Topology of transmembrane segments 1-4 in the human chloride/bicarbonate anion exchanger 1 (AE1) by scanning N-glycosylation mutagenesis

Abstract

Human AE1 (anion exchanger 1), or Band 3, is an abundant membrane glycoprotein found in the plasma membrane of erythrocytes. The physiological role of the protein is to carry out chloride/bicarbonate exchange across the plasma membrane, a process that increases the carbon-dioxide-carrying capacity of blood. To study the topology of TMs (transmembrane segments) 1-4, a series of scanning N-glycosylation mutants were created spanning the region from EC (extracellular loop) 1 to EC2 in full-length AE1. These constructs were expressed in HEK-293 (human embryonic kidney) cells, and their N-glycosylation efficiencies were determined. Unexpectedly, positions within putative TMs 2 and 3 could be efficiently glycosylated. In contrast, the same positions were very poorly glycosylated when present in mutant AE1 with the SAO (Southeast Asian ovalocytosis) deletion (DeltaA400-A408) in TM1. These results suggest that the TM2-3 region of AE1 may become transiently exposed to the endoplasmic reticulum lumen during biosynthesis, and that there is a competition between proper folding of the region into the membrane and N-glycosylation at introduced sites. The SAO deletion disrupts the proper integration of TMs 1-2, probably leaving the region exposed to the cytosol. As a result, engineered N-glycosylation acceptor sites in TM2-3 could not be utilized by the oligosaccharyltransferase in this mutant form of AE1. The properties of TM2-3 suggest that these segments form a re-entrant loop in human AE1.

Figure 1. Folding model of human AE1 and N-glycosylation mutants

Human AE1 consists of an N-terminal cytosolic domain and a membrane domain (12-TM model shown here). The endogenous N-glycosylation acceptor site is located in EC4, at Asn⁶⁴². Deletion of residues 400–408 in TM1 results in SAO. The N-glycosylation mutants used in the present study were created in a N642D background; individual N-glycosylation acceptor sites (N-X-S/T) were made in the region encompassing EC1–EC2.

Figure 2. Scanning N-glycosylation mapping results for AE1 constructs

HEK-293 cells transiently transfected with N-glycosylation mutants were labelled with [³⁵S]methionine for 2 h. This represents proteins localized mainly to the ER. The cells were then lysed, and the cell lysate was subjected to immunoprecipitation with anti-AE1 antibody. The samples were separated by lectin-shift SDS/PAGE and analysed by autoradiography. (A) AE1 constructs with TM2/3 glycosylation acceptor site mutation. (B) AE1 constructs with TM2/3 glycosylation acceptor site and I449T or V470T mutations. N-Glycosylation efficiencies were calculated by dividing the upper band (glycosylated) intensity by the total intensity of upper and lower (non-glycosylated) bands, and listed beneath each lane (±S.D.). ●, Glycosylated proteins; ○, non-glycosylated proteins.

Figure 3. N-Glycosylation efficiencies of AE1 constructs

The N-glycosylation efficiencies of various AE1 constructs expressed in HEK-293 cells are shown (±S.D.).

Figure 4. Scanning N-glycosylation mapping results for AE1 SAO constructs

Transiently transfected HEK-293 cells were radiolabelled with [³⁵S]methionine and lysed, and AE1 was immunoprecipitated. The samples were analysed by lectin-shift SDS/PAGE and autoradiography. N-Glycosylation efficiencies were calculated by dividing the upper band (glycosylated) intensity by the total intensity of upper and lower (non-glycosylated) bands, and listed beneath each lane (±S.D.). ●, Glycosylated proteins; ○, non-glycosylated proteins.

Figure 5. Refined model of the topology of TM1–4 in human AE1

The first four TMs of human AE1 are shown, with residues involved in SAO deletion. The results of scanning N-glycosylation mapping are summarized. Position 429 cannot be N-glycosylated, as shown previously [20]. Residues are shaded according to their N-glycosylation efficiencies. Using the ‘12+14 rule’ places the lumenal end of TM1 at Phe⁴²³ and the beginning of TM4 at Ile⁴⁸⁷.