Cloning and characterization of band 3, the human erythrocyte anion-exchange protein (AE1).

Abstract

The human erythrocyte anion-exchange protein (band 3 or AE1) was cloned from a fetal liver cDNA library. Three overlapping clones, encompassing 3637 nucleotides, were analyzed in detail. These encode a 911-amino acid protein (Mr 101,791) and detect a single 4.7-kilobase species in human reticulocyte RNA. The corresponding gene is located on chromosome 17. The protein is similar in structure to other anion exchangers and is divided into three regions: a hydrophilic, cytoplasmic domain that interacts with a variety of membrane and cytoplasmic proteins (residues 1-403); a hydrophobic, transmembrane domain that forms the anion antiporter (residues 404-882); and an acidic, C-terminal domain of unknown function (residues 883-911). The N-terminal domain contains several conserved sections (e.g., residues 57-86, 102-164, 219-347, and 375-403) cDNA Sequencing. Selected clones were sequenced either by "shotgun" (12) or exonuclease III/Sl nuclease deletion (13) methods. The deletion method was modified so that deletion plasmids of the desired size were isolated by electrophoresis in low-melting-point agarose, religated in agarose, and used to transform Escherichia coli NM522. Doublestranded plasmid templates were prepared by the method of Kraft et al. (14). In both cases sequencing reactions were performed by the dideoxy method as modified by Biggin et al. (15) and Bankier (16), using, as needed, Klenow or modified T7 DNA polymerase (Sequenase; United States Biochemical) for elongation reactions. 7-Deaza-GTP was substituted for GTP as needed for refractory sequences. Sequence data were compiled and analyzed using the University of Wisconsin Genetics Computer Group (UWGCG) programs.Chromosomal Location of Band 3 Gene. The EcoRV-Xba I fragment of pHB3-22 was labeled with [a-32P]dCTP (10) and hybridized to human chromosomes separated by fluorescence-activated cell sorting (17).Band 3 (anion exchanger 1 or AE1) ¶ is the major protein of the human erythrocyte membrane and one of the best studied of all membrane proteins. It is a chimeric molecule composed of two dissimilar and functionally distinct domains (1). The N-terminal portion forms a 43-kDa water-soluble cytoplasmic domain that has multiple binding functions, while the C-terminal transmembrane region is the physiologically important anion exchanger. Since the initial publication of mouse erythrocyte band 3 (AEl) in 1985 (1), the sequence of chicken erythrocyte AEl (2, 3), the sequences of truncated renal forms of mouse and rat AEl (4, 5), and the sequences of human (7) and mouse (8) band 3-related protein (AE2) have appeared. However, almost all functional and topographical studies of band 3 have been performed with proteins from human erythrocytes. In this paper we report the cloning and sequencing of a cDNA for human erythrocyte band 3.11Following completion of this work, Tanner et al. reported similar studies (6).MATERIALS AND METHODS cDNA Cloning. 32P-labeled cDNA probes were prepared by the random priming method (10) and used to screen human fetal liver...

Induction of anion exchanger-1 translation and its opposite roles in the carcinogenesis of gastric cancer cells and differentiation of K562 cells

“…The slc4a1 gene belongs to the family of anion exchangers encoding anion exchanger-1 (AE1, also called band-3), an erythroid-specific integral membrane protein (eAE1), which serves the dual roles of Cl À / HCO À 3 exchange and cytoskeletal anchorage to the red cell membrane (Lux et al, 1989). The gene has been mapped to chromosome 17q21-q22 and consists of 20 exons separated by 19 introns, spanning 17 kb in all (Showe et al, 1987;Sahr et al, 1994;Schofield et al, 1994).…”

In situ detection of AE2 anion-exchanger mRNA in the human liver

“…AE1 protein (also called ªband 3º) is the most abundant protein in the membranes of red blood cells (Fairbanks et al 1971). Human AE1 protein has been purified and characterized (Lukakovic et al 1981), and its cDNA has been cloned and sequenced as well (Tanner et al 1988;Lux et al 1989). AE2 and AE3 transcripts (and corresponding cDNAs) were identified by cross hybridization with AE1 cDNA probes (Demuth et al 1986;Alper et al 1988;Kopito et al 1989;Kudrycki et al 1990;Lindsey et al 1990).…”

Structure of Bor1 supports an elevator transport mechanism for SLC4 anion exchangers

“…In lungs, the partial pressure of CO 2 is lower and the process is reversed, thus driving cellular respiration. Decades of biochemical characterization have provided a wealth of information about Band 3 topology and multimerization (11)(12)(13)(14)(15), as well as the identification of amino acid residues likely involved in substrate transport (16)(17)(18)(19)(20)(21)(22)(23). However, our understanding of transport by SLC4 anion exchangers remains limited by a paucity of structural data, for the SLC4 family in general and Bor1 in particular.…”