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The neuropathic potential of anti-GM1 autoantibodies is regulated by the local glycolipid environment in mice
Kay N. Greenshields, … , Jaap J. Plomp, Hugh J. Willison
Kay N. Greenshields, … , Jaap J. Plomp, Hugh J. Willison
Published February 16, 2009
Citation Information: J Clin Invest. 2009;119(3):595-610. https://doi.org/10.1172/JCI37338.
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Research Article Neuroscience

The neuropathic potential of anti-GM1 autoantibodies is regulated by the local glycolipid environment in mice

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Abstract

Anti-GM1 ganglioside autoantibodies are used as diagnostic markers for motor axonal peripheral neuropathies and are believed to be the primary mediators of such diseases. However, their ability to bind and exert pathogenic effects at neuronal membranes is highly inconsistent. Using human and mouse monoclonal anti-GM1 antibodies to probe the GM1-rich motor nerve terminal membrane in mice, we here show that the antigenic oligosaccharide of GM1 in the live plasma membrane is cryptic, hidden on surface domains that become buried for a proportion of anti-GM1 antibodies due to a masking effect of neighboring gangliosides. The cryptic GM1 binding domain was exposed by sialidase treatment that liberated sialic acid from masking gangliosides including GD1a or by disruption of the live membrane by freezing or fixation. This cryptic behavior was also recapitulated in solid-phase immunoassays. These data show that certain anti-GM1 antibodies exert potent complement activation-mediated neuropathogenic effects, including morphological damage at living terminal motor axons, leading to a block of synaptic transmission. This occurred only when GM1 was topologically available for antibody binding, but not when GM1 was cryptic. This revised understanding of the complexities in ganglioside membrane topology provides a mechanistic account for wide variations in the neuropathic potential of anti-GM1 antibodies.

Authors

Kay N. Greenshields, Susan K. Halstead, Femke M.P. Zitman, Simon Rinaldi, Kathryn M. Brennan, Colin O’Leary, Luke H. Chamberlain, Alistair Easton, Jennifer Roxburgh, John Pediani, Koichi Furukawa, Keiko Furukawa, Carl S. Goodyear, Jaap J. Plomp, Hugh J. Willison

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Figure 5

Localization of GM1 and GD1a to raft fractions in PC12 cells.

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Localization of GM1 and GD1a to raft fractions in PC12 cells.
(A) PC12 c...
(A) PC12 cell immunostaining (original magnification, ×40). DG1 binding to PC12 cells (shown in phase contrast) is not detectable despite the presence of GM1, as shown by CTB staining of cells. Scale bars: 15 μm. (B) Effect of neuraminidase on DG1 and MOG35 binding to PC12 cells. Double staining reveals that control cells are positively stained with MOG35 (TRITC), with no binding of DG1 (FITC). Following neuraminidase treatment, MOG35 staining is diminished, with a concomitant increase in DG1 binding. Scale bars: 15 μm (images acquired at ×40 magnification). (C) GM1 and GD1a pixel-by-pixel colocalization. FITC and TRITC (anti-GM1 and anti-GD1a, respectively) images (×63 magnification) from double-stained PC12 cells, with colocalization appearing as yellow overlap. Plane-by-plane colocalization (linear scatter plot) shows strong colocalization. (D) Western blot of raft immunoprecipitation based on MOG35 binding, allowing isolation of GD1a-positive rafts by anti-mouse IgG–coated beads. In irrelevant antibody–incubated cells, no rafts were isolated by anti-mouse IgG–coated beads. Bound sample was concentrated (×10) to amplify any potentially weak signal. In MOG35-incubated cells, a population of rafts was isolated by the beads. Isolated fractions contained the raft-associated protein flotillin, but not SNAP25, which was taken as evidence that the raft extraction procedure did not lead to coalescence of the heterogeneous raft population. Bound fractions also contained both the light chain (LC) and heavy chain (HC) of the anti-GD1a antibody, and the isolated rafts were positive for both GM1 and GD1a. SM, starting material; UB, unbound; B, bound; BC, bound concentrated.

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