Characterization of glycolytic enzyme interactions with murine erythrocyte membranes in wild-type and membrane protein knockout mice

Abstract

Previous research has shown that glycolytic enzymes (GEs) exist as multienzyme complexes on the inner surface of human erythrocyte membranes. Because GE binding sites have been mapped to sequences on the membrane protein, band 3, that are not conserved in other mammalian homologs, the question arose whether GEs can organize into complexes on other mammalian erythrocyte membranes. To address this, murine erythrocytes were stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase and analyzed by confocal microscopy. GEs were found to localize to the membrane in oxygenated erythrocytes but redistributed to the cytoplasm upon deoxygenation, as seen in human erythrocytes. To identify membrane proteins involved in GE assembly, erythrocytes from mice lacking each of the major erythrocyte membrane proteins were examined for GE localization. GEs from band 3 knockout mice were not membrane associated but distributed throughout the cytoplasm, regardless of erythrocyte oxygenation state. In contrast, erythrocytes from mice lacking alpha-spectrin, ankyrin, protein 4.2, protein 4.1, beta-adducin, or dematin headpiece exhibited GEs bound to the membrane. These data suggest that oxygenation-dependent assembly of GEs on the membrane could be a general phenomenon of mammalian erythrocytes and that stability of these interactions depends primarily on band 3.

Figure 1

Alignment of the human, dog, mouse, rat, and cow NH₂-terminus of band 3. Alignment of the first 65 amino acids of human, dog, mouse, rat, and cow AE1 using the CLUSTALW program (http://align.genome.jp//). Asterisks (*) indicate complete sequence conservation; double and single dots, less conservation; (^) symbols, the 2 tyrosine phosphorylation sites at the NH₂-terminus of human band 3.

Figure 2

GE staining in oxygenated and in deoxygenated mouse erythrocytes. Confocal images of mouse erythrocytes after their staining for glycolytic enzymes under oxygenated (Air), deoxygenated with humidified argon (Argon), or deoxygenated with 10 mM of sodium dithionite (Dithionite) followed by humidified argon (“Preparation of cells”).

Figure 3

Localization of GEs in oxygenated band 3 knockout erythrocytes. Confocal images of band 3 null erythrocytes stained for both RNA (to identify reticulocytes, green stain) and GEs (red stain), as indicated. A merger of both stains is shown in the third row (overlay), and bright field images are displayed in the bottom row. Note that even the very few mature erythrocytes present (negative for SytoRNA stain, green color) do not show enzyme localization at the membrane.

Figure 4

Localization of glycolytic enzymes in murine erythrocytes resealed in the presence or absence of an antibody to the cytoplasmic domain of band 3. Freshly drawn murine erythrocytes were washed 3 times to remove serum and buffy coat, and divided into 2 aliquots. One suspension of erythrocytes was lysed and resealed in the presence of rabbit polyclonal antibody raised against cdb3. The other suspension (control) were similarly lysed and resealed in the absence of any antibody. After resealing, both erythrocyte preparations were similarly fixed with acrolein, permeabilized with Triton X-100 (“Preparation of cells”), and stained with the desired antibodies. (i) Antirabbit antibody conjugated to Cy2 (492 510) was used to identify cells containing entrapped anti-cdb3 antibody. (ii) Cells (lacking entrapped antibody) were first stained with the same rabbit anti-cdb3 antibody and then labeled with Cy2-conjugated antirabbit IgG. Membrane staining in this panel demonstrates the specificity of the antibody for band 3. (iii,iv) Stained first with goat antibodies specific for either aldolase (A) or pyruvate kinase (B) and then with antigoat antibody conjugated to Cy5 (650 670). (v,vi) Overlays of subpanels i and iii, and ii and iv, respectively. (vii,viii) Bright field images of subpanels i, iii, v, and ii, iv, vi, respectively.

Figure 5

Localization of glycolytic enzymes in murine erythrocytes resealed in the presence of the cytoplasmic domain of murine band 3 (residues 1-398). Murine wild-type cdb3 was dialyzed against 5 mM of potassium phosphate containing 160 mM of sodium chloride, pH 7.4, and concentrated to 6.8 mg/mL using a centricon-30 (Millipore, Billerica, MA). Freshly drawn mouse erythrocytes were washed in the above buffer and resuspended at 50% hematocrit in the same buffer containing mouse cdb3. The suspension was introduced into mini dialysis units, and lysed and resealed as described by Campanella et al. After resealing, erythrocytes were fixed, permeabilized, and stained for GAPDH, aldolase, LDH, and PK and visualized with secondary antibody conjugated with Cy2 (492 510). Control erythrocytes were lysed and resealed in the absence of cdb3, but otherwise treated identically. Glycolytic enzymes are displaced from the membrane in cells containing resealed murine cdb3.

Figure 6

Distribution of aldolase in erythrocytes from mice deficient in major erythrocyte membrane proteins. Intact erythrocytes from wild-type mice and mice deficient in protein 4.2, β-adducin, protein 4.1, dematin headpiece domain, ankyrin (nb/nb), α-spectrin (sph/sph), and band 3 were fixed, permeabilized, and stained for aldolase and RNA, as described in “Methods.”

Figure 7

Distribution of GAPDH in erythrocytes from mice deficient in major erythrocyte membrane proteins. All conditions are identical to those in Figure 6, except that anti-GAPDH was used to stain the cells rather than antialdolase.