Binding affinity and specificity of neuromyelitis optica autoantibodies to aquaporin-4 M1/M23 isoforms and orthogonal arrays
ABSTRACT Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for the neuroinflammatory disease neuromyelitis optica (NMO). We measured the binding of NMO autoantibodies to AQP4 in human astrocyte-derived U87MG cells expressing M1 and/or M23 AQP4, or M23 mutants that do not form orthogonal array of particles (OAPs). Binding affinity was quantified by two-color fluorescence ratio imaging of cells stained with NMO serum or a recombinant monoclonal NMO autoantibody (NMO-rAb), together with a C terminus anti-AQP4 antibody. NMO-rAb titrations showed binding with dissociation constants down to 44 ± 7 nm. Different NMO-rAbs and NMO patient sera showed a wide variation in NMO-IgG binding to M1 versus M23 AQP4. Differences in binding affinity rather than stoichiometry accounted for M1 versus M23 binding specificity, with consistently greater affinity of NMO-IgG binding to M23 than M1 AQP4. Binding and OAP measurements in cells expressing different M1:M23 ratios or AQP4 mutants indicated that the differential binding of NMO-IgG to M1 versus M23 was due to OAP assembly rather than to differences in the M1 versus M23 N termini. Purified Fab fragments of NMO-IgG showed similar patterns of AQP4 isoform binding, indicating that structural changes in the AQP4 epitope upon array assembly, and not bivalent cross-linking of whole IgG, result in the greater binding affinity to OAPs. Our study establishes a quantitative assay of NMO-IgG binding to AQP4 and indicates remarkable, OAP-dependent heterogeneity in NMO autoantibody binding specificity.
- SourceAvailable from: Tobias J Moritz
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- "OAP formation by AQP4 is of central importance in the pathogenesis of the multiple sclerosis-like disease NMO, where binding of autoantibodies to AQP4 on astrocytes in the central nervous system causes cytotoxicity, inflammation, myelin loss and neurological deficit (Papadopoulos and Verkman, 2012). We found previously that immunoglobulin G autoantibodies against AQP4, NMO-IgG, generally bind with much greater affinity to array-assembled AQP4 than to AQP4 tetramers (Crane et al., 2011). We recently reported a second, major effect of AQP4 OAP assembly in NMO involving complement-dependent cytotoxicity, in which binding of the multivalent complement protein C1q is greatly enhanced when NMO-IgG is clustered on AQP4 OAPs (Phuan et al., 2012). "
ABSTRACT: Aquaporin-4 (AQP4) is a water channel expressed in astrocytes, skeletal muscle and epithelial cells that forms supramolecular aggregates in plasma membranes called orthogonal arrays of particles (OAPs). AQP4 is expressed as a short isoform (M23) that forms large OAPs, and a long isoform (M1) that does not form OAPs by itself but can co-mingle with M23 to form relatively small OAPs. AQP4 OAPs were imaged with ∼20 nm spatial precision by photoactivation localization microscopy (PALM) in cells expressing chimeras of M1- or M23-AQP4 with photoactivatable fluorescent proteins. Native AQP4 was imaged by direct stochastic optical reconstruction microscopy (dSTORM) using primary anti-AQP4 antibody and fluorescent secondary antibodies. We found that OAP area increased from 1878±747 to 3647±958 nm(2) with decreasing M1M23 ratio from 11 to 13, and became elongated. Two-color dSTORM indicated that M1 and M23 co-assemble in OAPs with a M1-enriched periphery surrounding a M23-enriched core. Native AQP4 in astrocytes formed OAPs of area 2142±829 nm(2), which increased to 5137±1119 nm(2) with 2-bromopalmitate. PALM of AQP4 OAPs in live cells showed slow diffusion (average D ∼ 10(-12) cm(2)/s) and reorganization. OAP area was not altered by anti-AQP4 IgG autoantibodies (NMO-IgG) that cause the neurological disease neuromyelitis optica. Super-resolution imaging allowed elucidation of novel nanoscale structural and dynamic features of OAPs.Journal of Cell Science 06/2012; 125(18). DOI:10.1242/jcs.109603 · 5.33 Impact Factor
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- "These isoforms aggregate to form structures known as orthogonal arrays of particles (OAPs), the size of which is determined by their concentration ratio (Verbavatz et al., 1997; Furman et al., 2003). It is still controversial whether NMO-IgG specifically binds to OAP assemblies or M1 and/or M23 isoforms (Marnetto et al., 2009; Nicchia et al., 2009; Tani et al., 2009; Mader et al., 2010; Crane et al., 2011). Several lines of evidence indicate that NMO-IgG not only serves as a L. Melamud and J.M. Fernández contributed equally to this work. "
ABSTRACT: NMO-IgG autoantibody selectively binds to aquaporin-4 (AQP4), the most abundant water channel in the central nervous system and is now considered a useful serum biomarker of neuromyelitis optica (NMO). A series of clinical and pathological observations suggests that NMO-IgG may play a central role in NMO physiopathology. The current study evaluated, in well-differentiated astrocytes cultures, the consequences of NMO-IgG binding on the expression pattern of AQP4 and on plasma membrane water permeability. To avoid or to facilitate AQP4 down-regulation, cells were exposed to inactivated sera in two different situations (1 hr at 4°C or 12 hr at 37°C). AQP4 expression was detected by immunofluorescence studies using a polyclonal anti-AQP4 or a human anti-IgG antibody, and the water permeability coefficient was evaluated by a videomicroscopy technique. Our results showed that, at low temperatures, cell exposure to either control or NMO-IgG sera does not affect either AQP4 expression or plasma membrane water permeability, indicating that the simple binding of NMO-IgG does not affect the water channel's activity. However, at 37°C, long-term exposure to NMO-IgG induced a loss of human IgG signal from the plasma membrane along with M1-AQP4 isoform removal and a significant reduction of water permeability. These results suggest that binding of NMO-IgG to cell membranes expressing AQP4 is a specific mechanism that may account for at least part of the pathogenic process.Journal of Neuroscience Research 06/2012; 90(6):1240-8. DOI:10.1002/jnr.22822 · 2.73 Impact Factor
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- "We found by analysis of Mz truncation mutants that residues located between 12–22 in Mz AQP4 are involved in the relatively poor binding of NMO-IgG to Mz AQP4. Our prior data indicated that differences in binding affinity of NMO-IgG for AQP4 isoforms is due to differences in the AQP4 epitope, not to bivalent binding or AQP4 crosslinking by NMO-IgG (Crane et al., 2011). Therefore, we speculate that the poor binding of NMO-IgG to Mz is likely due to structural differences in the extracellular domain of the AQP4 tetramer. "
ABSTRACT: Water channel aquaporin-4 (AQP4) is expressed in astrocytes throughout brain and spinal cord. Two major AQP4 isoforms are expressed, M1 and M23, having different translation initiation sites. A longer isoform (Mz) has been reported in rat with translation initiation 126-bp upstream from that of M1. By immunoblot analysis of SDS and native gels probed with a C-terminus anti-AQP4 antibody, Mz was detected in rat brain as a distinct band of size ∼39 kDa. Mz was absent in human and mouse brain because of in-frame stop codons. The ability of rat Mz to form orthogonal arrays of particles (OAPs) was investigated by single particle tracking and native gel electrophoresis. We found that Mz, like M1, diffused rapidly in the cell plasma membrane and did not form OAPs. However, when co-expressed with M23, Mz associated in OAPs by forming heterotetramers with M23. Unexpectedly, Mz-expressing cells bound neuromyelitis optica autoantibodies (NMO-IgG) poorly, <5-fold compared with M1-expressing cells. Truncation analysis suggested that the poor NMO-IgG binding to Mz involves residues 31-41 upstream of Met-1. We conclude that Mz AQP4 is (a) present at low level in rat but not human or mouse brain, (b) unable to form OAPs on its own but able to associate with M23 AQP4 in heterotetramers, and (c) largely unable to bind NMO-IgG because of N-terminus effects on the structure of the AQP4/NMO-IgG binding site.Glia 07/2011; 59(7):1056-63. DOI:10.1002/glia.21177 · 6.03 Impact Factor