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 domain, one or more constant immunoglobulin-like domains, a membrane anchor, and often a cytoplasmic domain that carries signaling elements, for example, CEACAMs (carcinoembryonic antigen related cell adhesion molecules) and Siglecs (Sialic acid-binding immunoglobulin- type lectins). 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. Understanding these interactions can provide opportunities to intervene when normal signaling and regulatory events fail as in the case of malignancy or when pathogens hijack interaction pathways to invade cells or evade immune response. Attaining this understanding has, however, been challenging because of the difficulty in dealing with the heterogeneity of glycans and the lack of methodology for the preparation and structural characterization of appropriately glycosylated samples. This project develops protein expression and glycan engineering technology to prepare molecular systems appropriate for structural characterization, and it develops NMR technology to carry out structural characterization. Development is driven primarily by application to terminal domains of CEACAMs and one of their well characterized glycan interaction partners, the carbohydrate recognition domain of DC-SIGN (Dendritic Cell-Specific Intracellular adhesion molecule 3 (ICAM-3)-Grabbing Nonintegrin). Their interaction is a well-recognized element in immune response and CEACAMs are among important biomarkers for cancer where variations in levels and glycosylation are believed to contribute to metastasis. To characterize interaction the following aims are pursued: (1) Properly glycosylated CEACAM domains are expressed in mammalian cells (HEK293) using recently developed expression and purification technology. (2) The expressed constructs are structurally characterized using long range constraints from NMR observation of a sparse set of isotopically labeled sites. (3) Glycans are reengineered to carry isotopically labeled epitopes known to interact with DC-SIGN. (4) NMR methodology is developed to assign resonances from multiple glycosylation sites and provide information to guide molecular dynamics simulations of glycan conformation and motion. And (5) the interaction energetics and conformation of DC-SIGN-CEACAM complexes are determined by NMR and SPR. It is hypothesized that relative domain orientations is key in the modulation of cell-cell interactions and the initiation of intracellular signals. Understanding how glycan interactions contribute to preferred orientation will open new opportunities for intervention in human disease.

Public Health Relevance

The vast majority of proteins on mammalian cell surfaces are glycoproteins and the glycans (carbohydrates) on these proteins are known to influence cell-cell interactions that range from metastasis in cancer to colonization of pathogens. Structural characterization of these interactions can open opportunities to intervene in human disease. This project develops methodology for preparing well defined glycoprotein systems and nuclear magnetic resonance (NMR) methodology for characterizing glycan mediated interactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM033225-29A1
Application #
8652560
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
1984-07-01
Project End
2018-02-28
Budget Start
2014-03-10
Budget End
2015-02-28
Support Year
29
Fiscal Year
2014
Total Cost
$368,242
Indirect Cost
$118,242
Name
University of Georgia
Department
None
Type
Organized Research Units
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W et al. (2016) Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1). J Biol Chem 291:20085-95
Chalmers, G; Glushka, J N; Foley, B L et al. (2016) Direct NOE simulation from long MD trajectories. J Magn Reson 265:1-9
Pederson, Kari; Mitchell, Daniel A; Prestegard, James H (2014) Structural characterization of the DC-SIGN-Lewis(X) complex. Biochemistry 53:5700-9
Frank, Martin; Walker, Ross C; Lanzilotta, William N et al. (2014) Immunoglobulin G1 Fc domain motions: implications for Fc engineering. J Mol Biol 426:1799-811
Barb, Adam W; Wang, Xu; Prestegard, James H (2013) Refolded recombinant Siglec5 for NMR investigation of complex carbohydrate binding. Protein Expr Purif 88:183-9
Barb, Adam W; Ho, Tienhuei Grace; Flanagan-Steet, Heather et al. (2012) Lanthanide binding and IgG affinity construct: potential applications in solution NMR, MRI, and luminescence microscopy. Protein Sci 21:1456-66
Barb, Adam W; Meng, Lu; Gao, Zhongwei et al. (2012) NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation. Biochemistry 51:4618-26
Barb, Adam W; Prestegard, James H (2011) NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic. Nat Chem Biol 7:147-53
Barb, Adam W; Glushka, John N; Prestegard, James H (2011) Kinetics of Neuraminidase Action on Glycoproteins by 1D and 2D NMR. J Chem Educ 88:95-97
Barb, Adam W; Freedberg, Darón I; Battistel, Marcos D et al. (2011) NMR detection and characterization of sialylated glycoproteins and cell surface polysaccharides. J Biomol NMR 51:163-71

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