Short Disordered Epitope of CRTAM Ig-Like V Domain as a Potential Target for Blocking Antibodies.

Class-I Restricted T Cell-Associated Molecule (CRTAM) is a protein that is expressed after T cell activation. The interaction of CRTAM with its ligand, nectin-like 2 (Necl2), is required for the efficient production of IL-17, IL-22, and IFNγ by murine CD4 T cells, and it plays a role in optimal CD8 T and NK cell cytotoxicity. CRTAM promotes the pro-inflammatory cytokine profile; therefore, it may take part in the immunopathology of autoimmune diseases such as diabetes type 1 or colitis. Thus, antibodies that block the interaction between CRTAM and Necl2 would be useful for controlling the production of these inflammatory cytokines. In this work, using bioinformatics predictions, we identified three short disordered epitopes (sDE1-3) that are located in the Ig-like domains of murine CRTAM and are conserved in mammalian species. We performed a structural analysis by molecular dynamics simulations of sDE1 (QHPALKSSKY, Ig-like V), sDE2 (QRNGEKSVVK, Ig-like C1), and sDE3 (CSTERSKKPPPQI, Ig-like C1). sDE1, which is located within a loop of the contact interface of the heterotypic interaction with Nectl2, undergoes an order-disorder transition. On the contrary, even though sDE2 and sDE3 are flexible and also located within loops, they do not undergo order-disorder transitions. We evaluated the immunogenicity of sDE1 and sDE3 through the expression of these epitopes in chimeric L1 virus-like particles. We confirmed that sDE1 induces polyclonal antibodies that recognize the native folding of CRTAM expressed in activated murine CD4 T cells. In contrast, sDE3 induces polyclonal antibodies that recognize the recombinant protein hCRTAM-Fc, but not the native CRTAM. Thus, in this study, an exposed disordered epitope in the Ig-like V domain of CRTAM was identified as a potential site for therapeutic antibodies.

Neuromyelitis optica spectrum disorder in three generations of a Chinese family.

Neuromyelitis optica spectrum disorder is an inflammatory demyelinating disease that is largely sporadic. Familial disease has been reported in one or two generations, although its basis remains unknown. We report here three subjects meeting diagnostic criteria for NMOSD in one family: a father and son, and the maternal aunt of the father. Anticipation, of 27 years, was apparent in transmission from father to son. Aquaporin-4 antibodies were observed in the aunt but not the father and son, nor in other family members. A putative pathogenic mutation in the NECL2 gene was not found in this pedigree. This first report of NMOSD in three generations of one family underlines the heterogeneity of familial NMOSD.

Mutation of the cellular adhesion molecule NECL2 is associated with neuromyelitis optica spectrum disorder.

AIMS: To investigate the association of the Nectin/Necl family genes with the risk of developing NMOSD. METHODS: Whole-exome sequencing was performed on two familial NMOSD cases and two unaffected family members. Additionally, 106 patients with sporadic NMOSD and 212 healthy controls (HCs) underwent screening for mutant Necl2. Finally, the molecular weight and cellular localization of mutant NECL2 was examined in transfected HeLa cells. RESULTS: We identified a novel deletion mutation in Necl2 (c.1052_1060delCCACCACCA; p. Thr351_Thr353del), which was associated with disease manifestation in the NMOSD familial cases. The frequency at which the mutation occurred in patients with sporadic NMOSD was significantly higher than for HCs (5.7% and 0, respectively; p<0.01). The mutation was located in the extracellular domain close to the transmembrane region, at a point in the protein sequence characterized by threonine enrichment. The mutant NECL2 had a lower molecular weight and exhibited defective trafficking to the cell surface. CONCLUSIONS: Our results suggest that the Necl2 mutation identified herein may be associated with the risk of developing NMOSD. Furthermore, mutated NECL2 may play a role in the pathogenesis of the disease, potentially through its roles in axonal regeneration and/or via neuron-glia interactions that are relevant to myelination.

Class I-Restricted T Cell-Associated Molecule Is a Marker for IFN-γ-Producing iNKT Cells in Healthy Subjects and Patients with Type 1 Diabetes.

Class I-restricted T cell-associated molecule (CRTAM) is an activation marker expressed on the cell surface of activated invariant natural killer T (iNKT) cells, CD8+ T cells, and a small subset of CD4+ T cells. CRTAM has also been associated with a proinflammatory profile in murine CD4+ T cells. However, CRTAM has not been thoroughly explored in human cells. This work focused on evaluating CRTAM expression in human iNKT lymphocytes after activation with α-galactosylceramide, its widely used specific glycolipid antigen. We also analyzed the involvement of costimulatory molecules in CRTAM expression and whether CRTAM expression is associated with a specific effector cytokine profile. We found that the signal produced by invariant T cell receptor (iTCR) engagement with α-galactosylceramide is sufficient to trigger CRTAM expression on human iNKT cells after 18 h of stimulation. Moreover, we observed a clear association between CRTAM expression and IFN-γ production in iNKT cells from healthy subjects and patients with type 1 diabetes. However, blocking the engagement of costimulatory molecules, such as CD40, CD80, and CD86, did not modify CRTAM expression. These results indicate that CRTAM may also play a role in triggering the production of IFN-γ in human iNKT cells and that CRTAM could be used as a marker to identify these inflammatory cells.

Necl-2/CADM1 interacts with ErbB4 and regulates its activity in GABAergic neurons.

The neuronal network is tightly regulated by a large variety of locally connected GABAergic neurons. Neuregulin1 (Nrg1) and its receptor ErbB4 are master regulators in the morphological and functional development of excitatory synapses in GABAergic neurons. We previously showed that the immunoglobulin-like cell adhesion molecule, nectin-like molecule-2 (Necl-2)/CADM1, interacts with the ErbB3 and ErbB4 receptors, and that the interaction of Necl-2 with ErbB3 inhibits the Nrg1-induced ErbB3/ErbB2 signaling in epithelial cells. Here, we examined the role of the interaction of Necl-2 with ErbB4 in GABAergic neurons. Necl-2 was co-expressed with ErbB4 in parvalbumin-positive GABAergic neurons in the mouse hippocampus and co-localized with ErbB4 at excitatory synapses. Necl-2 knockdown enhanced the Nrg1-induced phosphorylation of ErbB4. Moreover, overexpression of PTPN13, which is a tyrosine phosphatase bound to the cytoplasmic tail of Necl-2, suppressed the Nrg1-induced development of excitatory synapses in GABAergic neurons through the inhibition of ErbB4 activity. These results indicate that Necl-2 interacts with ErbB4 and regulates the development of excitatory synapses via the regulation of ErbB4 activity in GABAergic neurons.

Adhesion molecule CADM1 contributes to gap junctional communication among pancreatic islet α-cells and prevents their excessive secretion of glucagon.

Cell adhesion molecule-1 (CADM1) is a recently identified adhesion molecule of pancreatic islet α-cells that mediates nerve-α-cell interactions via trans-homophilic binding and serves anatomical units for the autonomic control of glucagon secretion. CADM1 also mediates attachment between adjacent α-cells. Since gap junctional intercellular communication (GJIC) among islet cells is essential for islet hormone secretion, we examined whether CADM1 promotes GJIC among α-cells and subsequently participates in glucagon secretion regulation. Dye transfer assays using αTC6 mouse α-cells, which endogenously express CADM1, supported this possibility; efficient cell-to-cell spread of gap junction-permeable dye was detected in clusters of αTC6 cells transfected with nonspecific, but not with CADM1-targeting, siRNA. Immunocytochemical analysis of connexin 36, a major component of the gap junction among αTC6 cells, revealed that it was localized exclusively to the cell membrane in CADM1-non-targeted αTC6 cells, but diffusely to the cytoplasm in CADM1-targeted cells. Next, we incubated CADM1-targeted and non-targeted αTC6 cells in a medium containing 1 mM glucose and 200 mM arginine for 30 min to induce glucagon secretion, and found that the targeted cells secreted three times more glucagon than did the non-targeted. We conducted similar experiments using pancreatic islets that were freshly isolated from wild-type and CADM1-knockout mice, and expressed glucagon secretion as ratios relative to baseline values. The increase in ratio was larger in CADM1-knockout islets than in wild-type islets. These results suggest that CADM1 may serve as a volume limiter of glucagon secretion by sustaining α-cell attachment necessary for efficient GJIC.