Abnormalities of erythrocyte membrane proteins in Korean patients with hereditary spherocytosis. (33/741)

Hereditary spherocytosis (HS) is a common inherited erythrocyte membrane disorder characterized by chronic hemolytic anemia. Clinical manifestations and biochemical abnormalities of HS are heterogeneous. In this study, we investigated erythrocyte membrane protein defects in 27 Korean HS cases. Utilizing both the Fairbanks system and the Laemmli system, sodium dodecyl sulfate polyacrylamide gel electrophoresis of erythrocyte membrane proteins was performed. Proteins were stained with Coomassie brilliant blue and gels were scanned using a densitometer. We detected spectrin deficiency in 7.4% of cases (2/27), ankyrin deficiency in 29.6% (8/27), combined spectrin and ankyrin deficiency in 3.7% (1/27), band 3 deficiency in 11.1% (3/27) and protein 4.2 deficiency in 14.8% (4/27). Membrane protein deficiencies were not observed in nine cases (33.3%, 9/27). Members of two of seven families tested showed the same protein defects as the proband. Ankyrin deficiency alone and combined with spectrin deficiency accounted for 33.3% of cases (9/27), and they were the most common biochemical defects in Korean HS cases. Protein 4.2 deficiency caused HS more frequently in Koreans than in Caucasians.  (+info)

The human ankyrin-1 gene is selectively transcribed in erythroid cell lines despite the presence of a housekeeping-like promoter. (34/741)

To begin to study the sequence variations identified in the 5' flanking genomic DNA of the ankyrin gene in ankyrin-deficient hereditary spherocytosis patients and to provide additional insight into our understanding of the regulation of genes encoding erythrocyte membrane proteins, we have identified and characterized the erythroid promoter of the human ankyrin-1 gene. This compact promoter has characteristics of a housekeeping gene promoter, including very high G+C content and enzyme restriction sites characteristic of an HTF-island, no TATA, InR, or CCAAT consensus sequences, and multiple transcription initiation sites. In vitro DNAseI footprinting analyses revealed binding sites for GATA-1, CACCC-binding, and CGCCC-binding proteins. Transfection of ankyrin promoter/reporter plasmids into tissue culture cell lines yielded expression in erythroid, but not muscle, neural, or HeLa cells. Electrophoretic mobility shift assays, including competition and antibody supershift experiments, demonstrated binding of GATA-1, BKLF, and Sp1 to core ankyrin promoter sequences. In transfection assays, mutation of the Sp1 site had no effect on reporter gene expression, mutation of the CACCC site decreased expression by half, and mutation of the GATA-1 site completely abolished activity. The ankyrin gene erythroid promoter was transactivated in heterologous cells by forced expression of GATA-1 and to a lesser degree BKLF.  (+info)

AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide. (35/741)

In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. The ankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location of ankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is approximately 65% alpha-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katB and ankB are part of a small operon whose transcription is induced dramatically by H(2)O(2), and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of an ankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H(2)O(2), phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H(2)O(2) detoxification.  (+info)

Associations and interactions between bare lymphocyte syndrome factors. (36/741)

The bare lymphocyte syndrome, a severe combined immunodeficiency due to loss of major histocompatibility complex (MHC) class II gene expression, is caused by inherited mutations in the genes encoding the heterotrimeric transcription factor RFX (RFX-B, RFX5, and RFXAP) and the class II transactivator CIITA. Mutagenesis of the RFX genes was performed, and the properties of the proteins were analyzed with regard to transactivation, DNA binding, and protein-protein interactions. The results identified specific domains within each of the three RFX subunits that were necessary for RFX complex formation, including the ankyrin repeats of RFX-B. DNA binding was dependent on RFX complex formation, and transactivation was dependent on a region of RFX5. RFX5 was found to interact with CIITA, and this interaction was dependent on a proline-rich domain within RFX5. Thus, these studies have defined the protein domains required for the functional regulation of MHC class II genes.  (+info)

ankA: an Ehrlichia phagocytophila group gene encoding a cytoplasmic protein antigen with ankyrin repeats. (37/741)

Human granulocytic ehrlichiosis (HGE) is a potentially fatal, tick-borne disease caused by a bacterium related or identical to Ehrlichia phagocytophila. To identify and characterize E. phagocytophila group-specific protein antigen genes, we prepared and screened HGE agent and Ehrlichia equi genomic DNA expression libraries using polyclonal equine E. equi antibodies. Two clones, one each from HGE agent and E. equi, that were recognized specifically by antibodies to the E. phagocytophila group ehrlichiae had complete open reading frames of 3,693 and 3,615 nucleotides, respectively. The two clones were 96.6% identical and predicted a protein with at least 11 tandemly repeated ankyrin motifs. Thus, the gene was named ank (for ankyrin). When the encoded protein, named AnkA, was expressed in Escherichia coli, it was recognized by antibodies from rabbits and mice immunized with the HGE agent, sera from humans convalescent from HGE, and sera from horses convalescent from HGE and E. equi infection. Monospecific AnkA antibodies reacted with proteins in HGE agent immunoblots, and AnkA monoclonal antibodies detected cytoplasmic antigen in E. phagocytophila group bacteria and also detected antigen associated with chromatin in infected but not uninfected HL-60 cell cultures. These results suggest that this Ehrlichia protein may influence host cell gene expression.  (+info)

Ankyrins. (38/741)

This review is focused on ankyrin which is a protein linker between the integral membrane proteins and spectrin-based cytoskeleton. Structure and distribution of different ankyrin isoforms that are products of alternative-spliced genes are described. Interaction of ankyrins with various membranes is considered. Special attention is paid to ankyrin participation in signal transduction and in assembly of integral membrane proteins in specialized membrane domains.  (+info)

Crystallographic structure and functional interpretation of the cytoplasmic domain of erythrocyte membrane band 3. (39/741)

The red blood cell membrane (RBCM) is a primary model for animal cell plasma membranes. One of its major organizing centers is the cytoplasmic domain of band 3 (cdb3), which links multiple proteins to the membrane. Included among its peripheral protein ligands are ankyrin (the major bridge to the spectrin-actin skeleton), protein 4. 1, protein 4.2, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, deoxyhemoglobin, p72syk protein tyrosine kinase, and hemichromes. The crystal structure of cdb3 is reported at 0.26 nm (2.6 A) resolution. A tight symmetric dimer is formed by cdb3; it is stabilized by interlocked dimerization arms contributed by both monomers. Each subunit also includes a larger peripheral protein binding domain with an alpha(+) beta-fold. The binding sites of several peripheral proteins are localized in the structure, and the nature of the major conformational change that regulates membrane-skeletal interactions is evaluated. An improved structural definition of the protein network at the inner surface of the RBCM is now possible.  (+info)

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane. (40/741)

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.  (+info)