CRTAM is expressed post-activation on CD8 T cells, NKT cells, NK cells, and a subpopulation of CD4T cells[24–26]. As an adhesion molecule, CRTAM expressed on activated CD8 T cells established heterotyphic trans-interaction with Necl2 expressed on CD8 dendritic cells of lymph nodes [27, 28]. For this in vivo interaction, the extracellular domains of both molecules are implicated [11, 29].
In this study, we described that recombinant CRTAMEC and CRTAMIgC-stalk, after refolding, were purified by gel chromatography within their expected dimeric molecular weight. We observe the dimmer of CRTAMEC or CARTAMIgC. There is a free sulfhydryl group of Cys 177, a conserved residue in CTRAM of different species, but not in other nectin-like molecules (Supplementary Figs. 5 and 6). Therefore, one might expect the formation of a disulfide bond among two CRTAM molecules. Nonetheless, on activated human CD8 cells, only a tiny fraction of surface-expressed CRTAM is detected as a dimer[10]. On a T cell hybridoma transfected with CRTAM gen, dimers were detected if the cells were treated with a cross-linker Bis(sulfosuccinimidyl)suberate [27]. Those results suggest weak interactions are involved in dimer formation instead of a disulfide bond. Thus, a hydrophobic region with aromatic residues as phenylalanine, proline and tyrosine are involved to stabilize the cis-interaction of CRTAM molecules. This kind of interaction is possible because the Ig-like domains of CRTAM line up along their longitudinal axis as other Ig-like molecules. However, this point will be solved until it crystalizes the whole CRTAM molecule. On the other hand, dimers, and monomers of CRTAMIgV were recovered, implying the contribution of the Ig-V domain for the cis-interaction, probably through hydrophobic residues as was reported [13].
We found that the affinity of the CRTAMEC-NECL2EC interaction was 2.16 nM. This high affinity drops to 0.97 µM when the constant domain of CRTAM is not present, and the interaction dissociates rapidly (Fig. 3B). These results agree with the affinities of heterophilic interaction between Nectin members and Nectin-like families that they are in the nanomolar range while the homophilic ones are in micromolar values [6, 8, 9, 14]. The result also indicates the participation of the constant domain during the interaction; most likely, it might bind to the constant domain 2 of nectin-like2. However, further experiments are needed to define this matter.
Takeshi Ito et al [30]. reported the interaction of CRTAM-NECL2 with a value of 3.3 nM (Kon= 3.1x104 M− 1s− 1, Koff=1.1x10− 4s− 1), which is quite close to our results (Kd = 2.16 nM, Kon=6.04x104M− 1S− 1, Koff=1.30x10− 4s− 1). They used the Fc-chimeric molecules, which could cause a background, while our kinetics involved chimeric-free recombinants [30]. However, the similarity of these results confirms that the interaction between CRTAM and Necl2 is very stable and dissociates very slowly. Takeshi Ito et al. speculate on the possible participation of N-glycosylation in the interaction of those molecules, but for our analysis, we used recombinant proteins produced in E. coli. Thus, it is not the glycosylation, but the IgC domain that is essential to establish a high-affinity interaction of those molecules.
The results from the SRP technology were confirmed with cells transfected with genes encoding the CRTAM or Necl2. A stable cell-cell interaction was observed between cells that expressed the two extracellular CRTAM domains and the extracellular domains of Necl2, but not if the cells expressed only CRTAM IgV or the IgC domain. Another critical point is that surface adhesion is independent of scaffolding proteins that might be binding intracellularly through the CRTAM-ESIV motif and NECL2-EYFI motif. These results suggest that nonbinding of the receptor CRTAM and the ligand Necl2 is expressed as a homodimer on the cell surface (Fig. 7a), but when the opposite cells interact, the homodimer of CRTAM is switched to heterodimer with Necl2 where the constant domain of CRTAM probably bind by proximity to the first constant domain of Necl2 (Fig. 7b). Thus, this surface interactions occurs first, followed by the recruitment of other intracellular proteins, such as Scrib.