scholarly journals Monoclonal antibodies recognizing epitopes on the extracellular face and intracellular N-terminus of the human erythrocyte anion transporter (band 3) and their application to the analysis of South East Asian ovalocytes

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2929-2936 ◽  
Author(s):  
JS Smythe ◽  
FA Spring ◽  
B Gardner ◽  
SF Parsons ◽  
PA Judson ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Erythrocyte ◽  
East Asian ◽  
Band 3 ◽  
Extracellular Loop ◽  
Binding Studies ◽  
Cooperative Interactions ◽  
The Third ◽  
Anion Transporter

This report describes the production and characterization of 13 rodent monoclonal antibodies to the human erythrocyte anion transport protein AE1 (syn. band 3). Eleven antibodies (4 murine and 7 rat) recognize epitopes dependent on the integrity of the third extracellular loop of the protein. Two antibodies (1 murine and 1 rat) recognize epitopes on the N-terminal cytoplasmic domain. Quantitative binding studies using radioiodinated IgG and Fab fragments of antibodies to extracellular epitopes on AE1 ranged from 77,000 to 313,000 (IgG) and from 241,000 to 772,000 (Fab) molecules bound at saturation. The results indicate that the epitopes recognized by different antibodies vary in their accessibility and suggest that there is heterogeneity in the organization of individual AE1 molecules in the red blood cell membrane. Quantitative binding studies on South East Asian ovalocytes using several antibodies to AE1 and an anti-Wrb show a marked reduction in the number of antibody molecules bound at saturation. These results are consistent with the existence of highly cooperative interactions between transmembrane domains of AE1 in normal erythrocytes and the disruption of these interactions in the variant AE1 found in South East Asian ovalocytes.

Synthesis and Characterization of a Novel Spin-Labeled Affinity Probe of Human Erythrocyte Band 3:  Characteristics of the Stilbenedisulfonate Binding Site†

Biochemistry ◽  
1996 ◽  
Vol 35 (21) ◽  
pp. 6931-6943 ◽  
Author(s):  
Douglas J. Scothorn ◽  
Walter E. Wojcicki ◽  
Eric J. Hustedt ◽  
Albert H. Beth ◽  
Charles E. Cobb
Keyword(s):  
Binding Site ◽  
Human Erythrocyte ◽  
Band 3 ◽  
Synthesis And Characterization ◽  
Affinity Probe

Characterization of monoclonal antibodies assigned to the CD45 subgroup of the Third International Swine CD Workshop

2001 ◽  
Vol 80 (1-2) ◽  
pp. 165-174 ◽  
Author(s):  
F.A. Zuckermann ◽  
W.M. Schnitzlein ◽  
E. Thacker ◽  
J. Sinkora ◽  
K. Haverson
Keyword(s):  
Monoclonal Antibodies ◽  
The Third

scholarly journals Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 inEscherichia coli

1992 ◽  
Vol 1 (9) ◽  
pp. 1206-1214 ◽  
Author(s):  
Cheng C. Wang ◽  
Ryuichi Moriyama ◽  
Philip S. Low ◽  
Janine A. Badylak ◽  
Jack E. Dixon ◽  
...  
Keyword(s):  
Human Erythrocyte ◽  
Cytoplasmic Domain ◽  
Band 3 ◽  
Inescherichia Coli ◽  
Purification And Characterization

scholarly journals Alternative primary structures in the transmembrane domain of the chicken erythroid anion transporter

1988 ◽  
Vol 8 (3) ◽  
pp. 1327-1335
Author(s):  
J V Cox ◽  
E Lazarides
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Cytoplasmic Domain ◽  
Band 3 ◽  
Transporter Protein ◽  
Isolation And Characterization ◽  
Anion Transporter ◽  
Primary Structures ◽  
Membrane Spanning

Isolation and characterization of the chicken erythroid anion transporter (band 3) cDNA clone, pCHB3-1, revealed that the chicken erythroid band 3 polypeptide is 844 amino acids in length with a predicted mass of 109,000 daltons. This polypeptide is composed of a hydrophilic N-terminal cytoplasmic domain and a hydrophobic C-terminal transmembrane domain. The approximately 90 N-terminal amino acids of the human and murine erythroid band 3 polypeptides are absent in the predicted sequence of the chicken erythroid band 3 polypeptide. The absence of this very acidic N-terminal region is consistent with the lack of binding of glyceraldehyde-3-phosphate dehydrogenase to chicken erythroid band 3, as well as the relatively basic isoelectric point observed for this molecule. The remainder of the cytoplasmic domain shows little similarity to the cytoplasmic domain of the murine and human erythroid band 3, with the exception of the putative ankyrin-binding site, which is highly conserved. In contrast, the transmembrane domain of the chicken band 3 polypeptide is very similar to that of the murine erythroid and human nonerythroid band 3 polypeptides. The transmembrane domain contains 10 hydrophobic regions that could potentially traverse the membrane 12 to 14 times. In addition, a variant of chicken erythroid band 3, pCHB3-2, was cloned in which one of the hydrophobic regions of pCHB3-1 is lacking. The transcript complementary to pCHB3-2 accumulated in chicken erythroid cells in a similar manner as the transcript complementary to pCHB3-1 during embryonic development. This is the first example of a transporter protein or ion channel with alternative primary structures in its membrane-spanning segments.

Freeze-fracture Electron Microscopic observations on the effects of sulphydryl group reagents on human erythrocyte membranes

1990 ◽  
Vol 48 (3) ◽  
pp. 524-525 ◽  
Author(s):  
Gheorghe Benga ◽  
Anthony Brain ◽  
Victor I. Pop ◽  
John Wrigglesworth
Keyword(s):  
Human Erythrocyte ◽  
Freeze Fracture ◽  
Band 3 ◽  
Erythrocyte Membranes ◽  
Electron Microscopic ◽  
Experimental Conditions ◽  
Water Permeation ◽  
Human Erythrocyte Membranes ◽  
Erythrocyte Membrane Proteins

The intra-membrane particles (IMPs) observed on the fracture face of frozen erythrocyte membranes are thought to correspond primarily to “band 3” tetramers or dimers. Some recent studies correlating the inhibition of water diffusion in erythrocytes by p-chloromercuribenzene sulfonate (PCMBS) with the binding of 203Hg to erythrocyte membrane proteins has enabled band 3 and the polypeptides in band 4.5 to be identified as the proteins associated with the channels for water permeation in human erythrocytes. A further characterization of the effects of the incubation of human erythrocyte membranes with PCMBS and N-ethylmaleimide (NEM) has been performed as previously described. Experimental conditions have been previously described.A comparison was made of the appearance of freeze-etched membranes of control erythrocytes and erythrocytes with the sulphydryl reagents. It was found that on many of the control and NEM-treated cells, small (50-100 nm) elevated patches could be seen (Fig. 1, 2 and 3). These are present on both fracture and etch faces and are devoid of any intramembrane particles. The patch elevations were never observed in the membranes of PCMBS-treated cells (Fig. 4).

scholarly journals Characterization of the deoxyhemoglobin binding site on human erythrocyte band 3: implications for O2 regulation of erythrocyte properties

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 932-938 ◽  
Author(s):  
Haiyan Chu ◽  
Andrew Breite ◽  
Peter Ciraolo ◽  
Robert S. Franco ◽  
Philip S. Low
Keyword(s):  
Binding Site ◽  
Human Erythrocyte ◽  
Band 3 ◽  
Major Protein ◽  
Human Erythrocyte Membrane ◽  
Functional Studies ◽  
High Sequence Homology ◽  
Affinity Interaction ◽  
Related Proteins

Band 3, the major protein of the human erythrocyte membrane, associates with multiple metabolic, ion transport, and structural proteins. Functional studies demonstrate that the oxygenation state of the erythrocyte regulates cellular properties performed by these and/or related proteins. Because deoxyhemoglobin, but not oxyhemoglobin, binds band 3 reversibly with high affinity, these observations raise the hypothesis that hemoglobin might regulate erythrocyte properties through its reversible, oxygenation-dependent association with band 3. To explore this hypothesis, we have characterized the binding site of deoxyHb on human erythrocyte band 3. We report that (1) deoxyHb binds to residues 12-23 of band 3; (2) mutation of residues on either side of this sequence greatly enhances affinity of deoxyHb for band 3, suggesting that evolution of a higher affinity interaction would have been possible had it been beneficial for survival; (3) Hb does not bind to 2 other sequences in band 3 despite their high sequence homology to residues 12-23, and (4) the Hb binding site on band 3 lies proximal to binding sites for glycolytic enzymes, band 4.1 and ankyrin, suggesting possible mechanisms through which multifarious erythrocyte properties might be regulated by the oxygenation state of the cell.

Mapping of the Glycolytic Enzyme Binding Sites on Human Erythrocyte Membrane Band 3.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1668-1668
Author(s):  
Haiyan Chu ◽  
Philip S. Low
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Binding Sites ◽  
Troponin T ◽  
Band 3 ◽  
Glycolytic Enzyme ◽  
Human Erythrocyte Membrane ◽  
Binding Studies ◽  
Red Cell Membrane ◽  
N Terminus

Abstract Glycolytic enzymes (GEs) including aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are known to associate with the inner surface of the human erythrocyte membrane. Previous studies have suggested that the N-terminus of the cytoplasmic domain of band 3 (cdb3) constitutes the likely binding site for aldolase, GAPDH and PFK, however, the membrane docking sites for PK and LDH have not been elucidated. In this study, we demonstrate that PK and LDH exhibit no affinity for band 3, regardless of whether the association is measured by co-immunoprecipitation assay, binding competition studies, or catalytic inhibition analyses. We further find that the binding sites for GAPDH, aldolase and PFK on band 3 are distinct but partially overlapping, as evidenced by the fact that: 1) deletion of residues 1–11 of cdb3 eliminates the binding of aldolase, but not PFK or GAPDH, 2) fusion of thioredoxin (Trx) to the N-terminus of residues cdb3 blocks aldolase binding, but not the association of GAPDH or PFK, 3) deletion of sequences 1–50, 1–40, 1–31, or 1–23 of cdb3 blocks cdb3 association with all three GEs, whereas deletion of residues 12-23 only abrogates aldolase binding (while reducing the affinity of PFK and GAPDH), 4) the presence of both sequences, 6–DDYED-10 and 19-EEYED-23, are necessary for cdb3 association with aldolase, whereas the presence of either sequence alone (especially 19-EEYED-23) is sufficient to maintain association with GAPDH and PFK, 5) mutation of all of the acidic residues in the above two sequences to their corresponding amides (E→Q and D→N) results in loss of affinity for all GEs. Because i) kidney cdb3 (which lacks residues 1-65 of cdb3) shows no affinity for any of the GEs, ii) residues 1–55 of cdb3 show near normal affinity for aldolase, GAPDH and PFK, and iii) GAPDH, aldolase, and PFK all compete with each other for cdb3, we conclude that the binding sites for aldolase, GAPDH and PFK are all located within the first 23 residues of cdb3, with the docking site for aldolase likely residing somewhat more N-terminal than the binding sites for GAPDH and PFK. Finally, because each band 3 monomer contains three homologous sequences (6-DDYED-10, 19-EEYED-23 and 902-DEYDE-906) that are found in other proteins that bind GEs (e.g. actin, β-tubulin, troponin T), and since the first two of these sequences were shown to be required for cdb3 binding, the question naturally arose whether different GEs might also associate with residues 902-DEYDE-906 at the extreme C-terminus of band 3. Similar binding studies demonstrate that such an interaction does not occur. Thus, our data show that GAPDH, aldolase, and PFK all bind near the N-terminus of band 3 and that PK and LDH must dock somewhere else on the red cell membrane.

Sign in / Sign up

Export Citation Format

Share Document