Analysis of complementary DNA for human erythroid ankyrin indicates that the mature protein contains 1,880 amino acids comprising an N-terminal domain binding integral membrane proteins and tubulin, a central domain binding spectrin and vimentin, and an acidic C-terminal 'regulatory' domain containing an alternatively spliced sequence missing from ankyrin variant 2.2. The N-terminal domain is almost entirely composed of 22 tandem 33-amino-acid repeats. Similar repeats are found in yeast and invertebrate proteins involved in cell-cycle control and tissue differentiation.

The red blood cell (RBC) membrane protein AE1 provides high affinity binding sites for the membrane skeleton, a structure critical to RBC integrity. AE1 biosynthesis is postulated to be required for terminal erythropoiesis and membrane skeleton assembly. We used targeted mutagenesis to assess AE1 function in vivo. RBCs lacking AE1 spontaneously shed membrane vesicles and tubules, leading to severe spherocytosis and hemolysis, but the levels of the major skeleton components, the synthesis of spectrin in mutant erythroblasts, and skeletal architecture are normal or nearly normal. The results indicate that AE1 does not regulate RBC membrane skeleton assembly in vivo but is essential for membrane stability. We postulate that stabilization is achieved through AE1-lipid interactions and that loss of these interactions is a key pathogenic event in hereditary spherocytosis.

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...

Abstract. We cloned a novel ankyfin, Ank3, from mouse kidney cDNA. The full-length transcript is predicted to encode a 214-kD protein containing an 89 kD, NH2 terminal "repeat" domain; a 65 kD, central "spectrin-binding" domain; and a 56 kD, COOH-terminal

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Spectrin is an important structural component of the plasma membrane skeleton. Heretoforeunidentified isoforms of spectrin also associate with Golgi and other organelles. We have discovered another member of the ␤-spectrin gene family by homology searches of the GenBank databases and by 5 rapid amplification of cDNA ends of human brain cDNAs. Collectively, 7,938 nucleotides of contiguous clones are predicted to encode a 271,294-Da protein, called ␤III spectrin, with conserved actin-, protein 4.1-, and ankyrin-binding domains, membrane association domains 1 and 2, a spectrin dimer self-association site, and a pleckstrinhomology domain. ␤III spectrin transcripts are concentrated in the brain and present in the kidneys, liver, and testes and the prostate, pituitary, adrenal, and salivary glands. All of the tested tissues contain major 9.0-kb and minor 11.3-kb transcripts. The human ␤III spectrin gene (SPTBN2) maps to chromosome 11q13 and the mouse gene (Spnb3) maps to a syntenic region close to the centromere on chromosome 19. Indirect immunof luorescence studies of cultured cells using antisera specific to human ␤III spectrin reveal a Golgiassociated and punctate cytoplasmic vesicle-like distribution, suggesting that ␤III spectrin associates with intracellular organelles. This distribution overlaps that of several Golgi and vesicle markers, including mannosidase II, p58, transGolgi network (TGN)38, and ␤-COP and is distinct from the endoplasmic reticulum markers calnexin and Bip. Liver Golgi membranes and other vesicular compartment markers cosediment in vitro with ␤III spectrin. ␤III spectrin thus constitutes a major component of the Golgi and vesicular membrane skeletons.

Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic anaemias. HS red cells from both autosound dominant and recessive variants are spectrin-deficient, which correlates with the severity of the disease. Some patients with recessive HS have a mutation in the spectrin alpha-2 domain (S.L.M. et al., unpublished observations), and a few dominant HS patients have an unstable beta-spectrin that is easily oxidized, which damages the protein 4.1 binding site and weakens spectrin-actin interactions. In most patients, however, the cause of spectrin deficiency is unknown. The alpha- and beta-spectrin loci are on chromosomes 1 and 14 respectively. The only other genetic locus for HS is SPH2, on the short arm of chromosome 8 (8p11). This does not correspond to any of the known loci of genes for red cell membrane proteins including protein 4.1 (1p36.2-p34), the anion exchange protein (AE1, band 3; 17q21-qter), glycophorin C (2q14-q21), and beta-actin (7pter-q22). Human erythrocyte ankyrin, which links beta-spectrin to the anion exchange protein, has recently been cloned. We now show that the ankyrin gene maps to chromosome 8p11.2, and that one copy is missing from DNA of two unrelated children with severe HS and heterozygous deletions of chromosome 8 (del(8)(p11-p21.1)). Affected red cells are also ankyrin-deficient. The data suggest that defects or deficiency or ankyrin are responsible for HS at the SPH2 locus.

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