Characterization of the calcyclin (S100A6) binding site of annexin XI-A by site-directed mutagenesis.
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Residues in annexin XI-A essential for binding of calcyclin (S100A6) were examined by site-directed mutagenesis. GST fusion proteins with the calcyclin binding site of annexin XI-A, GST-AXI 34-62 and GST-AXI 49-77 bound to calcyclin-Sepharose Ca2+-dependently. The mutants GST-AXI L52E, M55E, A56E and M59E lost the binding ability, whereas GST-AXI A57E retained the ability. These results demonstrate that the hydrophobic residues L52, M55, A56 and M59 on one side surface of the alpha-helix are critical for the binding. Assays with GST fusion proteins and synthesized peptides corresponding to the calcyclin binding site indicated that other regions around the calcyclin binding site are important to stabilize the conformation. (+info)
GTPase activity and biochemical characterization of a recombinant cotton fiber annexin.
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A cDNA encoding annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. The cDNA was expressed in Escherichia coli, and the resultant recombinant protein was purified. We then investigated some biochemical properties of the recombinant annexin based on the current understanding of plant annexins. An "add-back experiment" was performed to study the effect of the recombinant annexin on beta-glucan synthase activity, but no effect was found. However, it was found that the recombinant annexin could display ATPase/GTPase activities. The recombinant annexin showed much higher GTPase than ATPase activity. Mg2+ was essential for these activities, whereas a high concentration of Ca2+ was inhibitory. A photolabeling assay showed that this annexin could bind GTP more specifically than ATP. The GTP-binding site on the annexin was mapped into the carboxyl-terminal fourth repeat of annexin from the photolabeling experiment using domain-deletion mutants of this annexin. Northern-blot analysis showed that the annexin gene was highly expressed in the elongation stages of cotton fiber differentiation, suggesting a role of this annexin in cell elongation. (+info)
Transcription, biochemistry and localization of nematode annexins.
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The transcription of three annexin genes in the nematode, Caenorhabditis elegans, was detected by reverse transcriptase/polymerase chain reaction amplification of messenger RNAs. The highest level of expression was from the nex-1 gene, with lower levels detected for the nex-2 and nex-3 genes. The expression of nex-1 was reduced in the Dauer larval stage relative to the other annexins, correlating with the absence of the spermathecal valves, a major site of nex-1 protein localization. Recombinant nex-1 protein was expressed in yeast, isolated by calcium-dependent binding to acidic phospholipids, and its membrane binding and aggregating activities characterized using bovine chromaffin granules as a representative intracellular substrate. Binding to granule membranes was promoted by calcium with half-maximal binding seen at 630 microM calcium. Chromaffin granule aggregation was similarly promoted by the nex-1 protein at 630 microM calcium. This low sensitivity to calcium suggests the annexin can only be activated in vivo near the plasma membrane or other sources of calcium. Sequences including the nex-1 promoter were fused to the gene for green fluorescent protein and this construct was introduced into nematodes by microinjection. Examination of transgenic offspring revealed specific nex-1 promoter activity in the pharynx, the hypodermal cells, the vulva, and the spermathecal valve, locations in which the annexin may function in collagen secretion/deposition and membrane-membrane interactions. A sensitive anti-nex-1 antibody labelled with rhodamine was injected into body cavities of the nematode but did not detect extracellular nex-1 protein. Therefore, this annexin is apparently cytosolic and may function on the cytoplasmic side of the plasma membrane of the spermathecal valve to chaperon the folding of this membrane during the opening and closing of the valve. (+info)
Annexin VII and annexin XI are tyrosine phosphorylated in peroxovanadate-treated dogs and in platelet-derived growth factor-treated rat vascular smooth muscle cells.
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The intraperitoneal administration of peroxovanadate results in the rapid accumulation of many tyrosine-phosphorylated proteins in the liver and kidney of treated animals. The availability of large pools of tyrosine-phosphorylated proteins derived from normal tissues facilitates the purification and identification of previously unknown targets for cellular tyrosine kinases. Using this procedure, we have thus far identified four proteins in the liver and kidney of peroxovanadate-treated dogs. Two of these, annexin VII and annexin XI, were novel and had not been previously reported to be substrates of tyrosine kinases while the remaining two, ezrin and clathrin, have been reported to be tyrosine phosphorylated in some cell culture systems. In the present study, isolated proteins were identified both by sequence analysis and immunological methods. Annexin VII and annexin XI are present in cultured rat vascular smooth muscle cells and both were tyrosine phosphorylated in response to a physiological ligand, platelet-derived growth factor-BB (PDGF-BB). Furthermore, the extent of tyrosine phosphorylation in response to PDGF-BB was augmented by the co-addition of peroxovanadate to cell cultures. In vitro phosphorylation assays showed that PDGF receptor, calcium-dependent tyrosine kinase (CADTK/Pyk-2), Src kinase, and epidermal growth factor receptor all were able to phosphorylate purified annexin VII and XI on tyrosine residues. These findings confirm the usefulness of phosphatase inhibition by peroxovanadate as a tool for identifying previously unknown physiological targets for cellular protein tyrosine kinases. (+info)
Immunological development and cardiovascular function are normal in annexin VI null mutant mice.
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Annexins are calcium-binding proteins of unknown function but which are implicated in important cellular processes, including anticoagulation, ion flux regulation, calcium homeostasis, and endocytosis. To gain insight into the function of annexin VI, we performed targeted disruption of its gene in mice. Matings between heterozygous mice produced offspring with a normal Mendelian pattern of inheritance, indicating that the loss of annexin VI did not interfere with viability in utero. Mice lacking annexin VI reached sexual maturity at the same age as their normal littermates, and both males and females were fertile. Because of interest in the role of annexin VI in cardiovascular function, we examined heart rate and blood pressure in knockout and wild-type mice and found these to be identical in the two groups. Similarly, the cardiovascular responses of both sets of mice to septic shock were indistinguishable. We also examined components of the immune system and found no differences in thymic, splenic, or bone marrow lymphocyte levels between knockout and wild-type mice. This is the first study of annexin knockout mice, and the lack of a clear phenotype has broad implications for current views of annexin function. (+info)
Annexins VII and XI are present in a human macrophage-like cell line. Differential translocation on FcR-mediated phagocytosis.
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We have studied the divalent cation-dependent association of proteins to subcellular fractions of human macrophage-like cells before and after FcR-mediated phagocytosis. Among these proteins we have identified annexins VII and XI for the first time in these cells, along with annexins I, III, and VI. Although all of these annexins are present in the cytosolic fraction, the extent of their association to membrane and phagosome fractions from resting and stimulated cells is variable. Annexin VII translocates from cytosolic to membrane fractions after phagocytic stimulation, along with annexin I, III, and VI. Annexins VII and XI are found associated with purified phagosomes along with I, III, and VI, and this association is greater after a 24-h chase period. Our results show differences in the intracellular distribution of different annexins in macrophage-like cells on phagocytosis. Annexins VII, VI, III, and I respond to particle ingestion by translocating to phagosomes and other cell membrane structures, whereas annexin XI translocates predominantly to phagosomes, suggesting dissimilarities in their function. (+info)
Cellubrevin is present in the basolateral endocytic compartment of hepatocytes and follows the transcytotic pathway after IgA internalization.
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The endocytic compartment of polarized cells is organized in basolateral and apical endosomes plus those endocytic structures specialized in recycling and transcytosis, which are still poorly characterized. The complexity of the various populations of endosomes has been demonstrated by the exquisite repertoire of endogenous proteins. In this study we examined the distribution of cellubrevin in the endocytic compartment of hepatocytes, since its intracellular location and function in polarized cells are largely unknown. Highly purified rat liver endosomes were isolated from estradiol-treated rats, and the early/sorting endosomal fraction was further subfractionated in a multistep sucrose density gradient, and studied. Analysis of dissected endosomal fractions showed that cellubrevin was located in early/sorting endosomes, with Rab4, annexins II and VI, and transferrin receptor, but in a specific subpopulation of these early endosomes with the same density range as pIgA and Raf-1. Interestingly, only in those isolated endosomal fractions, endosomes enriched in transcytotic structures (of livers loaded with IgA), the polymeric immunoglobulin receptor specifically co-immunoprecipitated with cellubrevin. In addition, confocal and immuno-electron microscopy identification of cellubrevin in tubular structures underneath the sinusoidal plasma membrane together with the re-organization of cellubrevin, in the endocytic compartment, after the IgA loading, strongly suggest the involvement of cellubrevin in the transcytosis of pIgA. (+info)
Annexin 24 from Capsicum annuum. X-ray structure and biochemical characterization.
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This work provides the first three-dimensional structure of a member of the plant annexin family and correlates these findings with biochemical properties of this protein. Annexin 24(Ca32) from Capsicum annuum was purified as a native protein from bell pepper and was also prepared by recombinant techniques. To overcome the problem of precipitation of the recombinant wild-type protein in crystallization trials, two mutants were designed. Whereas an N-terminal truncation mutant turned out to be an unstable protein, the N-terminal His-tagged annexin 24(Ca32) was crystallized, and the three-dimensional structure was determined by x-ray diffraction at 2. 8 A resolution. The structure refined to an R-factor of 0.216 adopts the typical annexin fold; the detailed structure, however, is different from non-plant annexins, especially in domains I and III and in the membrane binding loops on the convex side. Within the unit cell there are two molecules per asymmetric unit, which differ in conformation of the IAB-loop. Both conformers show Trp-35 on the surface. The loop-out conformation is stabilized by tight interactions of this tryptophan with residue side chains of a symmetry-related molecule and enforced by a bound sulfate. Characterization of this plant annexin using biophysical methods revealed calcium-dependent binding to phospholipid vesicles with preference for phosphatidylcholine over phosphatidylserine and magnesium-dependent phosphodiesterase activity in vitro as shown with adenosine triphosphate as the substrate. A comparative unfolding study of recombinant annexin 24(Ca32) wild type and of the His-tag fusion protein indicates higher stability of the latter. The effect of this N-terminal modification is also visible from CD spectra. Both proteins were subjected to a FURA-2-based calcium influx assay, which gave high influx rates for the wild-type but greatly reduced influx rates for the fusion protein. We therefore conclude that the N-terminal domain is indeed a major regulatory element modulating different annexin properties by allosteric mechanisms. (+info)