Specific contactin N-glycans are implicated in neurofascin binding and autoimmune targeting in peripheral neuropathies

Abstract

Cell adhesion molecules (CAMs) play a crucial role in the formation of the nodes of Ranvier and in the rapid propagation of the nerve impulses along myelinated axons. These CAMs are the targets of autoimmunity in inflammatory neuropathies. We recently showed that a subgroup of patients with aggressive chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) shows autoantibodies to contactin (1). The complex of contactin·Caspr·neurofascin-155 (NF155) enables the formation of paranodal junctions, suggesting that antibody attack against paranodes may participate in the severity of CIDP. In the present study, we mapped the molecular determinants of contactin targeted by the autoantibodies. In three patients, immunoreactivity was directed against the Ig domains of contactin and was dependent on N-glycans. The serum of one patient was selectively directed against contactin bearing mannose-rich N-glycans. Strikingly, the oligomannose type sugars of contactin are required for association with its glial partner NF155 (2). To investigate precisely the role of contactin N-glycans, we have mutated each of the nine consensus N-glycosylation sites independently. We found that the mutation of three sites (N467Q/N473Q/N494Q) in Ig domain 5 of contactin prevented soluble NF155-Fc binding. In contrast, these mutations did not abolish cis-association with Caspr. Next, we showed that the cluster of N-glycosylation sites (Asn-467, Asn-473, and Asn-494) was required for immunoreactivity in one patient. Using cell aggregation assays, we showed that the IgGs from the four CIDP patients prevented adhesive interaction between contactin·Caspr and NF155. Importantly, we showed that the anti-contactin autoantibodies induced alteration of paranodal junctions in myelinated neuronal culture. These results strongly suggest that antibodies to CAMs may be pathogenic and induce demyelination via functional blocking activity.

Keywords: Cell Adhesion; Cell Surface Protein; Glycoprotein; Myelin; Neuroinflammation.

FIGURE 1.

CIDP autoantibodies against contactin are of the IgG4 subclass. Shown is isotype analysis of the sera from patients 1, 2, and 4. ELISA plates were coated with human recombinant contactin (CNTN1). Optical density was measured at 450 nm after incubation with serial dilutions of CIDP sera and peroxidase-conjugated anti-human IgG1–4 secondary antibodies and reaction with tetramethylbenzidine solution.

FIGURE 2.

CIDP autoantibodies against contactin display functional blocking activity. Shown are cell aggregation assays using N2a cells co-transfected with Caspr-GFP and contactin (A) or transfected with NF155-mcherry (B). Cells were harvested and incubated on a rotary shaker for 2 h at 37 °C. As a negative control, cells transfected with Caspr2-GFP and NF155-mcherry did not form mixed aggregates (C). Caspr-GFP/contactin- and NF155-mcherry-expressing cells formed mixed aggregates in the presence of control IgGs diluted to 1:50 (D). IgGs from patients 1, 3, and 4 prevented the formation of mixed aggregates at a 1:200 dilution (F, H, and I). Incubation with IgGs from patient 2 blocked the formation of mixed aggregates at a 1:50 (E) but not at a 1:200 dilution (G). C–I, phase-contrast and fluorescence overlay (left) and fluorescence overlay (right). Bar, 40 μm. J, mixed aggregates were defined as described under “Experimental Procedures” after counting the green and red cells and the number of heterotypic contacts (white bars), as illustrated. 10 fields at the ×20 objective were analyzed for each condition. Shown is the mean number ± S.E. (error bars) of cells recruited in mixed aggregates per field. *, significant difference (p < 0.01) by comparison with control IgGs using analysis of variance and Fisher's test.

FIGURE 3.

CIDP autoantibodies against contactin induce paranodal alteration in myelinated DRG neurons in culture. DRG neuron/Schwann cell mixed cultures were induced for myelination for 7 weeks in vitro. A–C, serum IgGs from CIDP patients 1, 3, and 4 binds the paranodes (green). Cells were fixed with 1% paraformaldehyde and permeabilized with 0.1% Triton X-100 before immunostaining with IgGs from CIDP patients diluted 1:500 (green), rabbit anti-Caspr (red), and rat anti-MBP (blue) as a myelin marker. In functional assays, cells were incubated with control (D and F) or patient 4 (E and G) IgGs diluted 1:200 for 36 h. Cells were fixed with methanol and immunostained with rabbit anti-Caspr (red), mouse anti-pan Nav (green), and rat anti-MBP (blue). Note that IgGs from CIDP patient 4 induced alteration of paranodal immunostaining and enlargement of the nodal region. H, quantitative analysis of nodal and paranodal lengths and of the ratio between Caspr and Nav integrated fluorescence intensity after incubation with IgGs of control and CIDP patients 1 and 4. More than 70 nodes were analyzed under each condition. * and **, significant difference (p < 0.05 and p < 0.01, respectively) in comparison with control IgGs using analysis of variance and Fisher's test. Bar in A–C, F, and G, 5 μm; bar in D and E, 10 μm. Error bars, S.E.

FIGURE 4.

Autoantibodies from CIDP patients 1, 2, and 4 are targeted against the N-glycosylated Ig domains of contactin. N2a cells were transfected with contactin (A and C), contactin-Ig showing a deletion of the four Fn domains (B and D), or HA-tagged contactin-Fn showing a deletion of the six Ig domains (E and F). Surface labeling indicated that IgGs from CIDP patients 1 and 2 recognized contactin and contactin-Ig but not contactin-Fn. Double staining of contactin-Fn using anti-HA mAb (green) in E and F. G–L, N2a cells transfected with contactin-GFP were untreated (G–I) or treated (J–L) with tunicamycin (2 μg/ml). Cells were fixed and permeabilized before immunostaining with IgGs from CIDP patients 1, 2, and 4 (red). Unglycosylated contactin-GFP was retained in the ER and was not recognized by the autoantibodies. M and N, the sera from patients 1 and 2 recognized human CNTN1 transfected in HEK cells. Bars in A–F, M, and N, 10 μm; bars in G–L, 20 μm.

FIGURE 5.

Autoantibodies from CIDP patient 3 specifically recognize contactin bearing mannose-rich N-glycans. N2a cells were co-transfected with contactin-GFP and Caspr (A) or transfected with contactin-GFP alone (B) and surface-labeled with purified IgGs from CIDP patient 3 (red). CHO Lec1 mutant cells transfected with contactin (C) or contactin-Ig (D) were surface-labeled with IgGs from CIDP patient 3 (C and D, red) and rabbit anti-contactin antibody (green). CHO Lec1 cells were transfected with contactin and surface-labeled with IgGs from patient 1 (E) and patient 2 (F). As schematized, contactin co-expressed with Caspr in N2a cells or contactin expressed in Lec1 cells bears mannose-rich N-glycans. In contrast, contactin expressed in N2a cells bears complex N-glycans. Only mannose-rich contactin was targeted by autoantibodies from CIDP patient 3. G and H, N2a transfected with Caspr and contactin (G) and Lec1 cells transfected with contactin alone (H). Preincubation of IgGs from patient 3 with 20 μm oligomannose-5 did not prevent surface labeling. Bar, 10 μm.

FIGURE 6.

Antibodies from CIDP patient 3 are directed against N-glycosylated Caspr. N2a cells were transfected with Caspr (A), CasprΔ1 (B), or CasprΔ4 (C), which are deleted from the N- or C-region of the ectodomain, respectively. Cells were fixed with methanol and double-stained with IgGs from patient 3 (left) and rabbit anti-Caspr antibodies (right). N2a cells co-transfected for Caspr and contactin were untreated (D) or treated (E) with tunicamycin and fixed with methanol. Note that Caspr was expressed at the plasma membrane only when co-expressed with contactin (compared D with A). The autoantibodies from patient 3 reacted against full-length N-glycosylated Caspr. Bar, 10 μm.

FIGURE 7.

Selective mutations of N-glycosylation sites of contactin prevent its trans-interaction with NF155. Lec1 cells were transfected with contactin-GFP (A and B) or contactin-GFP mutated for its N-glycosylation sites (D and E) as schematized, with the mutated sites indicated in red. All of the mutated constructs were detected at the cell surface using mouse anti-GFP and Alexa-568-conjugated secondary antibodies (A and D). C, quantitative analysis of the cell surface expression of contactin-GFP mutants using cell ELISA. B and E, cells were incubated with 50 nm NF155-Fc chimera and Alexa-568-conjugated anti-human antibodies (red). Note that mutation of the three sites Asn-457, Asn-473, and Asn-494 in the Ig5 domain of contactin prevented NF155-Fc binding. F, Western blot analysis of contactin-GFP mutants expressed in N2a cells and detected using mouse anti-GFP mAb. Note the lower apparent molecular weight of the N457Q/N473Q/N494Q mutant by comparison with the other mutants and control contactin-GFP (Cont). Bar, 10 μm. Error bars, S.E. N_521,593Q, N521Q/N593Q; N_457,473,494Q, N457Q/N473Q/N494Q.

FIGURE 8.

Mutations of N-glycosylation sites of contactin do not prevent its cis-association with Caspr. N2a cells were transfected with Caspr alone or co-transfected with Caspr and contactin-GFP or with Caspr and contactin-GFP mutated for its N-glycosylation sites. Cells were fixed with methanol and immunostained with rabbit anti-Caspr antibodies and Alexa-568-conjugated secondary antibodies. The co-expression of wild-type or mutated contactin-GFP induces ER exit and cell surface targeting of Caspr. Bar, 5 μm. N_521,593Q, N521Q/N593Q; N_457,473,494Q, N457Q/N473Q/N494Q.

FIGURE 9.

The N-glycosylation sites of the Ig5 domain of contactin are selectively required for immune reactivity of CIDP patient 2. N2a cells (A and B) or Lec1 cells (C) were transfected with contactin-GFP or contactin-GFP mutated for its N-glycosylation sites. Cells were fixed with paraformaldehyde and immunostained with IgGs from CIDP patient 1 (A), patient 2 (B), and patient 3 (C) and Alexa-568-conjugated anti-human antibodies (red). Note that mutation of the three sites Asn-457, Asn-473, and Asn-494 in contactin Ig5 prevented immunoreactivity with IgGs from patient 2 but not from patients 1 and 3. Bar, 10 μm. N_521,593Q, N521Q/N593Q; N_457,473,494Q, N457Q/N473Q/N494Q.