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

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 α-spectrin, ankyrin, protein 4.2, protein 4.1, β-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.

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Cytosolic protein binding to band-3 protein inhibits endocytosis of isolated human erythrocyte membranes

Biochemical Journal ◽

10.1042/bj2070595 ◽

1982 ◽

Vol 207 (3) ◽

pp. 595-598 ◽

Cited By ~ 1

Author(s):

K A Cordes ◽

J M Salhany

Keyword(s):

Membrane Protein ◽

Integral Membrane Protein ◽

Band 3 ◽

Erythrocyte Membranes ◽

Band 3 Protein ◽

High Affinity ◽

Human Erythrocyte Membranes ◽

Affinity Interaction ◽

Cytoplasmic Side

Recent studies of haemoglobin binding to the cytoplasmic side of the erythrocyte membrane have shown that the predominant high-affinity interaction occurs with the major integral membrane protein known as band-3 protein and that this interaction may occur within the intact erythrocyte in a manner regulated by cell pH. We report here that haemoglobin and glyceraldehyde 3-phosphate dehydrogenase binding to band-3 protein in isolated membranes can inhibit endocytosis during vesiculation in vitro. The specificity of this effect was demonstrated by showing that myoglobin, which has an affinity for the membrane fully one to two orders of magnitude lower than that for haemoglobin, does not inhibit endocytosis.

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Modulation of red cell glycolysis: interactions between vertebrate hemoglobins and cytoplasmic domains of band 3 red cell membrane proteins

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00060.2004 ◽

2004 ◽

Vol 287 (2) ◽

pp. R454-R464 ◽

Cited By ~ 46

Author(s):

Roy E. Weber ◽

Wolfgang Voelter ◽

Angela Fago ◽

Hartmut Echner ◽

Estela Campanella ◽

...

Keyword(s):

Membrane Proteins ◽

Binding Site ◽

Protein Band ◽

Band 3 ◽

Glycolytic Enzyme ◽

Pentose Phosphate ◽

Dependent Manner ◽

Red Cell ◽

Red Cell Membrane ◽

Vital Functions

Several vital functions/physical characteristics of erythrocytes (including glycolysis, the pentose phosphate pathway, ion fluxes, and cellular deformability) display dependence on the state of hemoglobin oxygenation. The molecular mechanism proposed involves an interaction between deoxyhemoglobin and the cytoplasmic domain of the anion-exchange protein, band 3 (cdB3). Given that band 3 also binds to membrane proteins 4.1 and 4.2, several kinases, hemichromes, and integral membrane proteins, and at least three glycolytic enzymes, it has been suggested that the cdB3-deoxyhemoglobin interaction might modulate the pathways mediated by these associated proteins in an O2-dependent manner. We have investigated this mechanism by synthesizing 10-mer peptides corresponding to the NH2-terminal fragments of various vertebrate cdB3s, determining their effects on the oxygenation reactions of hemoglobins from the same and different species and examining binding of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase to the erythrocytic membrane of mouse erythrocytes. The cdB3 interaction is strongly dependent on pH and the number of negative and positive charges of the peptide and at the effector binding site, respectively. It lowers the O2association equilibrium constant of the deoxygenated (Tense) state of the hemoglobin and is inhibited by magnesium ions, which neutralize cdB3's charge and by 2,3-diphosphoglycerate, which competes for the cdB3-binding site. The interaction is stronger in humans (whose erythrocytes derive energy predominantly from glycolysis and exhibit higher buffering capacity) than in birds and ectothermic vertebrates (whose erythrocytes metabolize aerobically and are poorly buffered) and is insignificant in fish, suggesting that its role in the regulation of red cell glycolysis increased with phylogenetic development in vertebrates.

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Characterization of the autologous antibodies that opsonize erythrocytes with clustered integral membrane proteins

Blood ◽

10.1182/blood.v81.11.3146.3146 ◽

1993 ◽

Vol 81 (11) ◽

pp. 3146-3152 ◽

Cited By ~ 39

Author(s):

F Turrini ◽

F Mannu ◽

P Arese ◽

J Yuan ◽

PS Low

Keyword(s):

Membrane Protein ◽

Leading Edge ◽

Integral Membrane Protein ◽

Protein Band ◽

Band 3 ◽

Erythrocyte Membranes ◽

Immune Recognition ◽

High Molecular Weight Complex ◽

A Minor ◽

Autologous Antibodies

Abstract In earlier studies we presented evidence that the clustering of the integral membrane protein, band 3, can serve as a signal for immune recognition and clearance of senescent or abnormal erythrocytes from circulation. In this study, we have exploited the capacity of 1 mmol/L Zn+2 to mildly and reversibly cluster band 3 in situ to characterize the nature of the autologous antibodies specific for the clustered state. We report that the autologous IgG elute almost exclusively in a high molecular weight complex with other proteins when C12E8 detergent extracts of Zn clustered membranes are chromatographed on Sepharose CL- 6B. The complex was also seen to contain complement component C3, hemoglobin, and a cross-linked oligomer of band 3. Autologous IgG and complement were virtually absent from all other fractions. When the band 3 clusters were disaggregated by removal of the Zn+2, the autologous IgG eluted from the erythrocyte surface. Collection of this IgG and use of the antibody in immunoblots of erythrocyte membranes showed that the band 3 monomer, dimer, and oligomers were the major antigenic species. Except for a minor unidentified band at approximately 78,000 d, no other proteins were significantly stained. Curiously, band 3 showed an uneven staining pattern, with oligomers and the leading edge of the monomers appearing more intensely than expected from their abundances in the Coomassie blue-stained gels. Typing of the same autologous IgG with monoclonal antibodies specific for the different subclasses of IgG showed the presence of only subtypes 2 and 3. Taken together, these data suggest that a specific population of autologous IgG recognizes sites of integral membrane protein clustering (a common lesion in senescent and abnormal red blood cells) and that the antigen within these clusters involves an aggregated state of band 3.

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The nature of human erythrocyte receptors for Neisseria gonorrhoeae

Canadian Journal of Microbiology ◽

10.1139/m82-029 ◽

1982 ◽

Vol 28 (2) ◽

pp. 219-222 ◽

Cited By ~ 1

Author(s):

G. M. Wiseman ◽

P. McNicol

Keyword(s):

Neisseria Gonorrhoeae ◽

Human Erythrocyte ◽

Polyacrylamide Gel Electrophoresis ◽

Protein Band ◽

Band 3 ◽

Erythrocyte Membranes ◽

Glycophorin A ◽

Receptor Sites ◽

Human Erythrocyte Membranes ◽

Sepharose 4B

Normal and trypsinized human erythrocyte membranes were used as a model in the study of host cell receptors for Neisseria gonorrhoeae. Receptor sites were identified by adherence inhibition assays of fractions of membranes eluted from polyacrylamide gel electrophoresis columns. Results indicated that inhibition of gonococcus T1 and T4 adherence was associated with erythrocyte protein bands 3 and 4 and glycophorin A, the major sialoglycoprotein. Further investigation revealed that band 3 preparations isolated by affinity chromatography on concanavalin A – Sepharose 4B columns continued to inhibit T1 adherence to erythrocytes but did not inhibit adherence of T4 organisms. It is suggested that protein band 3 is the receptor on erythrocytes for T1 gonococci and that glycophorin A may be the receptor for T4 cells.

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Characterization of the autologous antibodies that opsonize erythrocytes with clustered integral membrane proteins

Blood ◽

10.1182/blood.v81.11.3146.bloodjournal81113146 ◽

1993 ◽

Vol 81 (11) ◽

pp. 3146-3152 ◽

Cited By ~ 1

Author(s):

F Turrini ◽

F Mannu ◽

P Arese ◽

J Yuan ◽

PS Low

Keyword(s):

Membrane Protein ◽

Leading Edge ◽

Integral Membrane Protein ◽

Protein Band ◽

Band 3 ◽

Erythrocyte Membranes ◽

Immune Recognition ◽

High Molecular Weight Complex ◽

A Minor ◽

Autologous Antibodies

In earlier studies we presented evidence that the clustering of the integral membrane protein, band 3, can serve as a signal for immune recognition and clearance of senescent or abnormal erythrocytes from circulation. In this study, we have exploited the capacity of 1 mmol/L Zn+2 to mildly and reversibly cluster band 3 in situ to characterize the nature of the autologous antibodies specific for the clustered state. We report that the autologous IgG elute almost exclusively in a high molecular weight complex with other proteins when C12E8 detergent extracts of Zn clustered membranes are chromatographed on Sepharose CL- 6B. The complex was also seen to contain complement component C3, hemoglobin, and a cross-linked oligomer of band 3. Autologous IgG and complement were virtually absent from all other fractions. When the band 3 clusters were disaggregated by removal of the Zn+2, the autologous IgG eluted from the erythrocyte surface. Collection of this IgG and use of the antibody in immunoblots of erythrocyte membranes showed that the band 3 monomer, dimer, and oligomers were the major antigenic species. Except for a minor unidentified band at approximately 78,000 d, no other proteins were significantly stained. Curiously, band 3 showed an uneven staining pattern, with oligomers and the leading edge of the monomers appearing more intensely than expected from their abundances in the Coomassie blue-stained gels. Typing of the same autologous IgG with monoclonal antibodies specific for the different subclasses of IgG showed the presence of only subtypes 2 and 3. Taken together, these data suggest that a specific population of autologous IgG recognizes sites of integral membrane protein clustering (a common lesion in senescent and abnormal red blood cells) and that the antigen within these clusters involves an aggregated state of band 3.

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Ultrastructure of the intact skeleton of the human erythrocyte membrane.

The Journal of Cell Biology ◽

10.1083/jcb.102.3.997 ◽

1986 ◽

Vol 102 (3) ◽

pp. 997-1006 ◽

Cited By ~ 127

Author(s):

B W Shen ◽

R Josephs ◽

T L Steck

Keyword(s):

Human Erythrocyte ◽

Actin Filaments ◽

Band 3 ◽

Carbon Films ◽

Erythrocyte Membranes ◽

Accessory Proteins ◽

Red Cell Membrane ◽

Human Erythrocyte Membranes ◽

Low Ionic Strength ◽

Triton X

Filamentous skeletons were liberated from isolated human erythrocyte membranes in Triton X-100, spread on fenestrated carbon films, negatively stained, and viewed intact and unfixed in the transmission electron microscope. Two forms of the skeleton were examined: (a) basic skeletons, stripped of accessory proteins with 1.5 M NaCl so that they contain predominantly polypeptide bands 1, 2, 4.1, and 5; and (b) unstripped skeletons, which also bore accessory proteins such as ankyrin and band 3 and small plaques of residual lipid. Freshly prepared skeletons were highly condensed. Incubation at low ionic strength and in the presence of dithiothreitol for an hour or more caused an expansion of the skeletons, which greatly increased the visibility of their elements. The expansion may reflect the opening of spectrin from a compact to an elongated disposition. Expanded skeletons appeared to be organized as networks of short actin filaments joined by multiple (5-8) spectrin tetramers. In unstripped preparations, globular masses were observed near the centers of the spectrin filaments, probably corresponding to complexes of ankyrin with band 3 oligomers. Some of these globules linked pairs of spectrin filaments. Skeletons prepared with a minimum of perturbation had thickened actin protofilaments, presumably reflecting the presence of accessory proteins. The length of these actin filaments was highly uniform, averaging 33 +/- 5 nm. This is the length of nonmuscle tropomyosin. Since there is almost enough tropomyosin present to saturate the F-actin, our data support the hypothesis that tropomyosin may determine the length of actin protofilaments in the red cell membrane.

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