Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)

Abstract

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (Ig V ) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the ␤-sandwich-like Ig V domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED ␤-strands and the other involving GFCCC؆ ␤-strands, with the former burying one prominent glycosylation site. These structures raise ques- The human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) 2 is involved in cell adhesion, proliferation, and immune response (1, 2). It also is implicated in cancer angiogenesis, progression, and metastasis (3). More specifically, it is known that CEACAM1 is a negative-regulator of cell proliferation and is down-regulated in some tumor cells (4 -10). Yet, CEACAM1 expression is reported to protect tumor cells from killing by immune cells (11)(12)(13)(14) and it has been found that the high expression level is associated with a number of other cancers (15)(16)(17)(18)(19). It is likely that this complex set of effects arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other members of the CEACAM superfamily. The N-terminal Ig variable (Ig V ) domain of CEACAM1 has been suggested to be the basis of cis and trans homo-dimer formation (20), as well as interactions with other molecules (21-24). There are crystal structures of the Ig V domain expressed in Escherichia coli, which have led to the suggestion of two distinct dimerization interfaces (24, 25). However, examination of the interfaces suggests that the extensive glycosylation found in native material would inhibit dimer formation in one of these cases. This raises questions about the actual type of dimer found in solution, with and without glycosylation. Here, we use NMR methods to examine dimer structures in solution using a non-glycosylated form expressed in E. coli, and several glycosylated variants expressed in HEK293 cells.Dimerization of cell surface molecules is a well accepted mechanism for transmitting signals from the cell surface to the interior (26). In the case of CEACAM1 it is believed that modulation of intercellular adhesion as well as transmission of signals to the cell interior involves switching between cis and trans dimerization interactions. The full-length CEACAM1 molecule is large, sharing a topology with many other cell-surface signaling molecules; the extracellular domain is composed of one Ig V -like domain and typically 3 Ig C2 -like dom...

CEACAM1 structure and function in immunity and its therapeutic implications

“…Our recent high resolution crystal structure (2.04 Å) of the unglycosylated hCEACAM1 IgV domain (PDB ID 4QXW) [15,27] confirmed this by featuring two IgV molecules in the crystallographic asymmetric unit anchored at their respective GFCC'C" surfaces and confirmed at atomic level resolution the participation of critical CC' and FG loop residues determined previously to be necessary for hCEACAM1 homodimerization [30][31][32]. Nuclear magnetic resonance studies (NMR) on unglycosylated hCEACAM1 IgV additionally demonstrated the presence of an exclusive GFCC'C"-mediated IgV dimer in solution [31] thus further reinforcing the significance of the GFCC'C" face in mediating hCEACAM1 IgV homo-oligomerization.…”

“…Independent mutagenesis analysis of hCEACAM1 corroborated these latter results by showing the critical role of the GFCC'C" interface in determining the high affinity homodimerization of the hCEACAM1 IgV domain (Kd ~ 450 nM) [29], and specifically highlighting the contribution of the CC' (residues Y34, V39, D40, R43, Q44) and FG (residues Q89, I91) loops [30][31][32]. Our recent high resolution crystal structure (2.04 Å) of the unglycosylated hCEACAM1 IgV domain (PDB ID 4QXW) [15,27] confirmed this by featuring two IgV molecules in the crystallographic asymmetric unit anchored at their respective GFCC'C" surfaces and confirmed at atomic level resolution the participation of critical CC' and FG loop residues determined previously to be necessary for hCEACAM1 homodimerization [30][31][32]. Nuclear magnetic resonance studies (NMR) on unglycosylated hCEACAM1 IgV additionally demonstrated the presence of an exclusive GFCC'C"-mediated IgV dimer in solution [31] thus further reinforcing the significance of the GFCC'C" face in mediating hCEACAM1 IgV homo-oligomerization.…”

Tool for Rapid Analysis of Glycopeptide by Permethylation via One-Pot Site Mapping and Glycan Analysis

“…These advances have been driven by the very low sample amounts obtainable from clinical sources, the extremely heterogeneous nature of glycan occupancies and structures, the need for high-throughput reliable analysis methods, and recent discoveries of more unusual glycoconjugates. 13–16 …”

Just a spoonful of sugar: Short glycans affect protein properties and functions

“…Likewise, the ligand binding affinity of notch receptor increases when GlcNAc is added to fucose at a specific Thr, and it is affected by O‐fucosylation at conserved sites . Also, dimer formation of carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1), and thus, probably, its interaction with other molecules, is altered by glycosylation . Even the addition of a single GlcNAc at each of the three native N‐glycosylation sites in the Ig variable domain of CEACAM1 inhibited its dimerization in in vitro studies.…”