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. 2011 May 6;286(18):16516-24.
doi: 10.1074/jbc.M111.227298. Epub 2011 Mar 21.

Binding affinity and specificity of neuromyelitis optica autoantibodies to aquaporin-4 M1/M23 isoforms and orthogonal arrays

Jonathan M Crane  1 , Chiwah Lam, Andrea Rossi, Tripta Gupta, Jeffrey L Bennett, A S Verkman
Affiliations

Binding affinity and specificity of neuromyelitis optica autoantibodies to aquaporin-4 M1/M23 isoforms and orthogonal arrays

Jonathan M Crane et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Schematic of the two-color ratio imaging method for quantitative measurement of NMO-IgG binding to AQP4 isoforms. A, AQP4 monomers (cylinders) are shown as assembling into tetramers (top) or OAPs (bottom). NMO-IgG (green) binds AQP4 at an extracellular domain, and a reference AQP4 antibody (red) binds on the cytoplasmic side. B, reference AQP4 antibody binds to the C terminus of AQP4, independent of the AQP4 N-terminal isoform and OAP formation.
FIGURE 2.
FIGURE 2.
Characterization of stably transfected, AQP4-expressing U87MG cells. A, confocal fluorescence images show U87MG cells stably expressing M1 (top) or M23 (bottom) and labeled with NMO-IgG (green) and C-terminal anti-AQP4 antibody (red). B, TIRF images show distinct OAPs in M23-expressing cells (bottom) and a smooth fluorescence staining pattern in M1-expressing cells (top). C, AQP4 immunoblot following Blue-Native-PAGE (top) and Tricine SDS-PAGE (bottom) of stable AQP4-expressing U87MG cell lysates is shown. D, measured G/R fluorescence ratios in U87MG cells after stable (gray) or transient (white) transfection with M1 or M23 AQP4 and labeled with the indicated recombinant monoclonal NMO-IgG (mean ± S.E. (error bars), n = 4) are shown.
FIGURE 3.
FIGURE 3.
Differential binding of NMO-IgG in NMO patient serum to M1 versus M23 AQP4. A, M1- and M23-expressing U87MG cells stained with 5% NMO serum (green) from four patients and with reference AQP4 antibody (red). B, binding curves for the NMO patient sera to M1 versus M23 AQP4 (mean ± S.E. (error bars), n = 5). Curves represent fits to single-site binding model.
FIGURE 4.
FIGURE 4.
Differential binding of purified monoclonal NMO-IgGs to M1 versus M23 AQP4. A, representative fluorescence micrographs for binding of rAb-53 and rAb-58 (green) as a function of concentration, together with reference AQP4 antibody (red). B, binding curves for rAb-53 (left), rAb-58 (middle), and rAb-186 (right) to M1 versus M23 AQP4 (mean ± S.E. (error bars), n = 5). Curves represent fits to a single-site binding model, with fitted KD = 44 ± 7 nm and 2.8 ± 1 μm (rAb-53), 68 ± 9 nm and 147 ± 20 nm (rAb-58), and 146 ± 25 nm and 8.1 ± 3 μm (rAb-186), each for M23 and M1, respectively.
FIGURE 5.
FIGURE 5.
Binding of NMO-IgG to mixtures of M1 and M23 AQP4 and to M23 mutants containing OAP-disrupting mutations. A, binding of rAb-53 (left) and cumulative distributions of diffusion range (right), measured by Qdot single-particle tracking, for M1 and M23 AQP4 mixtures at the indicated ratios (mean ± S.E. (error bars), n = 5). B, binding of rAb-53 (left) and diffusion range (right) for M23 AQP4 with M1 mutant CCA at the indicated ratios (mean ± S.E., n = 5). C, binding of rAb-53 (left) and diffusion range (right) for AQP4 mutants M23-F26Q (red) and M23-G28P (green) (mean ± S.E., n = 5).
FIGURE 6.
FIGURE 6.
Mechanism of increased NMO-IgG binding affinity to OAP-assembled AQP4. A, human IgG (Protein Data Bank ID code 1IGY) (38) and AQP4 (Protein Data Bank ID code 3GD8) (39) crystal structures showing relative size of the AQP4 tetramer compared with spacing between Fab binding sites in whole IgG. B, predictions of bivalent versus monovalent binding mechanisms. AQP4 monomers (cylinders) are shown as assembled in tetramers (M1) or OAPs (M23). NMO-IgG (green) binds either monovalently or bivalently to (unknown) extracellular domains on AQP4. C, binding of monoclonal mouse anti-Myc to cells expressing Myc-tagged M1 versus M23 AQP4. D, relative M1-to-M23 binding of whole IgG or purified Fab fragments of mouse anti-Myc (left), rAb-53 (middle), and rAb-58 (right) at a fixed concentration (mean ± S.E. (error bars), n = 5).
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