Single Particle Electron Microscopy Analysis of the Bovine Anion Exchanger 1 Reveals a Flexible Linker Connecting the Cytoplasmic and Membrane Domains

<div><p>Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO₂ transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.</p> </div

Fitting of the atomic structure of cytoplasmic domain

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055408#pone.0055408-Zhang1" target="_blank">[<b>14</b>] </a><b>and the 7.5 Å resolution 2D crystal structure of membrane domain </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055408#pone.0055408-Yamaguchi1" target="_blank">[<b>23</b>] </a><b>into our 3D map of full length AE1 dimer.</b> (<b>a</b>) Shaded surface views of the atomic structure of cytoplasmic domain (PDB ID: 1HYN) filtered to 2.4 nm resolution (green) compared to the corresponding views of cytoplasmic domain resolved in the EM single-particle reconstruction (gold) of full-length AE1 dimer. In the EM map, the membrane domain of AE1 dimer is removed for clarity. The two structures are similar in size and in having a double-humped shape on their cytoplasmic side. (<b>b</b>) Shaded surface views of AE1 membrane domain resolved from 2D crystals embedded in trehalose (EMDB ID: 1645) filtered to 2.4 nm resolution (blue), as compared to the corresponding views of membrane domain resolved in the EM single-particle reconstruction (gold). The extracellular and intracellular sides identified in the published 2D crystal structure were used to define the orientation for comparison. (<b>c</b>) Superposition of the two structures of membrane domains described in (<b>b</b>) viewed from the cytoplasmic side (top view). The EM single-particle reconstruction is rendered at higher density threshold to show the deep canyon, which is consistent with the membrane domain structure from 2D crystals. (<b>d</b>) Fitting the EM single-particle reconstruction of full-length AE1 dimer with the crystal structure of cytoplasmic domain (red and cyan) and 2D crystal structure of membrane domain (blue). The single-particle reconstruction is rendered in two density threshold values: at low threshold (gray mesh) and a high threshold (yellow). The approximate positions of N-terminus and C-terminus of the cytoplasmic domain are labeled with diamond and triangle, respectively.</p

Single-particle reconstruction of bovine AE1.

<p>(<b>a</b>) Schematic illustration of the OTR data collection method. For each target sample area, two micrographs were recorded with the grid tilted at −45° and +45°, respectively. (<b>b</b>) 3D map generated by averaging 25 OTR maps. Two orthogonal views, defined as front view (left panel) and side view (right panel), are shown. (<b>c</b>) Final map obtained by merging 174,197 particle images with single particle reconstruction method. The map in (<b>c</b>) is shown in the same orientations as in (<b>b</b>). (<b>d</b>) Fourier shell correlation (FSC) coefficient between two reconstructions obtained from even- and odd-numbered particle images. The effective resolution is estimated to be 2.4 nm using the 0.5 FSC cut-off. (<b>e</b>) Comparisons of the computed projection, class average, and raw particle. Four representative views (top, tilt, front, and side) are show from left to right, respectively. (<b>f</b>) Euler angle distribution of classified particles. The brightness of each point indicates the number of particles used in the class average in that orientation.</p

Purification and EM of bovine AE1.

<p>(<b>a</b>) SDS-PAGE of bovine AE1 after ion-exchange chromatography. Lane 1: Coomassie stained gel; Lanes 2,3: immunoblotting, 2: immune serum; 3: preimmune serum. (<b>b,c</b>) Size-exclusion chromatography examination of bovine AE1 sample after the ion-exchange chromatography step (top) and the dimer fraction after the size-exclusion chromatography step (<b>c</b>) showing a peak corresponding to dimeric AE1. (<b>d</b>) A representative area of transmission EM micrograph of bovine dimeric AE1 stained with 1% uranyl formate. (<b>e</b>) Representative class averages (CA) and the corresponding raw particle (RP) images of AE1. Each class average is obtained by averaging about 100 particle images. The side length of each box is 26 nm.</p

AnkT9W increases HbA synthesis to therapeutic levels in most of the thalassemic ErPCs.

<p>(A) Net increase of Hb A % (ΔHb A, calculated by subtracting Hb A after treatment from that following treatment) is plotted against VCN (on the X axis) in the three patients groups, β0/0, β0/+ and β+/+. The total Hb A% is also represented as percentage of Hb A before (light grey) and after transduction (dark grey) with AnkT9W in the same groups (B). Figures C and D show the concomitant percentages of Hb F and α-aggregates, both of which are reduced in thalassemic ErPCs after treatment with AnkT9W. The tie bars under the X axes group different specimens from the same individual. Several specimens were harvested and transduced at different times so as to expose the variability in such experiments. “*” Sign indicates specimens treated with AnkT9Wsil2.</p

Mutations in patients.

<p>List of β-thalassemia specimens analyzed in this study.</p

Sequences of exon 1 and 2 in AnkT9W and AnkT9Wsil1/2 silent mutants.

<p>Original and corresponding mutated bases of exons 1 and 2 inside constructs are in bold letters.</p

AnkT9W improves Hb synthesis and hematological parameters of thalassemic mice.

<p>Red cell hemolysates were analyzed monthly by HPLC for up to 6 months post BM transplant. Chimeric (A) and total absolute Hb content (B) in T9W-<i>Hbb^th3/+</i> and AnkT9W-<i>Hbb^th3/+</i> chimeras (n = 4 and 6, respectively). Hβ:mα Hb detected by HPLC and relative VCN (C and D) in mice chimeras obtained by transplanting <i>Hbb^th3+</i> BM treated with T9W or AnkT9W vectors into lethally irradiated <i>Hbb^th3+</i> recipient mice. (E, F) Hematologic parameters in WT, <i>Hbb^th3/+</i>, T9W-<i>Hbb^th3/+</i> or AnkT9W-<i>Hbb^th3/+</i> chimeras. On average, the hematocrits in AnkT9W-treated chimeras were 26% and 10% higher, the reticulocyte counts 71% and 49% lower, and the RDWs 35% and 22% lower than in <i>Hbb^th3/+</i> and T9W-<i>Hbb^th3/+</i> mice, respectively. For reticulocyte counts, Hb, RDW, p<0.0001,and for HCT, p = 0.0002.</p

The ankyrin insulator improves translation of the β-globin gene, and its sequence is conserved at the site of integration.

<p>(A) Expression of the chimeric Hb (expressed as a percentage) after HMBA differentiation, in MEL cell clones carrying one VCN of T9W or AnkT9W, and T9Ank2W. One way anova test, p = 0.048. (B) The β-globin mRNA (bottom panel), expressed by T9W (VCN = 0.87,1.4), AnkT9W (VCN = 0.39, 1.07, 1.47) and T9Ank2W (VCN = 1.26, 1.98) was not detected in non-erythroid cells (B-16 melanoma) but only in differentiated MEL cells (AnkT9W, VCN = 2), indicating that the transgene's transcription is tissue specific. (C) Two amplicons were amplified by PCR corresponding to the ankyrin sequence present, respectively, in the 5′ and 3′ viral LTRs. Oligonucleotides were designed to include the ankyrin element and selectively amplify fragments either from the 5′ or 3′ LTR. Only clones bearing a single AnkT9W integrant were used. The amplified fragments exhibited the expected size of 248 bp and 231 bp, indicating that the ankyrin element was faithfully replicated in both LTRs. (D) Southern blot assay of genomic DNA from single integrant clones of MEL cells carrying either T9W or AnkT9W or from MEL control. The genomic DNAs were digested with <i>Xmn</i>I restriction enzyme, which yielded the full β-globin cassette, identified by hybridization using a β-globin <i>BamH</i>I-<i>Nco</i>I probe. Clone #6 of the AnkT9W series was reloaded in higher quantity (left panel) to amplify the signal of the transgenic human β-globin gene, because it was insufficiently loaded and exhibited a weak signal on the first blot (right panel).</p

HPLC profiles of thalassemic specimens analyzed after differentiation from CD34-derived cultures.

<p>The HPLC profiles of the same thalassemic β0/0 (A) and β+/+ (B) specimens were analyzed at steady state (left) and following treatment with AnkT9W (right) after differentiation starting from either CD34⁺ cells (top) or ErPCs (bottom). In the same β0/0 specimen, AnkT9W contributes to increasing Hb A synthesis from from 0% to 62% (VCN = 0.92), in the CD34⁺-derived cells or to 73% (VCN = 0.97), in the ErPCs-derived cells. In the cells from the β+/+ patient, the net Hb increase was 35% (VCN = 0.88), if CD34⁺-derived or 23% (VCN = 0.94), if ErPCs-derived.</p