Formation of aquaporin-4 arrays is inhibited by palmitoylation of N-terminal cysteine residues

Tetramers of the mammalian water channel aquaporin-4 (AQP4) assemble into square arrays and mediate bidirectional water transport across the blood-brain interface. The aqp4 gene expresses two splicing isoforms. Only the shorter AQP4M23 isoform assembles into square arrays, while the longer AQP4M1 isoform interferes with array formation, presumably due to the additional 22 N-terminal residues. To understand why the N-terminus of AQP4M1 interferes with array formation, we constructed a series of N-terminal deletion mutants and examined their ability to form square arrays in Chinese hamster ovary (CHO) cells using SDS-digested freeze fracture replica labeling. Mutants with deletions of less than seventeen N-terminal residues did not form square arrays and showed dispersed immunogold labels against AQP4 molecules, whereas more deletions led to the formation of square arrays labeled with immunogolds. Furthermore, mutagenic substitution of the two cysteine residues at the position 13 and 17 in the N-terminus of AQP4M1 also resulted in the square array formation. Biochemical analysis and metabolic labeling of transfected CHO cells revealed that the two N-terminal cysteines of AQP4M1 are palmitoylated. These results suggest that palmitoylation of the N-terminal cysteines is the reason for the inability of AQP4M1 to form square arrays.     

Reversible, Temperature-Dependent Supramolecular Assembly of Aquaporin-4 Orthogonal Arrays in Live Cell Membranes

The shorter “M23” isoform of the glial cell water channel aquaporin-4 (AQP4) assembles into orthogonal arrays of particles (OAPs) in cell plasma membranes, whereas the full-length “M1” isoform does not. N-terminal residues are responsible for OAP formation by AQP4-M23 and for blocking of OAP formation in AQP4-M1. In investigating differences in OAP formation by certain N-terminus mutants of AQP4, as measured by freeze-fracture electron microscopy versus live-cell imaging, we discovered reversible, temperature-dependent OAP assembly of certain weakly associating AQP4 mutants. Single-particle tracking of quantum-dot-labeled AQP4 in live cells and total internal reflection fluorescence microscopy showed >80% of M23 in OAPs at 10-50°C compared to <10% of M1. However, OAP formation by N-terminus cysteine-substitution mutants of M1, which probe palmitoylation-regulated OAP assembly, was strongly temperature-dependent, increasing from <10% at 37°C to >70% at 10°C for the double mutant M1-C13A/C17A. OAP assembly by this mutant, but not by native M23, could also be modulated by reducing its membrane density. Exposure of native M1 and single cysteine mutants to 2-bromopalmitate confirmed the presence of regulated OAP assembly by S-palmitoylation. Kinetic studies showed rapid and reversible OAP formation during cooling and OAP disassembly during heating. Our results provide what to our knowledge is the first information on the energetics of AQP4 OAP assembly in plasma membranes.     

The molecular composition of square arrays

Square arrays are prominent structures in plasma membranes of brain, muscle, and kidneys with an unknown function. So far, the analysis of these arrays has been restricted to freeze fracture preparations, which have shown square arrays to contain the water channel Aquaporin-4 (AQP4). Using Blue-Native PAGE immunoblots, we provide evidence that higher-order AQP4 complexes correspond to square arrays, with the AQP4 isoform M23 playing a dominant role. Our data are consistent with the idea that square arrays consist of aggregates of AQP4 tetramers complexed with multiples of dimers. By comparison, Aquaporin-1 and Aquaporin-9 form tetramers, but not higher-order complexes. AQP4 square arrays are stable under several biochemical purification steps. Analyzing the internal composition of the higher-order complexes by 2D gels, we demonstrate that the square arrays in addition to M23 also invariably contain AQP4, M1, and a novel AQP4 isoform that we call Mz. The visualization AQP4 square arrays by a rapid, biochemical assay provides new insight in the molecular organization of square arrays and gives further proof of the heterogeneity of AQP4 square arrays in vivo.     

Heterotetrameric composition of aquaporin-4 water channels.

Aquaporin (AQP) water channel proteins are tetrameric assemblies of individually active approximately 30 kDa subunits. AQP4 is the predominant water channel protein in brain, but immunoblotting of native tissues has previously yielded multiple poorly resolved bands. AQP4 is known to encode two distinct mRNAs with different translation initiating methionines, M1 or M23. Using SDS-PAGE urea gels and immunoblotting with anti-peptide antibodies, four polypeptides were identified in brain and multiple other rat tissues with the following levels of expression: 32 kDa > 34 kDa > 36 kDa > 38 kDa. The 34 and 38 kDa polypeptides react with an antibody specific for the N-terminus of the M1 isoform, and 32 and 36 kDa correspond to the shorter M23 isoform. Immunogold electron microscopic studies with rat cerebellum cryosections demonstrated that the 34 kDa polypeptide colocalizes in perivascular astrocyte endfeet where the 32 kDa polypeptide is abundantly expressed. Velocity sedimentation, cross-linking, and immunoprecipitation analyses of detergent-solubilized rat brain revealed that the 32 and 34 kDa polypeptides reside within heterotetramers. Immunoprecipitation of AQP4 expressed in Xenopus laevis oocytes demonstrated that heterotetramer formation reflects the relative expression levels of the 32 and 34 kDa polypeptides; however, tetramers containing different compositions of the two polypeptides exhibit similar water permeabilities. These studies demonstrate that AQP4 heterotetramers are formed from two overlapping polypeptides and indicate that the 22-amino acid sequence at the N-terminus of the 34 kDa polypeptide does not influence water permeability but may contribute to membrane trafficking or assembly of arrays.     

Model of aquaporin-4 supramolecular assembly in orthogonal arrays based on heterotetrameric association of M1-M23 isoforms

Tetramers of aquaporin-4 (AQP4) water channels form supramolecular assemblies in cell membranes called orthogonal arrays of particles (OAPs). We previously reported evidence that a short (M23) AQP4 isoform produced by alternative splicing forms OAPs by an intermolecular N-terminus interaction, whereas the full-length (M1) AQP4 isoform does not by itself form OAPs but can coassemble with M23 in OAPs as heterotetramers. Here, we developed a model to predict number distributions of OAP size, shape, and composition as a function M23:M1 molar ratio. Model specifications included: random tetrameric assembly of M1 with M23; intertetramer associations between M23 and M23, but not between M1 and M23 or M1; and a free energy constraint limiting OAP size. Model predictions were tested by total internal reflection fluorescence microscopy of AQP4-green-fluorescent protein chimeras and native gel electrophoresis of cells expressing different M23:M1 ratios. Experimentally validated model predictions included: 1), greatly increased OAP size with increasing M23:M1 ratio; 2), marked heterogeneity in OAP size at fixed M23:M1, with increased M23 fraction in larger OAPs; and 3), preferential M1 localization at the periphery of OAPs. The model was also applied to test predictions about binding to AQP4 OAPs of a pathogenic AQP4 autoantibody found in the neuroinflammatory demyelinating disease neuromyelitis optica. Our model of AQP4 OAPs links a molecular-level interaction of AQP4 with its supramolecular assembly in cell membranes.     

Evidences for a Leaky Scanning Mechanism for the Synthesis of the Shorter M23 Protein Isoform of Aquaporin-4: IMPLICATION IN ORTHOGONAL ARRAY FORMATION AND NEUROMYELITIS OPTICA ANTIBODY INTERACTION*

Aquaporin-4 (AQP4) exists as two major isoforms that differ in the length of the N terminus, the shorter AQP4-M23 and the longer AQP4-M1. Both isoforms form tetramers, which can further aggregate in the plasma membrane to form typical orthogonal arrays of particles (OAPs) whose dimension depends on the ratio of the M1 and M23. In this study, we tested the hypothesis that the M23 isoform can be produced directly by the M1 mRNA. In cells transiently transfected with AQP4-M1 coding sequence we observed besides AQP4-M1 the additional presence of the AQP4-M23 isoform associated with the formation of typical OAPs observable by two-dimensional blue native/SDS-PAGE and total internal reflection microscopy. The mutation of the second in-frame methionine M23 in AQP4-M1 (AQP4-M1^M23I) prevented the expression of the M23 isoform and the formation of OAPs. We propose “leaky scanning” as a translational mechanism for the expression of AQP4-M23 protein isoform and that the formation of OAPs may occur even in the absence of AQP4-M23 mRNA. This mechanism can have important pathophysiological implications for the cell regulation of the M1/M23 ratio and thus OAP size. In this study we also provide evidence that AQP4-M1 is mobile in the plasma membrane, that it is inserted and not excluded into immobile OAPs, and that it is an important determinant of OAP structure and size.     

Identification of in vivo disulfide conformation of TRPA1 ion channel

TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 μM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.     

The primary structure of a minor isoform (H1.2) of histone H1 from the nematode Caenorhabditis elegans.

The complete amino acid sequence of a minor isoform (H1.2) of histone H1 from the nematode Caenorhabditis elegans was determined. The amino acid chain consists of 190 residues and has a blocked N-terminus. Histone subtype H1.2 is 17 residues shorter than the major isoform H1.1, mainly as the result of deletions of short peptide fragments. Considerable divergence from isoform H1.1 has occurred in the N-terminal domain and the very C-terminus of the molecule, but the central globular domain and most of the C-terminal domain, including two potential phosphorylation sites, have been well conserved. Secondary-structure predictions for both H1 isoforms reveal a high potential for helix formation in the N-terminal region 1-33 of isoform H1.1 whereas the corresponding region in isoform H1.2 has low probability of being found in alpha-helix. No major differences in secondary structure are predicted for other parts of both H1 subtypes. The aberrant conformation of isoform H1.2 may be indicative of a significantly different function.     

Aquaporin-4 dynamics in orthogonal arrays in live cells visualized by quantum dot single particle tracking

Freeze-fracture electron microscopy (FFEM) indicates that aquaporin-4 (AQP4) water channels can assemble in cell plasma membranes in orthogonal arrays of particles (OAPs). We investigated the determinants and dynamics of AQP4 assembly in OAPs by tracking single AQP4 molecules labeled with quantum dots at an engineered external epitope. In several transfected cell types, including primary astrocyte cultures, the long N-terminal "M1" form of AQP4 diffused freely, with diffusion coefficient approximately 5 x 10(-10) cm(2)/s, covering approximately 5 microm in 5 min. The short N-terminal "M23" form of AQP4, which by FFEM was found to form OAPs, was relatively immobile, moving only approximately 0.4 microm in 5 min. Actin modulation by latrunculin or jasplakinolide did not affect AQP4-M23 diffusion, but deletion of its C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding domain increased its range by approximately twofold over minutes. Biophysical analysis of short-range AQP4-M23 diffusion within OAPs indicated a spring-like potential, with a restoring force of approximately 6.5 pN/microm. These and additional experiments indicated that 1) AQP4-M1 and AQP4-M23 isoforms do not coassociate in OAPs; 2) OAPs can be imaged directly by total internal reflection fluorescence microscopy; and 3) OAPs are relatively fixed, noninterconvertible assemblies that do not require cytoskeletal or PDZ-mediated interactions for formation. Our measurements are the first to visualize OAPs in live cells.     

The binding property of a monoclonal antibody against the extracellular domains of aquaporin-4 directs aquaporin-4 toward endocytosis

Neuromyelitis optica (NMO), an autoimmune disease of the central nervous system, is characterized by an autoantibody called NMO-IgG that recognizes the extracellular domains (ECDs) of aquaporin-4 (AQP4). In this study, monoclonal antibodies (mAbs) against the ECDs of mouse AQP4 were established by a baculovirus display method. Two types of mAb were obtained: one (E5415A) recognized both M1 and M23 isoforms, and the other (E5415B) almost exclusively recognized the square-array-formable M23 isoform. While E5415A enhanced endocytosis of both M1 and M23, followed by degradation in cells expressing AQP4, including astrocytes, E5415B did so to a much lesser degree, as determined by live imaging using fluorescence-labeled antibodies and by Western blotting of lysate of cells treated with these mAbs. E5415A promoted cluster formation of AQP4 on the cell surface prior to endocytosis as determined by immunofluorescent microscopic observation of bound mAbs to astrocytes as well as by Blue native PAGE analysis of AQP4 in the cells treated with the mAbs. These observations clearly indicate that an anti-AQP4-ECDs antibody possessing an ability to form a large cluster of AQP4 by cross-linking two or more tetramers outside the AQP4 arrays enhances endocytosis and the subsequent lysosomal degradation of AQP4.     

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

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.     

Influence of the Cytoplasmic Domains of Aquaporin-4 on Water Conduction and Array Formation

Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the S180D mutant (AQP4M23S180D), which was generated to mimic phosphorylated Ser180, was determined to 2.8 Å resolution using electron diffraction patterns, it showed no significant differences from the structure of the wild-type channel. High-resolution density maps usually do not resolve protein regions that are only partially ordered, but these can sometimes be seen in lower-resolution density maps calculated from electron micrographs. We therefore used images of two-dimensional crystals and determined the structure of AQP4M23S180D at 10 Å resolution. The features of the 10-Å density map are consistent with those of the previously determined atomic model; in particular, there were no indications of any obstruction near the cytoplasmic pore entrance. In addition, water conductance measurements, both in vitro and in vivo, show the same water permeability for wild-type and mutant AQP4M23, suggesting that the S180D mutation neither reduces water conduction through a conformational change nor reduces water conduction by interacting with a protein that would obstruct the cytoplasmic channel entrance. Finally, the 10-Å map shows a cytoplasmic density in between four adjacent tetramers that most likely represents the association of four N termini. This finding supports the critical role of the N terminus of AQP4 in the stabilization of orthogonal arrays, as well as their interference through lipid modification of cysteine residues in the longer N-terminal isoform.     

Isolation and amino-acid sequence analysis of human sperm protamines P1 and P2. Occurrence of two forms of protamine P2

The two protamines of human sperm cell nuclei, P1 and P2, were isolated in pure form after extraction with 6M guanidine/5% mercaptoethanol and alkylation with vinyl pyridine by reversed-phase high-performance liquid chromatography. The amino-acid sequence of protamine P1 was determined by analysing the intact protein and the fragments obtained by cyanogen bromide cleavage. Out of the 50 amino-acid residues 24 are arginines and 6 are cysteines. The sequence of protamine P2 was determined by analysing the intact protein and the fragments resulting from cleavage with endoproteinase Lys-C and thermolysin. Protamine P2 was found to occur in two forms which only differ in their N-terminal regions. The form P2' is three amino-acid residues longer at the N-terminus than the form P2'. Out of the 57 amino-acid residues in the longer form 27 are arginines and 5 are cysteines. Human protamine P1 is highly homologous with the protamines isolated from bull, boar, ram and mouse sperm cells, but human protamine P2 shows a novel type of structure, although also here the dominant amino acids are arginine and cysteine.     

Live Cell Analysis of Aquaporin-4 M1/M23 Interactions and Regulated Orthogonal Array Assembly in Glial Cells

Aquaporin-4 (AQP4) can assemble into supramolecular aggregates called orthogonal arrays of particles (OAPs). In cells expressing single AQP4 isoforms, we found previously that OAP formation by AQP4-M23 requires N terminus interactions just downstream of Met-23 and that the inability of AQP4-M1 to form OAPs involves blocking by residues upstream of Met-23. Here, we studied M1/M23 interactions and regulated OAP assembly by nanometer-resolution tracking of quantum dot-labeled AQP4 in live cells expressing differentially tagged AQP4 isoforms and in primary glial cell cultures in which native AQP4 was labeled with a monoclonal recombinant neuromyelitis optica autoantibody. OAP assembly was assessed independently by Blue Native gel electrophoresis. We found that OAPs in native glial cells could be reproduced in transfected cells expressing equal amounts of AQP4-M1 and -M23. Mutants of M23 that do not themselves form OAPs, including M23-F26Q and M23-G28P, were able to fully co-associate with native M23 to form large immobile OAPs. Analysis of a palmitoylation-null M1 mutant (C13A/C17A) indicated palmitoylation-dependent OAP assembly only in the presence of M23, with increased M1 palmitoylation causing progressive OAP disruption. Differential regulation of OAP assembly by palmitoylation, calcium elevation, and protein kinase C activation was found in primary glial cell cultures. We conclude that M1 and M23 co-assemble in AQP4 OAPs and that specific signaling events can regulate OAP assembly in glial cells.     

Post-Golgi supramolecular assembly of aquaporin-4 in orthogonal arrays

The supramolecular assembly of aquaporin-4 (AQP4) in orthogonal arrays of particles (OAPs) involves N-terminus interactions of the M23-AQP4 isoform. We found AQP4 OAPs in cell plasma membranes but not in endoplasmic reticulum (ER) or Golgi, as shown by: (i) native gel electrophoresis of brain and AQP4-transfected cells, (ii) photobleaching recovery of green fluorescent protein-AQP4 chimeras in live cells and (iii) freeze-fracture electron microscopy (FFEM). We found that AQP4 OAP formation in plasma membranes, but not in the Golgi, was not related to AQP4 density, pH, membrane lipid composition, C-terminal PDZ domain interactions or α-syntrophin expression. Remarkably, however, fusion of AQP4-containing Golgi vesicles with (AQP4-free) plasma membrane vesicles produced OAPs, suggesting the involvement of plasma membrane factor(s) in AQP4 OAP formation. In investigating additional possible determinants of OAP assembly we discovered membrane curvature-dependent OAP assembly, in which OAPs were disrupted by extrusion of plasma membrane vesicles to ～110 nm diameter, but not to ～220 nm diameter. We conclude that AQP4 supramolecular assembly in OAPs is a post-Golgi phenomenon involving plasma membrane-specific factor(s). Post-Golgi and membrane curvature-dependent OAP assembly may be important for vesicle transport of AQP4 in the secretory pathway and AQP4-facilitated astrocyte migration, and suggests a novel therapeutic approach for neuromyelitis optica.     

Aggregation state determines the localization and function of M1– and M23–aquaporin-4 in astrocytes

The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23–AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1–AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23–AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1– and M23–AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1–AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23–AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state–dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1– and M23–AQP4.     

Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells

ExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N-terminus (residues 1-234) is a GTPase activating protein (GAP) for RhoGTPases, while the C-terminus (residues 232-453) encodes an ADP-ribosyltransferase. Utilizing a series of N-terminal deletion peptides of ExoS and an epitope-tagged full-length ExoS, two independent domains have been identified within the N-terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N-terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90-234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization. ExoS was present in CHO cells as a full-length form, which fractionated with membranes, and as an N-terminally processed fragment, which localized to the cytosol. Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP-ribosyltransferase activities.     

Cu1+, but not Cu2+ is capable of inhibition of AQP4 permeability in an in vitro CHO cell based model

Aquaporin 4 (AQP4) is an important water channel in the central nervous system which is implicated in several neurological disorders. Due to its significance, the identification of molecules which are able to modulate its activity is quite important for potential therapeutic applications. Here we used a novel screening method involving CHO cell lines which stably express AQP4 to test for potential molecules of interest. Using this method we identified a metal ion, Cu¹⁺, which is able to inhibit AQP4 activity in a cell model, an interaction which has not been previously described. This inhibition was effective at concentrations greater than 500 nM in the CHO cell model, and was confirmed in a proteoliposome based model. Furthermore, the binding sites for Cu¹⁺ inhibition of AQP4 are identified as cysteine 178 and cysteine 253 on the intracellular domain of the protein via the synthesis of AQP4 containing point mutations to remove these cysteines. These results suggest that Cu¹⁺ is able to access intracellular binding sites and inhibit AQP4 in a cell based model.