Coexpression of band 3 mutants and Rh polypeptides: differential effects of band 3 on the expression of the Rh complex containing D polypeptide and the Rh complex containing CcEe polypeptide.
K562 cells were stably transfected with cDNAs encoding the band 3 found in Southeast Asian ovalocytosis (B3SAO, deletion of residues 400-408), band 3 with a transport-inactivating E681Q point mutation (B3EQ), or normal band 3 (B3). Flow cytometric analysis and quantitative immunoblotting revealed that B3SAO expressed alone was translocated to the plasma membrane, at levels similar to B3 or B3EQ. Nine monoclonal antibodies that reacted with extracellular loops of B3 also reacted with B3SAO, although the affinity of most antibodies for the mutant protein was reduced. Both known Wr(b) epitopes were expressed on K562/B3SAO cells, demonstrating that B3SAO interacts with glycophorin A. The growth rates of K562 clones expressing equivalent amounts of B3 and B3EQ were the same, suggesting that the potentially toxic transport function of band 3 may be regulated in K562 cells. The band 3-mediated enhancement of Rh antigen reactivity and the depression of Rh epitopes on SAO erythrocytes were investigated by comparing the coexpression of B3, B3SAO, or B3EQ in K562 clones expressing exogenous RhcE or RhD polypeptides. The results are consistent with an interaction between band 3 and the Rh polypeptide-Rh glycoprotein (RhAG) complex, which may enhance translocation of the complex or affect its conformation in the plasma membrane. The data suggest that the interaction between band 3 and the RhD-RhAG complex is weaker than it is between band 3 and the RhCcEe-RhAG complex.
Beckmann
R
R
Department of Biochemistry, University of Bristol, and the Bristol Institute for Transfusion Sciences, United Kingdom.
Smythe
J S
JS
Anstee
D J
DJ
Tanner
M J
MJ
D001457:Anion Exchange Protein 1, Erythrocyte / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D001798:Blood Proteins; D015153:Blotting, Western; D002455:Cell Division; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D020869:Gene Expression Profiling; D015973:Gene Expression Regulation, Leukemic; D006021:Glycophorins / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells / Q000378:metabolism*; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012204:Rh-Hr Blood-Group System / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection; D014410:Tumor Stem Cell Assay
D001457:Anion Exchange Protein 1, Erythrocyte; D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D018076:DNA, Complementary; D000939:Epitopes; D006021:Glycophorins; D006023:Glycoproteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C498523:RHAG protein, human; C066468:RHCE protein, human; D011993:Recombinant Fusion Proteins; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen