Natural killer (NK) cells are a subpopulation of effector lymphocytes of the innate immune system with cytotoxic activity and cytokine-producing effector functions. Functions of NK cells include tumour elimination, engagement into and regulation of antiviral immune responses and regulation of other immune cells such as dendritic cells, T lymphocytes, and B lymphocytes by secretion of chemokines and cytokines [1].
CD69 is a type II C-type lectin-like transmembrane receptor expressed on cells of hematopoietic origin. It is a disulphide-linked homodimer with subunits of varying molecular mass depending on glycosylation degree. Recent evidence suggests the involvement of the early-activation antigen CD69 in the modulation of cytokine secretion as well as in the homing and migration of activated lymphocytes. Several protein ligands (galectin-1, S100A8/S100A9, S1P1R, MLC9 and MLC12) have been described for CD69; however, there is no detailed mechanistic and structural description of the proposed protein:protein complexes.
Galectin-1 (Gal-1) is a prototypical protein belonging to the family of galectins. Galectins are characterized by the presence of a common structural fold and a carbohydrate recognition domain with high affinity for β-galactosides. Expression of galectin-1 has been reported mostly in lymphoid organs, activated lymphocytes, and in immune-privileged sites. Physiologically, Gal-1 exists as a monomer and as a non-covalent homodimer. Gal-1 has been shown to mediate processes in cellular adhesion, motility and growth-regulation as well as in the immunoregulation of T lymphocyte homeostasis.
The interaction between CD69 and galectin-1 has been described in 2014 by de la Fuente [2]. The mechanisms associated with CD69:Gal-1 interaction along with the required biochemical and biophysical properties still remain to be elucidated. Our project aims at a better understanding of how the interaction occurs and if the interaction is protein- or glycosylation-specific. To answer such questions, we have employed MST and AUC techniques to assess the carbohydrate dependence of the binding. The obtained results show a considerable difference in the dissociation constants of the glycosylated CD69:Gal-1 and deglycosylated CD69:Gal-1 complexes, strongly indicating that the interaction is glycosylation-dependent. Unexpectedly, galectin-1 appears to be binding with similar or stronger affinity to other sequence- or family-related NK cell surface receptors, indicating that the interaction is not as specific as initially assumed.
To assess the specificity of the interaction and the biophysics involved in complex formation, complementary biophysical analyses (e.g., SPR, nanoDSF, and ITC) and protein crystallography are to be employed to provide a more detailed description of the interaction and structural mechanics of CD69:Gal-1 complex.
This study was supported by Czech Science Foundation (18-10687S) and Ministry of Education, Youth and Sports of the Czech Republic (LTC17065 in frame of the COST Action CA15126).