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. 2011 Jul;59(7):1056-63.
doi: 10.1002/glia.21177. Epub 2011 Apr 12.

Aquaporin-4 Mz isoform: brain expression, supramolecular assembly and neuromyelitis optica antibody binding

Andrea Rossi  1 , Jonathan M Crane, A S Verkman
Affiliations

Aquaporin-4 Mz isoform: brain expression, supramolecular assembly and neuromyelitis optica antibody binding

Andrea Rossi et al. Glia. 2011 Jul.

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.

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Figures

Figure 1
Figure 1
Sequence analysis of human, rat and mouse AQP4. A. Alignment of human, rat and mouse AQP4 sequences. Indicated in boxes are potential Mz, M1 and M23 translation initiation ATG sequences, and in-frame stop codons in the human (TAA) and mouse (TGA) sequences upstream of the M1 start codon. B. Kyte-Doolittle hydropathy plot of rat Mz AQP4 using a 5-residue running sum. Box at the bottom shows the amino acid sequence of Mz upstream from Met-1. C. AQP4 immunoblot of U87MG cell lysates following transfection with plasmids encoding rat, human and mouse AQP4 containing the Mz-initiation ATG.
Figure 2
Figure 2
Expression of AQP4 isoforms and OAP composition in the brain cortex. A. SDS-PAGE and AQP4 immunoblot of rat, human and mouse brain cortex homogenates. B. Two-dimensional BN/SDS-PAGE and AQP4 immunoblot of rat (top), human (middle) and mouse (bottom) brain cortex homogenates. Molecular sizes are shown on the left, with decreasing apparent OAP size shown from left-to-right.
Figure 3
Figure 3
OAP assembly and association by rat Mz AQP4. A. AQP4 schematic showing the positions of methionines corresponding to Mz, M1 and M23 in the cytoplasmic N-terminus, and the inserted Myc sequence (black) in the second extracellular loop. B. Representative single particle trajectories of AQP4 isoforms in U87MG cells transfected with Mz (left), M1 (middle) or M23 (right) AQP4. C. Cumulative probability distribution of ranges at 1 second [P(range)] of AQP4 isoforms in U87MG cells transfected with Mz, M1 or M23 alone (top), or in cells co-transfected with Mz+M23 (1:3) or M1+M23 (1:3) (bottom). P(range) for M23 and M1 AQP4 alone are shown for reference in the bottom panel. D. AQP4 immunoblot after BN-PAGE of lysates from U87MG cells transfected with Mz, M1 or M23 alone, or co-transfected with Mz+M23 (1:3) or M1+M23 (1:3) (left). BN-PAGE of lysates from cells co-transfected with Mz-myc+M23 (1:3) was probed with Myc and AQP4 antibodies (right).
Figure 4
Figure 4
Binding of NMO-IgG to Mz AQP4. A. Immunofluorescence of U87MG cells transfected with Mz-myc, M1-myc and M23-myc and stained with anti-Myc antibody (top). Scale bar: 20 µm. TIRF micrographs of Alexa488-labeled Mz, M1 or M23 AQP4 (bottom). Scale bar: 10 µm. B. AQP4 immunoblot after SDS-PAGE of lysates from U87MG cells transfected with Mz alone, Mz+M1 (1:1) or M1 alone. C. Representative immuofluorescence of U87MG cells expressing Mz (top), M1 (middle) or equimolar Mz+M1 (bottom) and stained with 10% NMO patient serum (red), and with reference AQP4 antibody (green). Scale bar: 20 µm. D. (left) Measured red-to-green fluorescence ratios (R/G) following immunostaining as shown in panel C for different NMO serum concentrations (mean ± S.E., n=4). U87MG cells were transfected with rat Mz (blue), M1 (red) or equimolar Mz+M1 (black). Curves represent fits to a single-site binding model. D. (right) R/G for three other NMO sera, each at 10% concentration (mean ± S.E., n=4).
Figure 5
Figure 5
Sequence dependence of poor NMO-IgG binding to Mz AQP4. A. N-terminal amino acid sequence of rat AQP4, with black arrows indicating sites of N-terminal truncations. B. Representative immuofluorescence from U87MG cells expressing AQP4 mutants Mz Δ12, Mz Δ22 and Mz Δ32. Cells were stained with 10% NMO patient serum (red) and reference AQP4 antibody (green). Scale bar: 20 µm. C. AQP4 immunoblot of lysates from U87MG cells transfected with Mz AQP4, and N-terminal deletion mutants Mz Δ12, Mz Δ22 and Mz Δ32. D. AQP4 immunoblot after BN-PAGE of lysates from U87MG cells transfected with full-length M23, M1 and Mz AQP4, and N-terminal Mz deletion mutants. E. Antibody-shift assay showing AQP4 immunoblot of U87MG cells transfected with Mz or M1 and incubated with purified NMO-IgG before BN-PAGE.
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