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.
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.
Beckmann
R
R
Department of Biochemistry, University of Bristol, UK; and the International Blood Group Reference Laboratory, Bristol, UK.
Smythe
J S
JS
Anstee
D J
DJ
Tanner
M J
MJ
D001457:Anion Exchange Protein 1, Erythrocyte / Q000096:biosynthesis* / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D020014:K562 Cells; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D012204:Rh-Hr Blood-Group System / Q000378:metabolism*; D014162:Transfection
D001457:Anion Exchange Protein 1, Erythrocyte; D018076:DNA, Complementary; D012204:Rh-Hr Blood-Group System