Monoclonal antibodies to the membrane domain of the human erythrocyte anion transport protein. Localization of the C-terminus of the protein to the cytoplasmic side of the red cell membrane and distribution of the protein in some human tissues

Abstract

(1) We have prepared murine monoclonal antibodies to the membrane domain of the human erythrocyte anion transport protein (band 3). (2) All of these antibodies react with regions of the protein located at the cytoplasmic surface of the red cell. (3) One of the antibodies reacts with an epitope present on a cytoplasmic loop of the protein located between the C-terminus and a point 168 amino acids from the C-terminus. The other antibodies recognize different epitopes on the C-terminal tail of the protein and the sequences likely to be involved in these epitopes are defined. (4) Our results show that the C-terminus of the red-cell anion transport protein is located on the cytoplasmic side of the red-cell membrane. (5) None of the antibodies inhibited sulphate exchange transport when introduced into resealed red-cell membranes; however, the bivalent form of one of the antibodies reduced the inhibitory potency of 4-acetamido-4'-isothiocyanatostilbene disulphonate on sulphate exchange transport in resealed erythrocyte membranes. (6) Immunostaining of human kidney sections with the antibodies showed strong staining of the basolateral membrane of some but not all of the epithelial cells of distal tubules and the initial connecting segment of collecting tubules. With human liver, only the haematopoeitic cells of fetal liver reacted with all the antibodies.

Novel Topology in C-terminal Region of the Human Plasma Membrane Anion Exchanger, AE1

“…This is consistent with results from epitope mapping (20) and carboxypeptidase Y digestion (34). The observed increase of biotinylation from V872C to C885 provides a high resolution determination of the last TM of AE1, which ends at Arg 871 .…”

“…Some mutants outside the C-terminal region were used as controls for the labeling protocol. Y555C was used as the extracellular surface control, since it is adjacent to the two chymotryptic cleavage sites in intact erythrocytes (33); K892C was used as intracellular surface control because it is located in the C-terminal tail of the protein, which previously mapped to the cytoplasm (20,34). Cys 201 , an endogenous cysteine residue located in the N-terminal cytoplasmic domain of AE1, was also cloned into AE1C…”

“…Hydropathy analysis of the region yields ambiguous results, with unclear transmembrane segments (10). Topology models have been proposed for this region based on experimental evidence including, epitope mapping (20), N-glycosylation-scanning mutagenesis (21,22), protease accessibility (23), and cysteine-scanning mutagenesis (24,25). In this report, we examined the topology of the C-terminal region of AE1, using cysteine-scanning mutagenesis and sulfhydryl-specific chemical labeling.…”

Investigating the key membrane protein changes during in vitro erythropoiesis of protein 4.2 (-) cells (mutations Chartres 1 and 2)

“…26,31 Immunoprecipitations and total cell lysates Cells were lysed as described 32 and quantified by Lowry assay (Biorad, Hercules, CA, USA). For immunoprecipitations 1.5×10 7 erythroblasts or 5×10 7 erythrocytes were pre-cleared (1h) with unbound protein-G-beads.…”

A Transport Metabolon

“…The membrane domain spans the lipid bilayer 12-14 times (6) and is responsible for Cl Ϫ /HCO 3 Ϫ exchange activity (7). The protein terminates with a cytoplasmic 33-amino acid carboxyl-terminal domain (8,9). A truncated form of human AE1 beginning at methionine 66 is found in kidney (10).…”