Glycosylation Affects Ligand Binding and Function of the Activating Natural Killer Cell Receptor 2B4 (CD244) Protein

Abstract

2B4 (CD244) is an important activating receptor for the regulation of natural killer (NK) cell responses.Here we show that 2B4 is heavily and differentially glycosylated in primary human NK cells and NK cell lines. The differential glycosylation could be attributed to sialic acid residues on N-and O-linked carbohydrates. Using a recombinant fusion protein of the extracellular domain of 2B4, we demonstrate that N-linked glycosylation of 2B4 is essential for the binding to its ligand CD48. In contrast, sialylation of 2B4 has a negative impact on ligand binding, as the interaction between 2B4 and CD48 is increased after the removal of sialic acids. This was confirmed in a functional assay system, where the desialylation of NK cells or the inhibition of O-linked glycosylation resulted in increased 2B4-mediated lysis of CD48-expressing tumor target cells. These data demonstrate that glycosylation has an important impact on 2B4-mediated NK cell function and suggest that regulated changes in glycosylation during NK cell development and activation might be involved in the regulation of NK cell responses.Natural killer cells are the first lymphoid subpopulation in the defense against tumors and viral infection. Their activity is regulated by the interplay between inhibitory receptors, most of which recognize MHC class I expression on target cells, and activating receptors, which are engaged by various ligands (1). Important inhibitory receptors are members of the killer cell immunoglobulin-like receptor family as well as CD94/NKG2 heterodimers (2). Examples for activating receptors are NKG2D (CD314), DNAM-1 (CD226), and the well characterized natural cytotoxicity receptors (NCRs) 2 NKp30 (CD337), NKp44 (CD336), and NKp46 (CD335) (2, 3). These receptors transduce their activation signal via small adaptor proteins like CD3zeta or DAP10 (4). The human activating NK cell receptor 2B4 (CD244) belongs to the SLAM-related receptor (5) and is a 365-amino acid type 1 transmembrane protein with a calculated molecular weight of 39 kDa and a pI of 9.16 (6). It has a large intracellular domain containing four immunoreceptor tyrosine-based switch motifs that recruit adaptor molecules like SAP (SH2D1A or DSHP) or EAT2 after phosphorylation (7-9). The extracellular part of 2B4 consists of an N-terminal V-set Ig domain, a membrane-proximal C2-set Ig domain, and several N-glycosylation sites (6). The ligand of 2B4 is CD48, a glycosyl-phosphatidylinositol-anchored molecule with broad expression in the hematopoietic system (10, 11). Engagement of 2B4 by its ligand results in the recruitment and clustering of the receptor into lipid rafts and phosphorylation of its immunoreceptor tyrosine-based switch motif domains (12, 13). This initiates a signaling cascade, leading to polarization and the release of cytolytic granules into the contact zone between the NK and target cell (14, 15). Although 2B4 stimulation alone is sufficient to activate IL-2 stimulated NK cells, it also serves as an important coactivatory signal for the stimulation of rest...

Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

“…2), indicating that changes in the biochemical property of this protein were due to addition of highly charged N-glycans. This is similar to previous data showing that glycosylation significantly alters the pI of other similar glycoproteins (20,21). Moreover, glycosylation can provide conformational and structural stability (22,23) and may facilitate correct folding (23,24) and importantly modulate ligand binding and functions (20,25,26).…”

Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

“…While we acknowledge that these murine studies are preliminary and do not examine the role of Tim-3 on NK cells in the Gal-9 KO mouse, they do support a role for Gal-9 in the inhibition of NK cells. Although the present study does not address the identification of the receptor on NK cells mediating inhibition by Gal-9, it is interesting to speculate that as Gal-9 recognizes carbohydrates, differential glycosylation of NK cell receptors is involved (26)(27)(28).…”

“…Currently, we do not know the effects of Tim-3-independent Gal-9 signaling in NK cells. As mentioned above, Gal-9 recognizes carbohydrates, and recent reports suggest that differential glycosylation of NK cell receptors represents an important receptor regulatory mechanism for control of NK cell function (26)(27)(28). In the current study, we investigated the effect of Gal-9 on human NK cell transcription and function and the NK cell phenotype and function of Gal-9 KO mice.…”

Signaling Lymphocytic Activation Molecule Family Receptor Homologs in New World Monkey Cytomegaloviruses

“…In this connection, elongated forms of CD48 have been shown to be profoundly inhibitory by disrupting the symmetry of the immunological synapse (50). Finally, carbohydrate modifications in cell surface receptors markedly change the properties and modulate the functions of these glycoproteins, as illustrated for CD244, where the sugar content has been reported to have an important impact on CD48 binding and NK cell responses (51). The fact that the viral SLAMF homologs are predicted to be more extensively glycosylated than the corresponding cellular SLAMF receptors may be of relevance not only to preserve their stability or prevent unwanted interactions but also for functional recognition.…”