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. 2013 Feb:40:21-7.
doi: 10.1016/j.jaut.2012.07.008. Epub 2012 Aug 18.

Astrocytic autoantibody of neuromyelitis optica (NMO-IgG) binds to aquaporin-4 extracellular loops, monomers, tetramers and high order arrays

Raffaele Iorio  1 , James P Fryer, Shannon R Hinson, Petra Fallier-Becker, Hartwig Wolburg, Sean J Pittock, Vanda A Lennon
Affiliations

Astrocytic autoantibody of neuromyelitis optica (NMO-IgG) binds to aquaporin-4 extracellular loops, monomers, tetramers and high order arrays

Raffaele Iorio et al. J Autoimmun. 2013 Feb.

Abstract

The principal central nervous system (CNS) water channel, aquaporin-4 (AQP4), is confined to astrocytic and ependymal membranes and is the target of a pathogenic autoantibody, neuromyelitis optica (NMO)-IgG. This disease-specific autoantibody unifies a spectrum of relapsing CNS autoimmune inflammatory disorders of which NMO exemplifies the classic phenotype. Multiple sclerosis and other immune-mediated demyelinating disorders of the CNS lack a distinctive biomarker. Two AQP4 isoforms, M1 and M23, exist as homotetrameric and heterotetrameric intramembranous particles (IMPs). Orthogonal arrays of predominantly M23 particles (OAPs) are an ultrastructural characteristic of astrocytic membranes. We used high-titered serum from 32 AQP4-IgG-seropositive patients and 85 controls to investigate the nature and molecular location of AQP4 epitopes that bind NMO-IgG, and the influence of supramolecular structure. NMO-IgG bound to denatured AQP4 monomers (68% of cases), to native tetramers and high order arrays (90% of cases), and to AQP4 in live cell membranes (100% of cases). Disease-specific epitopes reside in extracellular loop C more than in loops A or E. IgG binding to intracellular epitopes lacks disease specificity. These observations predict greater disease sensitivity and specificity for tissue-based and cell-based serological assays employing "native" AQP4 than assays employing denatured AQP4 and fragments. NMO-IgG binds most avidly to plasma membrane surface AQP4 epitopes formed by loop interactions within tetramers and by intermolecular interactions within high order structures. The relative abundance and localization of AQP4 high order arrays in distinct CNS regions may explain the variability in clinical phenotype of NMO spectrum disorders.

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Figures

Figure 1
Figure 1
A) Synthetic peptides corresponding to extracellular loops A (54–69), C (137–157) and E (207–231) of rat AQP4, and human AQP4 intracellular segments: N-terminal residues (1–22), loop B (94–114) and C-terminal residues (253–272). Extracellular loops A and C differ in rat and human AQP4 at 1 residue (highlighted in yellow); loop E is identical in rat and human AQP4. B) GST fusion proteins corresponding to extracellular loops A, C and E of human AQP4. Putative extracellular residues are bold and underlined.
Figure 2
Figure 2
A, B) Distribution of AQP4 immunoreactivity in plasma membranes of HEK293 cells transfected singly with plasmids encoding either M1 or M23 isoforms (imaged by confocal microscopy). M1 and M23 are identical in extracellular domain sequence. Their common C-terminal cytoplasmic segments were detected by a peptide-specific rabbit IgG (after fixation and permeabilization). AQP4 distribution is “continuous” in M1 membranes (note linear appearance at margins), but is discontinuous in M23 membranes. C, D) Freeze-fracture electron microscopic images of M1 and M23 membranes reveal singlet intramembranous particles in M1 cells. By contrast, most particles in M23 membranes appear as large lattices of orthogonal array-like assemblies. Note absence of lattices in M1 cells.
Figure 3
Figure 3
A) Indirect immunofluorescence reveals disease-specific IgG binding to the extracellular domain of AQP4 in live HEK293 cells transfected with M1 or M23, as illustrated by sera of 2 NMOSD patients. Healthy (and disease control) serum IgG does not bind. By contrast, rabbit IgG specific for cytoplasmic AQP4 epitopes (C-terminal residues 249–323) binds only to permeabilized cells. B) Western blot analysis of M1 and M23 cell lysate proteins separated in “native” form by BN-PAGE and in denatured form by SDS PAGE. IgG in serum of 2 illustrative NMOSD patients binds to AQP4 tetramers and intramembranous particles (M1 and M23) and to high order arrays (M23), and to monomers of each AQP4 isoform. IgG in a representative healthy control human serum does not bind to any AQP4 protein. The control rabbit IgG is specific for AQP4 C-terminal residues. Note: M1 cells lack evidence of an M23 polypeptide product.
Figure 4
Figure 4
A) Frequency of IgG reactive with peptides corresponding to AQP4 extracellular loops (detected by ELISA). The statistical significance of comparisons among groups is presented (cutoff for significance p=<0.05). B) Illustrative results of western blot analyses using human AQP4 extracellular loop GST fusion proteins. C) Schematic representation of human AQP4 residues constituting extracellular loops A, C and E.
Figure 5
Figure 5
Frequency of IgG reactive with peptides corresponding to AQP4 intracellular loops (detected by ELISA). The significance of comparisons among groups (p=<0.05) was determined by Fisher's Exact test.
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