and NMR determination of dimer structures and glycan interactions of targeted glycoforms using novel sparse-label methodology. Understanding how glycan interactions can alter structure and modulate signaling in the CEACAMs can impact directly on human health, but more importantly, the methods developed through work on CEACAMs can have a broader impact by allowing research on the many other glycosylated proteins found in the human body.
of parent grant: GM033225 This project will provide an improved structural understanding of the role of glycosylation in the function of CEACAMs, a family of cell-surface signaling and adhesion proteins. Since the abundance and specific forms of these proteins correlate with human diseases, including cancer, inflammatory disease and those of pathogen origin, this understanding can potentially facilitate the development of new diagnostics and treatment.
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