The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis

Abstract

Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation.

Comparison of the Proteome of Adult and Cord Erythroid Cells, and Changes in the Proteome Following Reticulocyte Maturation

“…Membranes were blocked with 10% milk powder for 1 h, followed by incubation with primary antibodies overnight at 4°C. Primary antibodies used were aquaporin I (CHIP28) and glycophorin A (CVDP) 1:1000 dilution, ankyrin 1 (BRIC274) 1:100 dilution, and band 3 (BRIC170) 1 (15,16).…”

Severe Ankyrin-R deficiency results in impaired surface retention and lysosomal degradation of RhAG in human erythroblasts

“…18 For assessment of ankyrin levels by flow cytometry, donor and patient erythrocytes were fixed with 1% paraformaldehyde + 0.0075% glutaraldehyde in PBS supplemented with 1 mg/mL BSA (PBSAG), 2 mg/mL glucose for 10 min at room temperature, permeabilized with 0.05% Triton X-100 for 2 min, centrifuged for 3 min at 500 G, washed once in PBSAG, re-suspended in PBSAG containing 4% BSA, and stained sequentially with BRIC272 (anti-ankyrin) and APC-conjugated polyclonal anti-mouse IgG (Biolegend), as described. 18 Pronase epitope recovery experiments were performed as previously described. 19 A list of antibodies used in this study is provided in the Online Supplementary Table S1.…”

“…20 By staining differentiating ankyrin deficient erythroblasts grown in our human in vitro culture system with Hoechst and using a recently published flow cytometry gating strategy, 18 protein partitioning profiles were determined for localization of specific membrane proteins within the plasma membrane of the reticulocyte and pyrenocyte (the extruded condensed nucleus encased by thin layer of cytoplasm and plasma membrane) derived from non-targeting control and ankyrin knockdown erythroblasts, respectively. The bar graphs in Figure 2A demonstrate that in the ankyrin deficient cultures there is an increase in the proportion of the ankyrin-R associated complex proteins band 3, GPA, RhAG, Rh and CD47 partitioning to the plasma membrane surrounding the extruded nucleus at this stage of differentiation.…”

“…After three days, 50 μL concentrated lentivirus was added per 1x10 6 cells in expansion medium -IMDMExp (Phase 2), as previously described, 18 supplemented with 8 μg/mL polybrene. After 20 h, the cells were washed 3 times in PBS and plated in fresh IMDMExp.…”

“…Erythroblasts were further expanded and differentiated (Phase 3), as previously described. 18 Lentiviral transduction of erythroblasts was performed as previously described using pLKO.1 vector with puromycin selection. Broad Institute Repository shRNA sequences TRCN0000083720 (shRNA 1) and TRCN0000083721 (shRNA 2) targeting ANK1 were used in preliminary experiments and TRCN0000083720 used unless otherwise stated.…”