Antibodies to heteromeric glycolipid complexes in Guillain-Barré syndrome

Abstract

Autoantibodies are infrequently detected in the sera of patients with the demyelinating form of Guillain-Barré syndrome most commonly encountered in the Western world, despite abundant circumstantial evidence suggesting their existence. We hypothesised that antibody specificities reliant on the cis interactions of neighbouring membrane glycolipids could explain this discrepancy, and would not have been detected by traditional serological assays using highly purified preparations of single gangliosides. To assess the frequency of glycolipid complex antibodies in a Western European cohort of patients GBS we used a newly developed combinatorial glycoarray methodology to screen against large range of antigens (11 gangliosides, 8 other single glycolipids and 162 heterodimeric glycolipid complexes). Serum samples of 181 patients from a geographically defined, Western European cohort of GBS cases were analysed, along with 161 control sera. Serum IgG binding to single gangliosides was observed in 80.0% of axonal GBS cases, but in only 11.8% of cases with demyelinating electrophysiology. The inclusion of glycolipid complexes increased the positivity rate in demyelinating disease to 62.4%. There were 40 antigens with statistically significantly increased binding intensities in GBS as compared to healthy control sera. Of these, 7 complex antigens and 1 single ganglioside also produced statistically significantly increased binding intensities in GBS versus neurological disease controls. The detection of antibodies against specific complexes was associated with particular clinical features including disease severity, requirement for mechanical ventilation, and axonal electrophysiology. This study demonstrates that while antibodies against single gangliosides are often found in cases with axonal-type electrophysiology, antibodies against glycolipid complexes predominate in cases with demyelinating electrophysiology, providing a more robust serum biomarker than has ever been previously available for such cases. This work confirms the activation of the humoral immune system in the dysimmune disease process in GBS, and correlates patterns of antigen recognition with different clinical features.

Figure 1. Diverse patterns of glycolipid binding in GBS sera.

(A,B) Representative 28x10 arrays. The antigen spotted at each grid location is revealed by combining the row and column headings given in the key below. Thus, locations (9,7) and (7,9) contain GA1:GM3 complexes. The line of ‘x’ across the centre of the membrane represents the negative control spots (methanol only spotted). The left most column and upper most row contain single antigens only. (A) An array probed with GBS serum showing both complex attenuated and enhanced binding patterns. GD1b binding (14) is completely attenuated in complex with GD2 (location 14,6). In contrast, despite no demonstrable binding to GA1, GM3 or GD1a in isolation, an intense spot is seen with both GA1:GM3 (location 9,7) and GA1:GD1a (location 9,13) complexes. This is complex enhanced/complex dependent binding. (B) Highly specific complex-dependent GBS sera. In this example, only the duplicate spots of GM1:phosphatidylserine complex (location 2,12) are bound. (C) Boxplot of the distribution of maximum binding intensity for GBS versus healthy control (HC) sera assessed for single gangliosides, single glycolipids, and glycolipid complexes. Maximum binding intensity is significantly higher for GBS sera versus healthy control (HC) sera for each antigen group (*p<0.05, **p<0.0001). GBS sera maximum binding intensity for complexes is significantly higher than for either single glycolipids or gangliosides (†=p<0.001), but HC sera maximum binding intensity is insignificantly different between the antigen groups (not marked). (all Kruskal-Wallis/Mann-Whitney). (D) Overview heatmap of raw, unclustered binding intensities for GBS (above horizontal white line) and HC (below horizontal white line) sera. Antigens to the right of the vertical white line are glycolipid complexes. The intensity of binding is given by the bar above the heatmap, ranging from black (negative) to red (most intense). A more detailed, zoomable version of this heatmap, allowing resolution of results for each antigen and each serum, is available online (Figure S1). Key: (1)Sphingomyelin, (2)Phosphatidylserine, (3)Globoside, (4)Cereamide trihexosides (CTH), (5) Sulfated glucuronyl paragloboside (SGPG), (6)GM2, (7)GM3, (8)GD2, (9)Asialo-GM1, (10)Galactocerebroside (GalC), (11)LM1, (12)GM1, (13)GD1a, (14)GD1b, (15)GD3, (16)GQ1b, (17)GT1b, (18)Sulfatide.

Figure 2. Complex enhanced binding.

(A) Receiver operator curves for single ganglioside antigens, single glycolipids, glycolipid complexes, and for all 181 antigens. Complex antigens (dashed green line) afford the best trade-off between sensitivity and specificity. (B) Individual value plots for GBS sera binding intensity to single sulfatide (sul), single CTH, the sum of these single intensities (sulfatide+CTH), and CTH:sulfatide complexes. (C) Comparison between the sum of single sulfatide and CTH binding intensities (sul+CTH) and binding intensity to CTH:sulfatide complexes in individual sera. Each line links the respective values in a single serum. Lines sloping upwards from left to right indicate complex enhanced binding with a single serum. (D) Overview heatmap of corrected complex binding intensities. The intensity value for each complex has been corrected by subtracting the binding intensities of each component glycolipid. Any residual intensity indicates complex enhanced binding. As before, GBS sera lie above and healthy control (HC) sera below the horizontal white line. The same colour scale as Figure 1 has been used. A more detailed, zoomable version of this heatmap, allowing resolution of results for each complex antigen and each serum, is available online (Figure S3).

Figure 3. Heatmap of glycolipid complex binding in GBS and control sera.

Clustered heatmap for the 35 antigens significantly associated with GBS (arranged on the x-axis), comparing the 181 GBS sera and 161 control sera (on the y-axis). Each row represents a single serum. The log transformed arbitrary binding intensity for each antigen is represented by the colour scale, from red (log transformed arbitrary intensity of 18, most intense) to the green background (log transformed arbitrary intensity of 0, no binding), as shown at the top of the figure. Each heatmap panel represents a separate group, as labelled. (GBS, Guillain-Barré syndrome; MS, Multiple Sclerosis; ONND, Other Non-Inflammatory Neurological Diseases; HC, Healthy Controls). Blue boxes delineate clusters of GM1 and sulfatide-complex antigens. Arrows mark sulfatide complexes which cluster separately.

Figure 4. Differing patterns of sulfatide complex enhanced binding in disease versus control sera.

(A) The receiver operator curve for the 8 antigen intensities significantly associated with GBS versus OND controls is insignificantly different to that produced by including all 35 antigens. (B) Individual value plots of binding intensity for GBS, healthy control (HC), and other neurological disease (OND) sera to sulfatide, CTH:sulfatifde complex, and GA1:sulfatide complex. (C) Mean change in complex compared to summed single antigen binding intensity for CTH:sulfatide and GA1:sulfatide. On average, GBS sera show complex enhancement, whereas HC and OND sera show complex attenuation.

Figure 5. GM1 complex antigens.

(A) Individual value plots for selected antigens. (B) Association between sulfatide:GM1 and GM1 binding intensities in individual sera. Points on the y-axis are GM1 negative, sulfatide:GM1 positive. Sera close to the diagonal blue line (joining points of identical binding intensity for both antigens) bind both GM1 and sulfatide:GM1 complex with similar intensity. (C) Association between phosphatidylserine:GM1 and GM1 binding intensities in individual sera. Points on the y-axis are GM1 negative, PS:GM1 positive. Sera on the x-axis are GM1 positive, PS:GM1 negative. (D) Association between phosphatidylserine:GM1 and sulfatide:GM1 binding intensities in individual sera.