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Reversible binding of hemoglobin to band 3 constitutes the molecular switch that mediates O2 regulation of erythrocyte properties

Haiyan Chu, Mary M. McKenna, Nathan A. Krump, Suilan Zheng, Laurel Mendelsohn, Swee Lay Thein, Lisa J. Garrett, David M. Bodine and Philip S. Low

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Design of transgenic mice with altered deoxyHb-binding sites on erythrocyte band 3. (A) The homologous recombination strategy to targeting the murine band 3 (Slc4A1) allele. The blue boxes represent the exons of human SLC4A1 knocked into the Slc4A1 locus. The deletion of the sequence encoding amino acids 12-23 is indicated by a smaller box. The red triangles represent loxP sites which flank a PGK-NEO gene to facilitate subsequent removal. (B) Schematic representation of erythrocyte band 3 proteins from normal human, wild-type mouse, and the 2 transgenic mice including 1 with humanized band 3 (w/ human Hb site) and the other lacking the deoxyHb-binding site on band 3 (w/o Hb site). w/, with; w/o, without.

  • Figure 2.

    Characterization of transgenic mouse erythrocytes with altered deoxyHb-binding sites on band 3. (A) Confocal immunofluorescence (top) and corresponding brightfield (bottom) images of murine RBCs stained with a monoclonal antibody to the NH2 terminus (residues 1-10) of human band 3. Note that wild-type mouse RBCs do not stain whereas both transgenic erythrocytes do. (B) Binding of deoxygenated murine Hb to KI-IOVs of normal and transgenic mouse erythrocytes. The y-axis shows the molecular ratio of deoxyHb to band 3. (C) Comparison of the osmotic fragility of wild-type and transgenic mouse erythrocytes. (D) Comparison of the deformability of wild-type and transgenic mouse erythrocytes by ektacytometry. The elongation index upon subjection of the erythrocytes to increasing shear stress is shown. The mean of maximal elongation index of 3 experiments is also shown.

  • Figure 3.

    Effect of deoxygenation on the localization of GEs on the membranes of wild-type and transgenic murine erythrocytes. Erythrocytes from (A) wild-type, (B) transgenic mice containing a human deoxyHb-binding site (w/ human Hb site), or (C) transgenic mice lacking a deoxyHb-binding site (w/o Hb site) were either oxygenated or deoxygenated prior to fixation and staining with antibodies against the indicated GEs.

  • Figure 4.

    Effect of deoxygenation on band 3 retention in membrane cytoskeletons isolated from wild-type and transgenic mouse erythrocytes. (A) Erythrocytes from wild-type (left panel), transgenic mice containing a human deoxyHb-binding site (middle panel), or transgenic mice lacking a deoxyHb-binding site (right panel) were either oxygenated or deoxygenated prior to solubilization in 1% Triton X-100. Detergent-insoluble membrane fractions containing the spectrin/actin membrane skeletons were separated by SDS-PAGE and immunoblotted with anti-cdb3 or antiactin antibodies. (B) Densitometric analysis of the relative amount of band 3 retained in the above cytoskeletal pellets using actin as a loading control. All values of band 3 retention in cytoskeletons derived from deoxygenated RBCs are relative to the amount of band 3 retained in the pellet of the corresponding oxygenated erythrocytes (mean of 3 experiments ± standard deviation [SD]).

  • Figure 5.

    Effect of deoxygenation on the accessibility of ankyrin epitopes in transgenic murine erythrocytes evaluated by both immunofluorescence staining and flow cytometry. (A) RBCs were oxygenated or deoxygenated, then fixed and stained with a monoclonal antibody to ankyrin. The stained RBCs were observed using a confocal fluorescence microscope (top) and further analyzed for antiankyrin fluorescence staining intensity by flow cytometry (bottom). (B) Mean fluorescence intensity (MFI) of antiankyrin staining in oxygenated and deoxygenated erythrocytes. The relative MFI of oxygenated erythrocytes was set at 1 and compared with the MFI of the deoxygenated RBCs (mean of 3 experiments ± SD).

  • Figure 6.

    Effect of deoxygenation on ATP release from wild-type and transgenic mouse erythrocytes. Washed RBCs were oxygenated or deoxygenated and kept for 30 minutes at room temperature, and gently pelleted. The supernatant was collected and evaluated for ATP and Hb content. The pelleted RBCs were then lysed with ddH2O and the Hb concentration was measured to calculate erythrocyte numbers. ATP arising from hemolysis was subtracted from the total ATP in supernatant to yield net ATP released by deoxygenation. Absolute numbers of ATP released from intact erythrocytes containing the murine deoxyHb-binding site (wild-type), the human deoxyHb-binding site (w/ human Hb site), and no deoxyHb site (w/o Hb site) in oxygenated vs deoxygenated condition are 44 ± 4.0 pmol/108 cells vs 67 ± 5.1 pmol/108 cells, 39 ± 3.1 pmol/108 cells vs 54 ± 1.4 pmol/108 cells, and 53 ± 1.5 pmol/108 cells vs 60 ± 3.0 pmol/108 cells.

Tables

  • Table 1.

    Transgenic murine hematologic indices

    Mean ± SD
    Wild-typew/ human Hb sitew/o Hb site
    n81215
    (4 female; 4 male)(6 female; 6 male)(7 female; 8 male)
     RBC, ×1012/L10.67 ± 0.6410.58 ± 0.258.91 ± 1.36*
     Hb, g/L160.3 ± 2.8159.8 ± 5.1153.7 ± 6.1*
     Hematocrit, %47.34 ± 2.0444.63 ± 1.40*44.06 ± 2.46*
     MCH15.33 ± 0.3915.36 ± 0.4216.77 ± 0.80*
     MCV, fL42.18 ± 1.6942.12 ± 0.4847.68 ± 2.27*
     Reticulocytes, %4.35 ± 0.294.62 ± 0.535.14 ± 0.78
     WBC, ×109/L7.00 ± 3.367.68 ± 0.93*11.86 ± 3.46*
     Platelet, ×109/L899.8 ± 110850.0 ± 104805.4 ± 111
    n545
     Total cells in marrow, ×1077.30 ± 0.628.38 ± 1.847.78 ± 1.65
     Ter119+ cells in marrow, ×1072.87 ± 0.393.43 ± 1.153.63 ± 1.07
     Ter119+ cells in marrow (%)39.3 ± 4.240.8 ± 10.546.6 ± 8.1*
     Total cells in spleen, ×10730.14 ± 5.8134.35 ± 7.3760.61 ± 9.38*
     Ter119+ cells in spleen, ×10714.88 ± 2.8319.98 ± 5.5550.49 ± 12.95*
     Ter119+ cells in spleen, %49.9 ± 3.157.8 ± 6.4*82.4 ± 12.4*
     Spleen weight, g0.11 ± 0.010.13 ± 0.040.24 ± 0.04*
    • SD, standard deviation; w/, with; w/o, without.

    • * P < .05 compared with the wild-type (C57BL/6) strain.

    • P < .05 compared with the transgenic strain with the human Hb site.