The effects of glycophorin A on the expression of the human red cell anion transporter (band 3) in Xenopus oocytes

1994 ◽  
Vol 140 (1) ◽  
Author(s):  
J.D. Groves ◽  
M.J.A. Tanner
Keyword(s):  
Xenopus Oocytes ◽  
Band 3 ◽  
Glycophorin A ◽  
Red Cell ◽  
Anion Transporter

scholarly journals Complementation studies with co-expressed fragments of human red cell band 3 (AE1): the assembly of the anion-transport domain in Xenopus oocytes and a cell-free translation system

1998 ◽  
Vol 332 (1) ◽  
pp. 161-171 ◽  
Author(s):  
Jonathan D. GROVES ◽  
Lin WANG ◽  
Michael J. A. TANNER
Keyword(s):  
Xenopus Oocytes ◽  
Band 3 ◽  
Translation System ◽  
Red Cell ◽  
Membrane Domain ◽  
Cytoplasmic Loop ◽  
Free System ◽  
Anion Transporter ◽  
Free Translation ◽  
The Individual

We examined the assembly of the membrane domain of the human red cell anion transporter (band 3; AE1) by co-expression of recombinant N- and C-terminal fragments in Xenopus oocytes and in cell-free translation with canine pancreatic microsomes. Co-immunoprecipitation was performed in non-denaturing detergent solutions using antibodies directed against the N- and C-termini of the membrane domain. Eleven of the twelve fragments were expressed stably in oocytes in the presence or absence of their respective partners. However, the fragment containing from putative span nine to the C-terminus could be detected in oocytes only when co-expressed with its complementary partner containing the first eight spans. Co-expression of pairs of fragments divided in the first, second, third and fourth exofacial loops and in the fourth cytoplasmic loop resulted in a concentration-dependent association, but a pair of fragments divided in the sixth cytoplasmic loop did not co-immunoprecipitate. When two complementary fragments were translated separately in the cell-free system and the purified microsomes were then mixed, co-immunoprecipitation was observed only if the membranes were first fused using polyethylene glycol. This shows that co-immunoprecipitation results from specific interactions within the membrane and is not an artefact of detergent solubilization or immunoprecipitation. We demonstrate that band 3 assembly can occur within the membrane after translation, insertion and initial folding of the individual fragments have been completed. We conclude that most band 3 fragments contain the necessary information to fold in the membrane and adopt a structure that is sufficiently similar to the native protein that it permits correct assembly with its complementary partner.

Hereditary Stomatocytosis Associated with a Loss of Function Mutation In Rh-Associated Glycoprotein (RhAG)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2040-2040
Author(s):  
Connie M Westhoff ◽  
Seth Alper
Keyword(s):  
Xenopus Oocytes ◽  
Collecting Duct ◽  
Monovalent Cation ◽  
Band 3 ◽  
Cell Permeability ◽  
Red Cell ◽  
Loss Of Function ◽  
Wild Type ◽  
Water Control ◽  
Glycophorin B

Abstract Abstract 2040 The erythroid Rh family of proteins includes RhCE and RhD which carry the common Rh antigens, and the related Rh-associated glycoprotein, RhAG. RhAG is required for trafficking of the blood group proteins to the membrane and forms the core of a macro-complex in the membrane which includes glycophorin B, Band 3, CD47, and LW. The Rh proteins are structurally and functionally related to the Amt superfamily of NH3/NH4+ transport proteins, and RhAG and its nonerythroid paralogs, RhCG and RhBG, have been shown to mediate NH3/NH4+ transport. RhCG is responsible for part of renal collecting duct epithelial cell NH3/NH4+ secretion, and Rhcg-/- mice exhibit incomplete distal renal tubular acidosis due to impaired urinary NH4+ excretion. The Rhag-/- mouse is grossly normal, and the significance of RhAG-mediated NH3/NH4+ transport in human erythrocytes remains unclear. Over-hydrated hereditary stomatocytosis (OHSt) is a rare dominant disorder characterized by moderate hemolytic anemia, increased mean red cell volumes, stomatocytes and echinocytes, and increased red cell permeability to the monovalent cations, Na+ and K+. Six of the seven OHSt kindred studied by Bruce et al. (Blood. 2009;113:1350) displayed a heterozygous Phe65Ser mutation in RhAG. Expression studies of the mutant 65Ser-RhAG in Xenopus oocytes induced a monovalent cation flux compatible with the cation leak seen in RBCs. The increased Na+ and decreased K+ contents of mutant RhAG-expressing oocytes suggested that F65S is a gain-of-function mutation that opens a cation leak, likely within the RhAG polypeptide. In this study the ammonia transport properties of the OHSt mutant 65Ser-RhAG were investigated. Xenopus oocytes were injected with cRNA encoding wild-type RhAG, the OHSt mutant 65Ser-RhAG, and 65Val-RhAG, an engineered mutation with a smaller hydrophobic side chain at position 65. Wild-type and mutant RhAG polypeptides were well-expressed in the oocyte membrane as measured by quantitative immunoblotting. Uptake of the NH3/NH4+ substrate analog 14C-methylammonium (MA), was assayed in oocytes previously injected with water (control) or with cRNA. Expression of wild-type RhAG mediated MA uptake at rates 6-fold greater than that of water-injected controls. Uptake of MA by oocytes expressing 65Val-RhAG was equivalent to that of wild type RhAG. However, MA uptake by oocytes expressing OHSt mutant 65Ser-RhAG was greatly reduced to less than 20% that of oocytes expressing wild-type RHAG or 65Val-RhAG, and was only 1.5-fold greater than that of water-injected control oocytes. Co-expression with other, individual Rh complex members glycophorin B, RhD, RhCE, or Band 3 did not alter MA-mediated uptake by RhAG-expressing oocytes. Importantly, this study reveals that the RhAG mutation Phe65Ser found in patients with type 1 over-hydrated stomatocytosis is a loss of function mutation. Further study is required to define the relationship between loss of NH3/NH4+ transport and erythrocyte Na+ and K+ cation content. Disclosures: Westhoff: Immucor: Scientific Advisor.

scholarly journals Red-cell glycophorin A–band 3 interactions associated with the movement of band 3 to the cell surface

2000 ◽  
Vol 350 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Mark T. YOUNG ◽  
Roland BECKMANN ◽  
Ashley M. TOYE ◽  
Michael J. A. TANNER
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Band 3 ◽  
Cell Surface Expression ◽  
Glycophorin A ◽  
Detergent Solution ◽  
Red Cell ◽  
Cell Clones ◽  
Absolute Requirement ◽  
Dimerization Interface

We have examined the mechanism by which glycophorin A (GPA) facilitates the movement of the human red-cell anion exchanger (band 3, AE1) to the cell surface. GPA itself forms stable dimers in membranes and detergent solution. Four mutants of human GPA with impaired dimerization were prepared (L75I, I76A, G79L and G83L). All four GPA mutants enhanced band 3 translocation to the Xenopus oocyte plasma membrane in the same way as wild-type GPA, showing that the GPA monomer is sufficient to mediate this process. Cell-surface expression of the natural band 3 mutant G701D has an absolute requirement for GPA. GPA monomers also rescued the cell-surface expression of this mutant band 3. Taking into account other evidence, we infer that the site of GPA interaction with band 3 is located outside the GPA dimerization interface but within the GPA transmembrane span. The results of examination of the cell-surface expression of GPA and band 3 in different K562 erythroleukaemia cell clones stably transfected with band 3 are consistent with the movement of GPA and band 3 to the cell surface together. We discuss the pathways by which band 3 moves to the cell surface in the presence and the absence of GPA, concluding that GPA has a role in enhancing the folding and maturation of band 3. We propose that GPA functions in erythroid cells to assist with the incorporation of large amounts of properly folded band 3 into the membrane within a limited time span during erythroid maturation.

scholarly journals Complete Deficiency of Glycophorin A in Red Blood Cells From Mice With Targeted Inactivation of the Band 3 (AE1) Gene

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 2146-2151 ◽  
Author(s):  
Hani Hassoun ◽  
Toshihiko Hanada ◽  
Mohini Lutchman ◽  
Kenneth E. Sahr ◽  
Jiri Palek ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Blood Cell ◽  
Red Blood Cells ◽  
Red Blood Cell ◽  
Blood Cells ◽  
Band 3 ◽  
Glycophorin A ◽  
Blood Group Antigens ◽  
Red Blood Cell Membrane ◽  
Anion Transporter

Abstract Glycophorin A is the major transmembrane sialoglycoprotein of red blood cells. It has been shown to contribute to the expression of the MN and Wright blood group antigens, to act as a receptor for the malaria parasite Plasmodium falciparum and Sendai virus, and along with the anion transporter, band 3, may contribute to the mechanical properties of the red blood cell membrane. Several lines of evidence suggest a close interaction between glycophorin A and band 3 during their biosynthesis. Recently, we have generated mice where the band 3 expression was completely eliminated by selective inactivation of the AE1 anion exchanger gene, thus allowing us to study the effect of band 3 on the expression of red blood cell membrane proteins. In this report, we show that the band 3 −/− red blood cells contain protein 4.1, adducin, dematin, p55, and glycophorin C. In contrast, the band 3 −/− red blood cells are completely devoid of glycophorin A (GPA), as assessed by Western blot and immunocytochemistry techniques, whereas the polymerase chain reaction (PCR) confirmed the presence of GPA mRNA. Pulse-label and pulse-chase experiments show that GPA is not incorporated in the membrane and is rapidly degraded in the cytoplasm. Based on these findings and other published evidence, we propose that band 3 plays a chaperone-like role, which is necessary for the recruitment of GPA to the red blood cell plasma membrane.

scholarly journals Red-cell glycophorin A‒band 3 interactions associated with the movement of band 3 to the cell surface

2000 ◽  
Vol 350 (1) ◽  
pp. 53 ◽  
Author(s):  
Mark T. YOUNG ◽  
Roland BECKMANN ◽  
Ashley M. TOYE ◽  
Michael J.A. TANNER
Keyword(s):  
Cell Surface ◽  
Band 3 ◽  
Glycophorin A ◽  
Red Cell

Inhibition of malarial invasion by intracellular antibodies against intrinsic membrane proteins in the red cell

Parasitology ◽  
1986 ◽  
Vol 93 (3) ◽  
pp. 427-431 ◽  
Author(s):  
A. R. Dluzewski ◽  
K. Rangachari ◽  
M. J. A. Tanner ◽  
D. J. Anstee ◽  
R. J. M. Wilson ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Transmembrane Proteins ◽  
Band 3 ◽  
Red Cells ◽  
Glycophorin A ◽  
Red Cell ◽  
Structural Effects ◽  
Membrane Cytoskeleton ◽  
Resealed Ghosts ◽  
Human Red Cells

SUMMARYIt has previously been shown that antibodies against the transmembrane proteins, band 3 and glycophorin A, inhibit entry of the merozoite into the red cell and, in the case of band 3, it was established that attachment of the parasite to the cell is not prevented. We have found that antibodies against the cytoplasmic domains of band 3 and of glycophorin A, when present in the interior of resealed ghosts of human red cells, also inhibit invasion by P. falciparum. It is inferred that attachment of the merozoite to the red cell causes structural effects that are transduced to the membrane cytoskeleton and the antibodies against transmembrane proteins interfere with the invasion sequence at this level.

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