Long-term expression of gamma-globin mRNA in mouse erythrocytes from retrovirus vectors containing the human gamma-globin gene fused to the ankyrin-1 promoter.
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Gene therapy for patients with hemoglobin disorders has been hampered by the inability of retrovirus vectors to transfer globin genes and their cis-acting regulatory sequences into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells due to position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Recently, we have shown that the human ankyrin (Ank) gene promoter directs position-independent, copy number-dependent expression of a linked gamma-globin gene in transgenic mice. We inserted the Ank/(A)gamma-globin gene into retrovirus vectors that could transfer one or two copies of the Ank/(A)gamma-globin gene to target cells. Both vectors were stable, transferring only intact proviral sequences into primary mouse hematopoietic stem cells. Expression of Ank/(A)gamma-globin mRNA in mature red blood cells was 3% (single copy) and 8% (double copy) of the level of mouse alpha-globin mRNA. We conclude that these novel retrovirus vectors may be valuable for treating a variety of red cell disorders by gene replacement therapy including severe beta-thalassemia if the level of expression can be further increased. (+info)
Calcium-independent phospholipase A(2): structure and function.
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The classical Ca(2+)-independent phospholipase A(2) enzyme, now known as Group VIA PLA(2), was initially purified and characterized from the P388D(1) macrophage-like cell line. The corresponding cDNA was subsequently cloned from a variety of sources, and it is now known that multiple splice variants of the enzyme are expressed, some of which may act as negative regulators of the active enzyme. Group VIA PLA(2) has a consensus lipase motif (GTSTG) containing the catalytic serine, is 85-88 kDa, and exists in an aggregated form. The enzyme contains multiple ankyrin repeats, which may play a role in oligomerization. The Group VIA enzyme exhibits lysophospholipase activity as well as phospholipase A(2) activity, and it is capable of hydrolyzing a wide variety of phospholipid substrates. A major function of Group VIA PLA(2) is to mediate phospholipid remodeling, but the enzyme may play other roles as well. Other Ca(2+)-independent PLA(2) enzymes have more recently been identified, and it may be possible to discriminate between the various Ca(2+)-independent PLA(2) enzymes based on sequence or inhibitor-sensitivity. However, the physiological functions of the newly identified enzymes have yet to be elucidated. (+info)
betaIV spectrin, a new spectrin localized at axon initial segments and nodes of ranvier in the central and peripheral nervous system.
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We report the identification of betaIV spectrin, a novel spectrin isolated as an interactor of the receptor tyrosine phosphatase-like protein ICA512. The betaIV spectrin gene is located on human and mouse chromosomes 19q13.13 and 7b2, respectively. Alternative splicing of betaIV spectrin generates at least four distinct isoforms, numbered betaIVSigma1-betaIVSigma4 spectrin. The longest isoform (betaIVSigma1 spectrin) includes an actin-binding domain, followed by 17 spectrin repeats, a specific domain in which the amino acid sequence ERQES is repeated four times, several putative SH3-binding sites and a pleckstrin homology domain. betaIVSigma2 and betaIVSigma3 spectrin encompass the NH(2)- and COOH-terminal halves of betaIVSigma1 spectrin, respectively, while betaIVSigma4 spectrin lacks the ERQES and the pleckstrin homology domain. Northern blots revealed an abundant expression of betaIV spectrin transcripts in brain and pancreatic islets. By immunoblotting, betaIVSigma1 spectrin is recognized as a protein of 250 kD. Anti-betaIV spectrin antibodies also react with two additional isoforms of 160 and 140 kD. These isoforms differ from betaIVSigma1 spectrin in terms of their distribution on subcellular fractionation, detergent extractability, and phosphorylation. In islets, the immunoreactivity for betaIV spectrin is more prominent in alpha than in beta cells. In brain, betaIV spectrin is enriched in myelinated neurons, where it colocalizes with ankyrin(G) 480/270-kD at axon initial segments and nodes of Ranvier. Likewise, betaIV spectrin is concentrated at the nodes of Ranvier in the rat sciatic nerve. In the rat hippocampus, betaIVSigma1 spectrin is detectable from embryonic day 19, concomitantly with the appearance of immunoreactivity at the initial segments. Thus, we suggest that betaIVSigma1 spectrin interacts with ankyrin(G) 480/270-kD and participates in the clustering of voltage-gated Na(+) channels and cell-adhesion molecules at initial segments and nodes of Ranvier. (+info)
Characterization of ANKRA, a novel ankyrin repeat protein that interacts with the cytoplasmic domain of megalin.
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Ankyrin-repeat family A protein (ANKRA) is a novel protein that interacts directly and specifically with the cytoplasmic tail of megalin in the yeast two-hybrid system and glutathione-S-transferase pull-down assays. ANKRA has three ankyrin repeats and shows 61% overall homology to regulatory factor X, ankyrin repeat-containing protein. Mapping studies show that the three ankyrin repeats and C-terminus of ANKRA are required for binding to a unique juxtamembrane, 19-amino acid sequence on the megalin tail. Point mutational analysis reveals that a proline-rich motif (PXXPXXP) within this region is the site of ANKRA binding. ANKRA interacts with megalin but not with low-density lipoprotein receptor related protein, in keeping with the fact that the sequence of the megalin tail is unique. By cell fractionation, ANKRA is found both in the cytosol and associated with membranes enriched in megalin in L2 cells and proximal tubule cells. By immunofluorescence, ANKRA is concentrated near megalin along the plasma membrane of L2 cells and in the kidney cortex is expressed in glomerular and proximal tubule epithelia which also express megalin. These observations suggest that ANKRA may play a unique role in megalin's function as a clearance receptor in the kidney and L2 cells. In addition, ANKRA may have other partners because northern blot analysis reveals that ANKRA is more broadly expressed than megalin, and by immunofluorescence ANKRA is also expressed in connecting tubule cells and principal cells of collecting ducts. (+info)
Interaction between the N-terminal domain of gastric H,K-ATPase and the spectrin binding domain of ankyrin III.
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We screened a cDNA bank of rabbit gastric fundic mucosa by two-hybrid assays looking for binding partners of the N-terminal domain of the rabbit gastric H,K-ATPase. We extracted five clones sharing more than 90% sequence identity. The longest clone codes for a protein sharing a high identity (96 and 96.8%, respectively) with a fragment of the membrane domain, from Arg-835 to Ser-873, plus the major part of the "spectrin binding domain" going from Glu-874 to Leu-1455 of human and mouse ankyrin III. We conclude that the membrane and spectrin binding domains of the rabbit ankyrin III are candidates for the binding partner of the N-terminal domain of the rabbit gastric H,K-ATPase. To validate the ankyrin-ATPase interaction and to test its specificity, we produced both domains in yeast and bacteria, coimmunoprecipitated them with an anti-ATPase antibody, and copurified them by affinity chromatography. The sequence of rabbit ankyrin III was not known, and this is the first report demonstrating that the ankyrin III and the H,K-ATPase interact with no intermediate. The interaction involves the N-terminal domain of the ATPase on one hand and the spectrin binding domain of the ankyrin on the other. (+info)
Regulating ankyrin dynamics: Roles of sigma-1 receptors.
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Ankyrin is a cytoskeletal adaptor protein that controls important cellular functions, including Ca(2+) efflux at inositol 1,4,5-trisphosphate receptors (IP(3)R) on the endoplasmic reticulum. The present study found that sigma-1 receptors (Sig-1R), unique endoplasmic reticulum proteins that bind certain steroids, neuroleptics, and psychotropic drugs, form a trimeric complex with ankyrin B and IP(3)R type 3 (IP(3)R-3) in NG-108 cells. The trimeric complex could be coimmunoprecipitated by antibodies against any of the three proteins. Sig-1R agonists such as pregnenolone sulfate and cocaine caused the dissociation of an ankyrin B isoform (ANK 220) from IP(3)R-3. This effect caused by Sig-1R agonists was blocked by a Sig-1R antagonist. The degree of dissociation of ANK 220 from IP(3)R-3 caused by Sig-1R ligands correlates excellently with the ligands' efficacies in potentiating the bradykinin-induced increase in cytosolic free Ca(2+) concentration. Immunocytohistochemistry showed that Sig-1R, ankyrin B, and IP(3)R-3 are colocalized in NG-108 cells in perinuclear areas and in regions of cell-to-cell communication. These results suggest that Sig-1R and associated ligands may play important roles in cells by controlling the function of cytoskeletal proteins and that the Sig-1R/ANK220/IP(3)R-3 complex regulating Ca(2+) signaling may represent a site of action for neurosteroids and cocaine. (+info)
Nodes of Ranvier form in association with ezrin-radixin-moesin (ERM)-positive Schwann cell processes.
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In the adult peripheral nerve, microvillous processes of myelinating Schwann cells project to the nodes of Ranvier; their composition and physiologic function have not been established. As the ezrin-radixin-moesin (ERM) proteins are expressed in the microvilli of many epithelial cells, we have examined the expression and distribution of these proteins in Schwann cells and neurons in vitro and in vivo. Cultured Schwann cells express high levels of all three proteins and the ezrin-binding protein 50, whereas neurons express much lower, although detectable, levels of radixin and moesin. Ezrin is specific for Schwann cells. All three ERM proteins are expressed predominantly at the membrane of cultured Schwann cells, notably in their microvilli. In vivo, the ERM proteins are concentrated strikingly in the nodal processes of myelinating Schwann cells. Because these processes are devoid of myelin proteins, they represent a unique compartment of the myelinating Schwann cell. During development, the ERM proteins become concentrated at the ends of Schwann cells before myelin basic protein expression, demonstrating that Schwann cells are polarized longitudinally at the onset of myelination. ERM-positive Schwann cell processes overlie and are associated closely with nascent nodes of Ranvier, identified by clusters of ankyrin G. Ankyrin accumulation at the node precedes that of Caspr at the paranodes and therefore does not depend on the presence of mature paranodal junctions. These results demonstrate that nodes of Ranvier in the peripheral nervous system form in contact with specialized processes of myelinating Schwann cells that are highly enriched in ERM proteins. (+info)
Na,K-ATPase in skeletal muscle: two populations of beta-spectrin control localization in the sarcolemma but not partitioning between the sarcolemma and the transverse tubules.
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We used immunological approaches to study the factors controlling the distribution of the Na,K-ATPase in fast twitch skeletal muscle of the rat. Both alpha subunits of the Na,K-ATPase colocalize with beta-spectrin and ankyrin 3 in costameres, structures at the sarcolemma that lie over Z and M-lines and in longitudinal strands. In immunoprecipitates, the alpha1 and alpha2 subunits of the Na,K-ATPase as well as ankyrin 3 associate with beta-spectrin/alpha- fodrin heteromers and with a pool of beta-spectrin at the sarcolemma that does not contain alpha-fodrin. Myofibers of mutant mice lacking beta-spectrin (ja/ja) have a more uniform distribution of both the alpha1 and alpha2 subunits of the Na,K-ATPase in the sarcolemma, supporting the idea that the rectilinear sarcomeric pattern assumed by the Na,K-ATPase in wild-type muscle requires beta-spectrin. The Na,K-ATPase and beta-spectrin are distributed normally in muscle fibers of the nb/nb mouse, which lacks ankyrin 1, suggesting that this isoform of ankyrin is not necessary to link the Na,K-ATPase to the spectrin-based membrane skeleton. In immunofluorescence and subcellular fractionation experiments, the alpha2 but not the alpha1 subunit of the Na,K-ATPase is present in transverse (t-) tubules. The alpha1 subunit of the pump is not detected in increased amounts in the t-tubules of muscle from the ja/ja mouse, however. Our results suggest that the spectrin-based membrane skeleton, including ankyrin 3, concentrates both isoforms of the Na,K-ATPase in costameres, but that it does not play a significant role in restricting the entry of the alpha1 subunit into the t-tubules. (+info)