Search PubMed⌕ Search

Biomedical subjects

S E Lux

Publications and source records attributed to S E Lux.

At least 37 records · Page 2Linked to original sources
PubMed1996

Isolated beta-globin chains reproduce, in normal red cell membranes, the defective binding of spectrin to alpha-thalassaemic membranes.

Alpha-thalassaemic erythrocytes develop a specific membrane skeletal defect that is manifest as a loss of normal spectrin-binding sites on the inner surface of the thalassaemic membranes. To test whether this lesion could be caused by the excess free beta-globin chains that accumulate in alpha-thalassaemic red cells, we incubated normal red cell membranes with native, haem-containing alpha or beta globin chains or with haemoglobin A. Spectrin-depleted inside-out membrane vesicles (IOVs) derived from membranes incubated with beta-globin chains bound only 9 +/- 3% as much spectrin as IOVs from control membranes incubated with bovine serum albumin. In contrast. IOVs from membranes incubated with alpha-globin chains or haemoglobin A were nearly normal (79 +/- 3% and 86 +/- 5% of controls, respectively). This differential effect of globin chains was not seen when membranes were first transformed into spectrin-depleted IOVs and then incubated with the isolated globin chains. Under these conditions, both alpha and beta globin chains reduced the spectrin-binding capacity of the IOVs by approximately 45% (alpha 46 +/- 7%, beta 43 +/- 6%) whereas haemoglobin A had no effect. Unlike IOVs, spectrin isolated from membranes exposed to alpha or beta globin chains bound normally to IOVs and to actin (in the presence of protein 4.1). These studies show that isolated beta-globin chains (but not alpha-globin chains) can produce a spectrin-binding defect in normal red cell membranes similar to that seen in alpha thalassaemia. The existence of similar defects in the membrane skeletons of red cells from other diseases with unstable beta globins suggests a common pathophysiology for the premature destruction of these cells.

Ankyrins↗
PubMed1995

Increased cation permeability in mutant mouse red blood cells with defective membrane skeletons.

Cellular cation homeostasis in mouse erythrocytes with defective membrane skeletons was examined in three mouse mutants, hemolytic anemia (sphha/sphha), spherocytosis (sph/sph), and normoblastosis (nb/nb), and compared with reticulocytes produced by repetitive bleeding of congenic normal mice. To assess reticulocyte maturity, nucleic acid and transferrin receptor contents were measured by fluorescence flow cytometry; mutant cells were somewhat more mature than normal reticulocytes by these criteria. Red blood cell (RBC) sodium contents (Nac+) in homozygous sphha/sphha, sph/sph, and nb/nb animals were 30.1 +/- 0.9, 28.9 +/- 0.3, and 26.9 +/- 1.5 mmol/L cell, respectively, whereas cellular potassium (Kc+) was 102 +/- 2.6, 101 +/- 7.8, and 97.4 +/- 3.0. Nac+ and Kc+ in normal reticulocyte preparations were 11.3 +/- 0.7 and 123 +/- 10, respectively. Net Na+ and K+ fluxes in the presence of ouabain were markedly increased in mutant RBCs. Sodium uptake was 14.8 +/- 1.6, 15.4 +/- 3.3, and 14.7 +/- 3.1 mmol/L cell/h in sphha/sphha, sph/sph, and nb/nb mutants, respectively, whereas K+ loss was 17.0 +/- 4.0, 15.0 +/- 3.8, and 14.1 +/- 2.6. Normal mouse reticulocytes gained Na+ at a rate of 3.9 +/- 1.0 mmol/L cell/h and lost K+ at 6.0 +/- 2.1, rates indistinguishable from those in mature mouse RBCs. Potassium loss from sphha/sphha and nb/nb cells was not dependent on the presence of a Na+ gradient, and net cation movements were insensitive to bumetanide (sphha/sphha and nb/nb RBCs) and to chloride replacement with sulfamate (nb/nb cells). We conclude that mutant mouse RBCs with dysfunctional membrane skeletons have increased passive permeability to monovalent cations. These findings support a role of the membrane skeleton in the maintenance of the membrane permeability barrier and suggest that the abnormal permeability associated with human hereditary spherocytosis and elliptocytosis may be a consequence of the membrane skeleton defects reported in these disorders.

Anemia, Hemolytic↗
PubMed1995

Characterization of the binary interaction between human erythrocyte protein 4.1 and actin.

The binary interaction between human erythrocyte protein 4.1 and rabbit skeletal muscle F-actin was examined by rapid pelleting of the binary complexes. The binding curves show that the reaction was saturable at approximately one protein 4.1 molecule/actin monomer. The reaction was highly co-operative, displaying a Hill coefficient close to 2. Using a fixed concentration of radiolabelled protein 4.1, and varying the concentration of F-actin, the apparent molar association constant, Ka, was observed to range from 5 x 10(4) M-1 to > 10(6) M-1. The binary interaction between erythrocyte spectrin and actin was also observed to be co-operative under the same conditions. The rate of reaction between protein 4.1 and actin was temperature sensitive in a manner consistent with a high energy of activation. The pelleting assay also showed that the concentration of actin was reduced in the supernatant in the presence of protein 4.1 compared with actin alone, indicating that the critical concentration of actin was lowered in the presence of protein 4.1. Polyvalent anions disrupted the binary interaction between F-actin and protein 4.1, the disruption being consistent with the number of negative charges on these anions at pH 7.5. We postulate that the co-operativity of the binding of protein 4.1 to actin results from a protein 4.1 molecule binding to a single monomer within the filament structure which then promotes conformational changes allowing further protein 4.1 binding. The demonstration of a specific binary association between protein 4.1 and actin suggests that this interaction contributes significantly to the stabilization of the spectrin-actin-protein-4.1 ternary complex.

Actins↗
PubMed1995

Ank3 (epithelial ankyrin), a widely distributed new member of the ankyrin gene family and the major ankyrin in kidney, is expressed in alternatively spliced forms, including forms that lack the repeat domain.

We cloned a novel ankyrin, 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 "regulatory" domain. The Ank3 gene maps to mouse Chromosome 10, approximately 36 cM from the centromere, a locus distinct from Ank1 and Ank2. Ank3 is the major kidney ankyrin. Multiple transcripts of approximately 7.5, 6.9, 6.3, 5.7, 5.1, and 4.6 kb are highly expressed in kidney where Ank1 and Ank2 mRNAs are barely detectable. The smaller mRNAs (< or = 6.3 kb) lack the entire repeat domain. These transcripts have a unique 5'untranslated region and NH2-terminal sequence and encode a predicted protein of 121 kD. Two small sequences of 21 and 18 amino acids are alternatively spliced at the junction of the repeat and spectrin-binding domains in the larger (> or = 6.9 kb) RNAs. Alternative splicing of a 588 bp sequence (corresponding to a 21.5-kD acidic amino acid sequence) within the regulatory domain also occurs. Ank3 is much more widely expressed than previously described ankyrins. By Northern hybridization or immunocytochemistry, it is present in most epithelial cells, in neuronal axons, in muscle cells, and in megakaryocytes/platelets, macrophages, and the interstitial cells of Leydig (testis). On immunoblots, an antibody raised to a unique regions of the regulatory domain detects multiple Ank3 isoforms in the kidney (215, 200, 170, 120, 105 kD) and in other tissues. The 215/200 kD and 120/105-kD kidney proteins are close to the sizes predicted for the 7.5/6.9- and 6.3/5.7-kb RNAs (with/without the 588-bp acidic insert). Interestingly, it appears that Ank3 exhibits a polarized distribution only in tissues that express the approximately 7.0-kb isoforms, the only isoforms in the kidney that contain the repeat domain. In tissues where smaller transcripts (< or = 6.3 kb) are expressed. Ank3 is diffusely distributed in some or all cells and may be associated with cytoplasmic structures. We conclude that Ank3 is a broadly distributed epithelial ankyrin and is the major ankyrin in the kidney and other tissues, where it plays an important role in the polarized distribution of many integral membrane proteins.

Alternative Splicing↗
PubMed1995

Differential expression of Na(+)-K(+)-ATPase, ankyrin, fodrin, and E-cadherin along the kidney nephron.

Ionic homeostasis in vertebrates is maintained by epithelial cells that line kidney nephrons. Transport of ions and solutes is coupled to Na+ reabsorption from the ultrafiltrate and requires specific subcellular distribution and activity of Na(+)-K(+)-ATPase along the nephron. Studies using cell culture models of renal epithelia indicate that the subcellular distribution of Na(+)-K(+)-ATPase is regulated by interactions with the submembrane cytoskeleton and E-cadherin-mediated adherens junctions. We have now examined the relevance of these in vitro observations to the subcellular organization of these proteins in different nephron segments of the adult mouse kidney using immunofluorescence microscopy. Our results demonstrate that segmental and subcellular distributions of Na(+)-K(+)-ATPase and the membrane-cytoskeletal proteins, ankyrin and fodrin, vary in parallel along the nephron and do not parallel variations in expression of the tight junction protein ZO-1 or E-cadherin. These data indicate that a mechanism for restricting Na(+)-K(+)-ATPase subcellular distributions through interactions with the membrane cytoskeleton is likely to be relevant in vivo.

Animals↗
PubMed1993

Combined spectrin and ankyrin deficiency is common in autosomal dominant hereditary spherocytosis.

The common autosomal dominant form of hereditary spherocytosis (HS) has been genetically linked to defects of the erythroid ankyrin gene in a few families; however, the frequency of ankyrin deficiency and its relationship to red blood cell (RBC) spectrin content are unknown. To test these questions, we measured RBC spectrin and ankyrin by radioimmunoassay in 39 patients from 20 families with dominant HS. Normal RBCs contained 242,000 +/- 20,500 spectrin heterodimers and 124,500 +/- 11,000 ankyrins per cell. In dominant HS, RBC spectrin and ankyrin ranged from about 40% to 100% of normal and were continuously distributed. Measurements in the same patient on different occasions were reproducible (+/- 5% to 10%) and RBCs from affected members of a kindred contained similar amounts of spectrin and ankyrin (+/- 3% to 4%). Spectrin and ankyrin levels were almost always less than the assay controls, but were less than the normal range in only 75% and 80% of kindreds, respectively. Remarkably, the degree of RBC spectrin and ankyrin deficiency was very similar in 19 of 20 HS kindreds. One otherwise typical family differed, with marked ankyrin deficiency (45% of control) and a relatively mild spectrin deficit (81%). We conclude that most patients with dominant HS have combined ankyrin and spectrin deficiency and that the two proteins are usually about equally deficient, suggesting that defects in ankyrin expression, ankyrin stability, or ankyrin band 3 (AE1) interactions may be common in dominant HS.

Ankyrins↗
PubMed1993

A highly conserved region of human erythrocyte ankyrin contains the capacity to bind spectrin.

Ankyrin has a spectrin-binding region within a central 62-kDa chymotryptic peptide. We examined the spectrin binding ability of a series of smaller ankyrin fragments and recombinant peptides within the 62-kDa domain using a ligand blot assay. The smallest proteolytic fragment that bound was a 12-kDa tryptic peptide starting at amino acid 1068. Peptides containing this region expressed as glutathione S-transferase fusion products also bound spectrin and suggested that residues 1101-1192 were important. In contrast, a fusion protein containing residues 826-898 did not bind spectrin, a surprising finding since this region is known to influence binding affinity. Proteins that bound spectrin on ligand blots also competed for binding in solution, but did so with one-tenth the affinity of the native peptide. Comparing the 62-kDa domains of erythrocyte and brain ankyrins (species that bind spectrin but with 10-fold differences in affinity), the NH2-terminal regions are 0-40% identical, while the regions (1136-1160) common to all binding peptides are 80-90% identical. We hypothesize that the highly conserved region contains an important spectrin-binding site, while the poorly conserved region controls the binding affinity. We speculate that this unique NH2-terminal region is what gives different members of the ankyrin family their signature set of affinities, and accordingly their distinctive cellular localization.

Amino Acid Sequence↗
PubMed1993

Complex patterns of sequence variation and multiple 5' and 3' ends are found among transcripts of the erythroid ankyrin gene.

The structural protein ankyrin functions in red blood cells to link the spectrin-based membrane skeleton to the plasma membrane. Ankyrin proteins are now known to occur in most cell types, and two distinct ankyrin genes have been identified (erythroid (Ank-1) and brain (Ank-2)). We have characterized transcripts of the mouse erythroid ankyrin gene by cDNA cloning and DNA sequencing. Ank-1 transcripts of 7.5 and 9.0 kilobases are found in erythroid tissues, and a 9.0-kilobase transcript is found in cerebellum. RNA hybridization blot analysis of 13 additional mouse tissues has detected four novel Ank-1 transcripts (5.0, 3.5, 2.0, and 1.6 kilobases in size). Sequencing of Ank-1 cDNA clones isolated from mouse reticulocyte, spleen, and cerebellar libraries has identified (i) multiple 5' ends that indicate possible multiple promoters; (ii) alternative polyadenylation sites that probably account for the 7.5- and 9.0-kilobase size difference; (iii) a variety of small insertions and deletions that could produce transcripts (and ultimately proteins) of nearly identical size, but different functions; and (iv) clones with large deletions of coding sequence that account for the smaller transcripts seen in spleen, skeletal muscle, and heart.

Amino Acid Sequence↗
PubMed1993

Mouse microcytic anaemia caused by a defect in the gene encoding the globin enhancer-binding protein NF-E2.

The nuclear DNA-binding protein NF-E2 is thought to mediate the powerful erythroid enhancer activity of the alpha- and beta-globin locus control regions and participates in the control of genes encoding two enzymes of haem biosynthesis (porphobilinogen deaminase and ferrochelatase). The major component of NF-E2 is a 45K polypeptide (designated p45 NF-E2) that belongs to the basic region-leucine zipper family of transcription factors. This subunit of NF-E2 is specifically expressed in haematopoietic progenitor cells and differentiated cells of the erythroid, megakaryocyte and mast cell lineages. The gene encoding p45 NF-E2 (murine gene Nfe2) has been mapped to mouse chromosome 15 near the mutation microcytosis (mk). Homozygous mk mice have severe hypochromic microcytic anaemia as a result of decreased globin synthesis and defects in intestinal and erythroid iron absorption. Here we investigate whether the mk mutation lies within Nfe2 by characterizing the p45 NF-E2 gene and determining its DNA sequence in wild-type and mk alleles. The mk allele carries a missense mutation that causes substitution of valine by alanine at amino acid 173 of the p45 NF-E2 protein. Expression of p45 NF-E2 messenger RNA was detected in erythroid tissues of normal mice and in the duodenum of normal and severely anaemic beta-thalassaemic (Hbbd-th3/Hbbd-th3) mice. We propose that the mk mutation results in an impaired form of NF-E2 which fails to regulate both globin production and iron metabolism properly.

Amino Acid Sequence↗
PubMed1993

Beta spectrin kissimmee: a spectrin variant associated with autosomal dominant hereditary spherocytosis and defective binding to protein 4.1.

We analyzed the DNA sequence of the cDNA encoding the NH2 terminal region of beta spectrin from members of a kindred with autosomal dominant hereditary spherocytosis associated with defective protein 4.1 binding. We found a point mutation at codon 202 within the 272 amino acid NH2-terminal region of beta spectrin. TGG was changed to CGG, resulting in the replacement of tryptophan by arginine. The base change eliminates a normally occurring PvuII restriction site and creates a new MspI site. This finding enabled rapid detection or exclusion of the mutation at the DNA level among the family members, including one member for whom this analysis was performed prenatally. The mutation was found only in the affected family members and occurred as a de novo mutation in the proband. It has not been found in 20 other kindreds. The recombinant peptide derived from the normal cDNA retains the capacity to sediment with protein 4.1 and F-actin. The mutant peptide spontaneously degrades. This variant represents both the first point mutation and the first beta spectrin mutation demonstrated in autosomal dominant hereditary spherocytosis. Furthermore, the mutation is located within a conserved sequence among spectrinlike proteins and may define an amino acid critical for protein 4.1 binding activity.

Adult↗
PubMed1992

Changing patterns in cytoskeletal mRNA expression and protein synthesis during murine erythropoiesis in vivo.

The major cytoskeletal proteins alpha-spectrin, beta-spectrin, and ankyrin are synthesized and assembled into a supportive membrane skeleton during erythroid differentiation. Information on the temporal appearance of mRNA and protein species is essential for understanding both the cytoskeletal assembly process and the function of various isoforms. We have isolated highly enriched populations of fetal erythroid cells at various stages of maturation. mRNAs for erythroid ankyrin, alpha-spectrin, and beta-spectrin were expressed at all stages but there were differences in transcript types and levels. The ratio of 9-kilobase (kb) to 7.5-kb erythroid ankyrin transcripts decreased markedly during differentiation, but there was no change in the ratio of the 10.1-kb and 9.3-kb erythroid beta-spectrin transcripts. The relative amounts of ankyrin, alpha-spectrin, and beta-spectrin mRNA increased during yolk sac cell differentiation, whereas only alpha-spectrin mRNA increased during differentiation of the fetal liver cells. The amounts of beta-spectrin mRNA exceeded the amounts of alpha-spectrin mRNA in the early precursors from both yolk sac and fetal liver; protein synthetic levels showed the same pattern. The 16-day fetal peripheral reticulocytes, on the other hand, had the adult mRNA and protein synthetic ratios with alpha/beta greater than 1. The data indicate that at least two mechanisms exist to meet changing erythroid membrane cytoskeletal requirements during development in utero: (i) stage-specific processing of the mRNA for the major cytoskeletal linker protein ankyrin and (ii) developmentally regulated alpha/beta-spectrin protein synthetic rates.

Animals↗
PubMed1992

Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli.

The cytoplasmic domain of the human erythrocyte membrane protein, band 3 (cdb3), contains binding sites for hemoglobin, several glycolytic enzymes, band 4.1, band 4.2, and ankyrin, and constitutes the major linkage between the membrane skeleton and the membrane. Although erythrocyte cdb3 has been partially purified from proteolyzed red blood cells, further separation of the water-soluble 43-kDa and 41-kDa proteolytic fragments has never been achieved. In order to obtain pure cdb3 for crystallization and site-directed mutagenesis studies, we constructed an expression plasmid that has a tandemly linked T7 promoter placed upstream of the N-terminal 379 amino acids of the erythrocyte band 3 gene. Comparison of several Escherichia coli strains led to the selection of the BL21 (DE3) strain containing the pLysS plasmid as the best host for efficient production of cdb3. About 10 mg of recombinant cdb3 can be easily purified from 4 L of E. coli culture in two simple steps. Comparison of cdb3 released from the red blood cell by proteolysis with recombinant cdb3 reveals that both have the same N-terminal sequence, secondary structure, and pH-dependent conformational change. The purified recombinant cdb3 is also a soluble stable dimer with the same Stokes radius as erythrocyte cdb3. The affinities of the two forms of cdb3 for ankyrin are essentially identical; however, recombinant cdb3 with its unblocked N-terminus exhibits a slightly lower affinity for aldolase.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1992

Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain.

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.

Amino Acid Sequence↗
PubMed1992

The murine pallid mutation is a platelet storage pool disease associated with the protein 4.2 (pallidin) gene.

Pallid is one of 12 independent murine mutations with a prolonged bleeding time that are models for human platelet storage pool deficiencies in which several intracellular organelles are abnormal. We have mapped the murine gene for protein 4.2 (Epb4.2) to chromosome 2 where it co-localizes with pallid. Southern blot analyses suggest that pallid is a mutation in the Epb4.2 gene. Northern blot analyses demonstrate a smaller than normal Epb4.2 transcript in affected pallid tissues, such as kidney and skin. This is the first gene defect to be associated with a platelet storage pool deficiency, and may allow the identification of a novel structure or biological pathway that influences granulogenesis.

Animals↗
PubMed1991

Isolation and chromosomal localization of a novel nonerythroid ankyrin gene.

Immunoreactive isoforms of erythrocyte ankyrin have been shown to be present in a variety of nonerythroid tissues. Isolation of the genes that encode these isoforms will clarify their relationship to erythrocyte ankyrin. Using an erythrocyte ankyrin cDNA clone as a hybridization probe, we screened a human genomic library and isolated a clone that hybridizes with the probe at low stringency but not at high stringency. Partial nucleotide sequence of the clone revealed the presence of a 99-bp segment that is homologous to an exon of the erythrocyte ankyrin gene. Northern analysis showed that a labeled fragment of the clone hybridized to a 7-kb message in RNA of fetal brain but not of erythroid cells, suggesting that this clone is part of a novel gene that is expressed predominantly in nonerythroid tissue. Comparison of the sequence of the genomic clone with that of a recently isolated cDNA clone for brain ankyrin (Otto et al., 1989) showed identity of 96 of 99 bp between the putative exon and a segment of the cDNA clone (V. Bennett, personal communication, 1991), suggesting that the genomic clone is part of a gene for nonerythroid ankyrin, which we have designated ANK2. By analysis of somatic cell hybrids and fluorescence in situ hybridization, we assigned ANK2 to human chromosome 4 at a position equivalent to bands 4q25-q27.

Amino Acid Sequence↗