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.

Interaction of anion exchanger 1 and glycophorin A in human erythroleukaemic K562 cells

2009 ◽  
Vol 421 (3) ◽  
pp. 345-356 ◽  
Author(s):  
Allison J. Pang ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Cell Surface ◽  
Anion Exchanger ◽  
K562 Cells ◽  
Surface Expression ◽  
Protein Band ◽  
Band 3 ◽  
Cell Surface Expression ◽  
Glycophorin A ◽  
Anion Exchanger 1 ◽  
Integral Proteins

AE1 [anion exchanger 1, also known as SLC4A1 (solute carrier family 4, anion exchanger, member 1) and band 3 (erythrocyte membrane protein band 3)] is a major membrane glycoprotein expressed in human erythrocytes where it mediates the exchange of chloride and bicarbonate across the plasma membrane. Glycophorin A (GPA) is a sialoglycoprotein that associates with AE1 in erythrocytes forming the Wrb (Wright b) blood group antigen. These two integral proteins may also form a complex during biosynthesis, with GPA facilitating the cell surface expression of AE1. This study investigates the interaction of GPA with AE1 in K562 cells, a human erythroleukaemic cell line that expresses GPA, and the role of GPA in the cell surface expression of AE1. In K562 cells, GPA was dimeric and N- and O-glycosylated similar to erythroid GPA. GPA was localized at the cell surface, but also localized to the Golgi. AE1 expressed in K562 cells contained both complex and high-mannose oligosaccharides, and co-localized with GPA at the cell surface and in the endoplasmic reticulum (ER). The Wrb antigen was detected at the cell surface of AE1-transfected K562 cells, indicating the existence of an AE1–GPA complex. Immunofluorescence and co-immunoprecipitation studies using AE1 and an ER-localized hereditary spherocytosis mutant (R760Q AE1) showed that GPA and AE1 could interact in the ER. GPA knockdown by shRNAs (small-hairpin RNAs), however, had no effect on the level of cell surface expression of AE1. The results indicate that AE1 and GPA form a complex in the ER of human K562 cells, but that both proteins can also traffic to the cell surface independently of each other.

scholarly journals The Level of CD81 Cell Surface Expression Is a Key Determinant for Productive Entry of Hepatitis C Virus into Host Cells

2006 ◽  
Vol 81 (2) ◽  
pp. 588-598 ◽  
Author(s):  
George Koutsoudakis ◽  
Eva Herrmann ◽  
Stephanie Kallis ◽  
Ralf Bartenschlager ◽  
Thomas Pietschmann
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Surface ◽  
Surface Expression ◽  
Hcv Infection ◽  
Host Cells ◽  
Productive Infection ◽  
Cell Surface Expression ◽  
Type I ◽  
Cell Clones

ABSTRACT Recently a cell culture model supporting the complete life cycle of the hepatitis C virus (HCV) was developed. Searching for host cell determinants involved in the HCV replication cycle, we evaluated the efficiency of virus propagation in different Huh-7-derived cell clones. We found that Huh-7.5 cells and Huh7-Lunet cells, two former replicon cell clones that had been generated by removal of an HCV replicon by inhibitor treatment, supported comparable levels of RNA replication and particle production, whereas virus spread was severely impaired in the latter cells. Analysis of cell surface expression of CD81 and scavenger receptor class B type I (SR-BI), two molecules previously implicated in HCV entry, revealed similar expression levels for SR-BI, while CD81 surface expression was much higher on Huh-7.5 cells than on Huh7-Lunet cells. Ectopic expression of CD81 in Huh7-Lunet cells conferred permissiveness for HCV infection to a level comparable to that for Huh-7.5 cells. Modulation of CD81 cell surface density in Huh-7.5 cells by RNA interference indicated that a certain amount of this molecule (∼7 × 104 molecules per cell) is required for productive infection with a low dose of HCV. Consistent with this, we show that susceptibility to HCV infection depends on a critical quantity of CD81 molecules. While infection is restricted in cells expressing very small amounts of CD81, susceptibility rapidly rises within a narrow range of CD81 levels, reaching a plateau where higher expression does not further increase the efficiency of infection. Together these data indicate that a high density of cell surface-exposed CD81 is a key determinant for productive HCV entry into host cells.

Functional Cell Surface Expression of Band 3, the Human Red Blood Cell Anion Exchange Protein (AE1), in K562 Erythroleukemia Cells: Band 3 Enhances the Cell Surface Reactivity of Rh Antigens

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4428-4438 ◽  
Author(s):  
Roland Beckmann ◽  
Jonathan S. Smythe ◽  
David J. Anstee ◽  
Michael J.A. Tanner
Keyword(s):  
Cell Surface ◽  
Chloride Transport ◽  
K562 Cells ◽  
Surface Expression ◽  
Band 3 ◽  
Surface Reactivity ◽  
Cell Surface Expression ◽  
Anion Exchanger 1 ◽  
Erythroleukemia Cells ◽  
Antigen Activity

Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBabe retroviral vector. Stable K562 clones expressing band 3 were isolated by flow cytometry, and surface expression was quantified by immunoblotting. The function of band 3 expressed at the cell surface was demonstrated in chloride transport assays. K562 cells expressing band 3 also displayed high levels of the Wrb blood group antigen, confirming the role of band 3 in Wrb expression, and an increase in the low levels of endogenous Rh antigen activity. We also performed coexpression experiments with K562 clones that had previously been transduced with cDNAs encoding RhD or RhcE polypeptides. The transfection and expression of band 3 in these clones substantially increased the levels of RhD and cE antigen activity expressed on the cells and also increased the reactivity of the cells with antibody to the endogenous Rh glycoprotein (RhGP, Rh50). The increased reactivity of Rh antigens may result from cell surface or intracellular interactions of band 3 with the protein complex which contains the Rh polypeptides and RhGP, or from indirect effects of band 3 on the membrane environment. This work establishes a system for cell surface expression of band 3 in a mammalian cell line, which will enable further studies of the protein and its interactions with other membrane components.

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

Functional Cell Surface Expression of Band 3, the Human Red Blood Cell Anion Exchange Protein (AE1), in K562 Erythroleukemia Cells: Band 3 Enhances the Cell Surface Reactivity of Rh Antigens

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4428-4438 ◽  
Author(s):  
Roland Beckmann ◽  
Jonathan S. Smythe ◽  
David J. Anstee ◽  
Michael J.A. Tanner
Keyword(s):  
Cell Surface ◽  
Chloride Transport ◽  
K562 Cells ◽  
Surface Expression ◽  
Band 3 ◽  
Surface Reactivity ◽  
Cell Surface Expression ◽  
Anion Exchanger 1 ◽  
Erythroleukemia Cells ◽  
Antigen Activity

Abstract Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBabe retroviral vector. Stable K562 clones expressing band 3 were isolated by flow cytometry, and surface expression was quantified by immunoblotting. The function of band 3 expressed at the cell surface was demonstrated in chloride transport assays. K562 cells expressing band 3 also displayed high levels of the Wrb blood group antigen, confirming the role of band 3 in Wrb expression, and an increase in the low levels of endogenous Rh antigen activity. We also performed coexpression experiments with K562 clones that had previously been transduced with cDNAs encoding RhD or RhcE polypeptides. The transfection and expression of band 3 in these clones substantially increased the levels of RhD and cE antigen activity expressed on the cells and also increased the reactivity of the cells with antibody to the endogenous Rh glycoprotein (RhGP, Rh50). The increased reactivity of Rh antigens may result from cell surface or intracellular interactions of band 3 with the protein complex which contains the Rh polypeptides and RhGP, or from indirect effects of band 3 on the membrane environment. This work establishes a system for cell surface expression of band 3 in a mammalian cell line, which will enable further studies of the protein and its interactions with other membrane components.

scholarly journals Recombinant Miltenberger I and II human blood group antigens: the role of glycosylation in cell surface expression and antigenicity of glycophorin A

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1913-1920 ◽  
Author(s):  
M Ugorski ◽  
DP Blackall ◽  
P Pahlsson ◽  
SH Shakin-Eshleman ◽  
J Moore ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Blood Group ◽  
Cho Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Glycophorin A ◽  
Blood Group Antigens ◽  
Wild Type

Abstract Glycophorin A is a heavily glycosylated glycoprotein (1 N-linked and 15 O-linked oligosaccharides) and is highly expressed on the surface of human red blood cells. It is important in transfusion medicine because it carries several clinically relevant human blood group antigens. To study further the role of glycosylation in surface expression of this protein, four mutations were separately introduced into glycophorin A cDNA by site-directed mutagenesis. Each of these mutations blocks N- linked glycosylation at Asn26 of this glycoprotein by affecting the Asn- X-Ser/Thr acceptor sequence. Two of these mutations are identical to the amino acid polymorphisms found at position 28 in the Mi.I and Mi.II Miltenberger blood group antigens. The mutated recombinant glycoproteins were expressed in transfected wild-type and glycosylation- deficient Chinese hamster ovary (CHO) cells. When expressed in wild- type CHO cells and analyzed on Western blots, each of the four mutants had a faster electrophoretic mobility than wild-type glycophorin A, corresponding to a difference of approximately 4 Kd. This change is consistent with the absence of the N-linked oligosaccharide at Asn26. Each of the four mutants was highly expressed on the surface of CHO cells, confirming that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not necessary for cell surface expression of this glycoprotein. To examine the role of O- linked glycosylation in this process, the Mi.I mutant cDNA was transfected into the IdlD glycosylation-deficient CHO cell line. When the transfected IdlD cells were cultured in the presence of N- acetylgalactosamine alone, only intermediate levels of cell surface expression were seen for Mi.I mutant glycophorin A containing truncated O-linked oligosaccharides. In contrast, when cultured in the presence of galactose alone, or in the absence of both galactose and N- acetylgalactosamine, Mi.I mutant glycophorin A lacking both N-linked and O-linked oligosaccharides was not expressed at the cell surface. This extends previous results (Remaley et al, J Biol Chem 266:24176, 1991) showing that, in the absence of O-linked glycosylation, some types of N-linked glycosylation can support cell surface expression of glycophorin A. The glycophorin A mutants were also used for serologic testing with defined human antisera. These studies showed that the recombinant Mi.I and Mi.II glycoproteins appropriately bound anti-Vw and anti-Hut, respectively. They also demonstrated that these antibodies recognized the amino acid polymorphisms encoded by Mi.I and Mi.II rather than cryptic peptide antigens uncovered by the lack of N- linked glycosylation.

scholarly journals Recombinant Miltenberger I and II human blood group antigens: the role of glycosylation in cell surface expression and antigenicity of glycophorin A

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1913-1920 ◽  
Author(s):  
M Ugorski ◽  
DP Blackall ◽  
P Pahlsson ◽  
SH Shakin-Eshleman ◽  
J Moore ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Blood Group ◽  
Cho Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Glycophorin A ◽  
Blood Group Antigens ◽  
Wild Type

Glycophorin A is a heavily glycosylated glycoprotein (1 N-linked and 15 O-linked oligosaccharides) and is highly expressed on the surface of human red blood cells. It is important in transfusion medicine because it carries several clinically relevant human blood group antigens. To study further the role of glycosylation in surface expression of this protein, four mutations were separately introduced into glycophorin A cDNA by site-directed mutagenesis. Each of these mutations blocks N- linked glycosylation at Asn26 of this glycoprotein by affecting the Asn- X-Ser/Thr acceptor sequence. Two of these mutations are identical to the amino acid polymorphisms found at position 28 in the Mi.I and Mi.II Miltenberger blood group antigens. The mutated recombinant glycoproteins were expressed in transfected wild-type and glycosylation- deficient Chinese hamster ovary (CHO) cells. When expressed in wild- type CHO cells and analyzed on Western blots, each of the four mutants had a faster electrophoretic mobility than wild-type glycophorin A, corresponding to a difference of approximately 4 Kd. This change is consistent with the absence of the N-linked oligosaccharide at Asn26. Each of the four mutants was highly expressed on the surface of CHO cells, confirming that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not necessary for cell surface expression of this glycoprotein. To examine the role of O- linked glycosylation in this process, the Mi.I mutant cDNA was transfected into the IdlD glycosylation-deficient CHO cell line. When the transfected IdlD cells were cultured in the presence of N- acetylgalactosamine alone, only intermediate levels of cell surface expression were seen for Mi.I mutant glycophorin A containing truncated O-linked oligosaccharides. In contrast, when cultured in the presence of galactose alone, or in the absence of both galactose and N- acetylgalactosamine, Mi.I mutant glycophorin A lacking both N-linked and O-linked oligosaccharides was not expressed at the cell surface. This extends previous results (Remaley et al, J Biol Chem 266:24176, 1991) showing that, in the absence of O-linked glycosylation, some types of N-linked glycosylation can support cell surface expression of glycophorin A. The glycophorin A mutants were also used for serologic testing with defined human antisera. These studies showed that the recombinant Mi.I and Mi.II glycoproteins appropriately bound anti-Vw and anti-Hut, respectively. They also demonstrated that these antibodies recognized the amino acid polymorphisms encoded by Mi.I and Mi.II rather than cryptic peptide antigens uncovered by the lack of N- linked glycosylation.

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