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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), some of which may contribute to binding sites for ankyrin, protein 4.1, or protein 4.2. The membrane domain is highly conserved with the exception of a single segment (residues 543-567) that contains several sites for cleavage of the protein by extracellular proteases. Based on hydropathy analyses and the wealth of available topographical and functional data, a model is proposed in which the protein crosses the membrane 14 times.

SUBMITTER: Lux SE

PROVIDER: S-EPMC298439 | biostudies-other | 1989 Dec

REPOSITORIES: biostudies-other

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

Lux S E SE John K M KM Kopito R R RR Lodish H F HF

Proceedings of the National Academy of Sciences of the United States of America 19891201 23

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 intera ...[more]

PMID: 2594752

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Project description:The human erythrocyte anion exchanger (AE1, Band 3) contains up to 14 transmembrane segments, with a single site of N-glycosylation at Asn642 in extracellular (EC) loop 4. Scanning and insertional N-glycosylation mutagenesis were used to determine the folding pattern of AE1 in the membrane. Full-length AE1, when expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells, retained a high-mannose oligosaccharide structure. Scanning N-glycosylation mutagenesis of EC loop 4 showed that N-glycosylation acceptor sites (Asn-Xaa-Ser/Thr) spaced 12 residues from the ends of adjacent transmembrane segments could be N-glycosylated. An acceptor site introduced at position 743 in intracellular (IC) loop 5 that could be N-glycosylated in a cell-free translation system was not N-glycosylated in transfected cells. Mutations designed to disrupt the folding of this loop enhanced the level of N-glycosylation at Asn743 in vitro. The results suggest that this loop might be transiently exposed to the lumen of the endoplasmic reticulum during biosynthesis but normally folds rapidly, precluding N-glycosylation. EC loop 4 insertions into positions 428, 484, 754 and 854 in EC loops 1, 2, 6 and 7 respectively were efficiently N-glycosylated, showing that these regions were extracellular. EC loop 4 insertions into positions 731 or 785 were poorly N-glycosylated, which was inconsistent with an extracellular disposition for these regions of AE1. Insertion of EC loop 4 into positions 599 and 820 in IC loops 3 and 6 respectively were not N-glycosylated in cells, which was consistent with a cytosolic disposition for these loops. Inhibitor-affinity chromatography with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate (SITS)-Affi-Gel was used to assess whether the AE1 mutants were in a native state. Mutants with insertions at positions 428, 484, 599, 731 and 785 showed impaired inhibitor binding, whereas insertions at positions 754, 820 and 854 retained binding. The results indicate that the folding of the C-terminal region of AE1 is more complex than originally proposed and that this region of the transporter might have a dynamic aspect.

Project description:The N-terminal 72 residues of an integral membrane fragment, P5, of the human erythrocyte anion-transport protein, which is known to be directly involved in the anion-exchange process, was shown to have the following amino acid sequence: Met-Val-Pro-Lys-Pro-Gln-Gly-Pro-Leu-Pro-Asn-Thr-Ala-Leu-Leu-Ser-Leu-Val-Leu-Met -Ala-Gly-Thr-Phe-Phe-Phe-Ala-Met-Met-Leu-Arg-Lys-Phe-Lys-Asn-Ser-Ser-Tyr-Phe-Pro-Gly-Lys-Leu-Arg-Arg-Val-Ile-Gly-Asp-Phe-Gly-Val-Pro-Ile-Ser-Ile-Leu-Ile-Met-Val-Leu-Val-Asp-Phe-Phe-Ile-Gln-Asp-Thr-Tyr-Thr-Gln- The structure of this fragment was analysed, with account being taken of the constraints that apply to the folding of integral membrane proteins and the topographical locations of various sites in the sequence. It was concluded that this sequence forms two transmembrane alpha-helices. These are probably part of a cluster of amphipathic transmembrane alpha-helices, which could comprise that part of the protein responsible for transport activity. The presently available evidence relating to the anion-exchange process was considered with the structural features noted in this study and a possible molecular mechanism is proposed. In this model the rearrangement of a network of intramembranous charged pairs mediates the translocation of an anion between anion-binding regions at each surface of the membrane, which are composed of clusters of positively charged amino acids. This model imposes a sequential exchange mechanism on the system. Supplementary material, including Tables and Figures describing the compositions of peptides determined by amino acid analysis and sequence studies, quantitative and qualitative data that provide a residue-by-residue justification for the sequence assignment and a description of modifications to and use of the solid-phase sequencer has been deposited as Supplementary Publication SUP 50123 (12 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained as indicated in Biochem. J. (1983) 209, 5.

Dataset Information

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

Publications

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

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