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At least 19 recordsLinked to original sources

PubMed1993

Annexin 5 as a potential regulator of annexin 1 phosphorylation by protein kinase C. In vitro inhibition compared with quantitative data on annexin distribution in human endothelial cells.

In vitro phosphorylation of annexin 1 by purified rat brain protein kinase C (PKC) has been studied in the presence of annexin 5, which is not a substrate for PKC. Annexin 5 promoted a dose-dependent inhibition of annexin 1 phosphorylation, which could be overcome by increasing the concentration of phosphatidylserine (PtdSer). In addition, a close relationship was found between the amount of PtdSer uncovered by annexin 5 and the residual phosphorylation of annexin 1. These data fit with the 'surface depletion model' explaining the antiphospholipase activity of annexins. In order to check the possibility that the in vitro effect of annexin 5 could be of some physiological relevance, annexins 1, 2, and 5, as well as the light chain of calpactin 1 (p11), have been quantified in human endothelial cells by measuring the radioactivity bound to the proteins after Western blotting with specific antibodies and 125I-labelled secondary antibody. Our data indicate that annexins 1 and 5, PKC and PtdSer are present in human endothelial cells in relative amounts very similar to those used in vitro under conditions permitting the detection of the inhibitory effect of annexin 5. Since annexin 1 remained refractory to PKC-dependent phosphorylation in intact cells, we suggest that annexin 5 might exert its inhibitory effect towards PKC in vivo, provided that its binding to phospholipids can occur at physiological (micromolar) concentrations of Ca2+. This was previously shown to occur in vitro using phosphatidylethanolamine/phosphatidic acid vesicles [Blackwood and Ernst (1990) Biochem. J. 266, 195-200]. Using identical assay conditions, which also allowed expression of PKC activity, annexin 5 again inhibited annexin 1 phosphorylation without interfering with PKC autophosphorylation. These data suggest that annexins 1 and 5 might interact with each other on the lipid surface, resulting in a specific inhibition of annexin 1 phosphorylation by PKC. Whether a similar mechanism also occurs in vivo remains to be determined.

Adenosine Triphosphate↗

PubMed1997

Voltage dependent binding of annexin V, annexin VI and annexin VII-core to acidic phospholipid membranes.

Annexin V, VI and VII-core (delta1-107) are members of the annexin protein family and bind to acidic phospholipid membranes in a calcium dependent manner. They also show ion channel activity under certain conditions. As annexins bind peripherally to lipid membranes, ion channel formation must consist of at least two steps: An adsorption reaction regulating the binding of annexin to the membrane surface and the opening and closing of the active species controlling the channel activity. By using the baseline current through the patch clamp seal as a probe for unoccupied binding sites at the membrane, we show that the adsorption of annexins to membranes is not only calcium dependent but also strongly voltage dependent. Whereas the free transfer energies at low calcium concentrations are similar for all three annexins, the binding of annexin V becomes much tighter with higher calcium levels, compared to annexin VI and VII-core. This correlates with the finding that annexin VI and VII-core display channel activity much more often than annexin V if one assumes that a high coverage of the membrane surface with annexins stabilizes the bilayer. At higher protein concentrations weaker binding is observed in agreement with the previously reported anti-cooperativity of membrane binding.

Adsorption↗

PubMed1994

Involvement of annexin I and annexin II in hepatocyte proliferation: can annexins I and II be markers for proliferative hepatocytes?

Annexin is the name of a new family of Ca(2+)-dependent membrane-binding proteins. Eleven types of its related proteins have been reported to date. Among those, annexin I and annexin II have been reported to possess many biological functions in vitro. Its actual role in vivo, however, is yet unknown. The involvement of annexin I and annexin II in the proliferation processes of hepatocytes was examined in the following aspects: (a) hepatocyte proliferation after carbon tetrachloride-induced liver damage, (b) hepatocyte regeneration after partial hepatectomy and (c) postnatal development of hepatocytes. These results showed collectively that annexin I and annexin II were increased in proliferative (or regenerative) hepatocytes, suggesting that both proteins play a certain role in the proliferation event. Furthermore, annexin I- and annexin II-positive hepatocytes always show a wider distribution than that of proliferating cell nuclear antigen or cytokeratin 7-positive hepatocytes, indicating that annexin I and annexin II may be useful markers for detecting not only actively proliferating hepatocytes but also hepatocytes in preproliferative and postproliferative stages.

Animals↗

PubMed1997

Effects of profilin-annexin I association on some properties of both profilin and annexin I: modification of the inhibitory activity of profilin on actin polymerization and inhibition of the self-association of annexin I and its interactions with liposomes.

We have previously shown that annexin I, a member of a family of calcium-dependent phospholipid and membrane binding proteins, interacts with profilin with high specificity and affinity. This finding further suggests that annexin I is involved through profilin in the regulation of membrane-cytoskeleton organization. We have investigated the consequences of a complex formed by these two proteins on the functions of both profilin and annexin I. Annexin I is able to modify the inhibitory effect of profilin on actin polymerization. This action is partial and the mechanism involved appears to be complex. On the other hand, the association between annexin I and profilin is sufficiently strong to inhibit the self-association of annexin I. The binding capacity of annexin I to liposomes containing phosphatidylserine, which mimics annexin I binding to membranes, is also decreased by profilin. This binding is nevertheless restored when phosphatidylinositol 4,5-biphosphate (PtdInsP2) is included in the liposomes. Finally, the capacity of annexin I to aggregate liposomes is also modified. It is worthwhile mentioning that the liposomes-binding and liposomes-aggregating activities of annexin I are independently regulated. The cell localization and functions of annexin I and profilin suggest that interaction between these two proteins may be directly implicated in the regulation of membrane-cytoskeleton. The phospholipid composition of membranes may be one of the modulating factors.

Actins↗

PubMed1998

Hinge-bending motions in annexins: molecular dynamics and essential dynamics of apo-annexin V and of calcium bound annexin V and I.

Annexins are homologous proteins that bind to membranes in a calcium dependent manner, but for which precise physiological roles have yet to be defined. Most annexins are composed of a planar array of four homologous repeats, each containing five alpha-helices and associated into two modules. Annexin V forms a voltage-gated calcium channel in phospholipid bilayers. It has been proposed that the hydrophilic pore in the centre of the molecule may represent the ion conduction pathway and that a hinge movement in annexin V causes a variation of the inter-module angle and opens the calcium ion path. Here we present the results of molecular dynamics simulations of apo-annexin V and of calcium-bound annexin V and annexin I. The three simulations show significant differences in conformation and dynamics. The essential dynamics method was used to study the essential subspace of annexin V and showed that one of the essential motions corresponds to the postulated hinge motion. The hinge residues were located between repeats but belong to helices rather than to the links between helices. Calcium binding to annexin V led to a limitation of this hinge motion with more open conformations being favoured.

Amino Acids↗

PubMed2007

Annexin 2-mediated enhancement of cytomegalovirus infection opposes inhibition by annexin 1 or annexin 5.

Biochemical studies have suggested that annexin 2 (A2) may participate in cytomegalovirus (CMV) infection. In the current work, effects of A2 monomer (p36) and heterotetramer (A2t; p36(2)p11(2)) were investigated. Demonstrating a role for endogenous A2, the four stages of infection that were followed were each inhibited by anti-p36 or anti-p11 at 37 degrees C. Immuno-inhibition was attenuated when the virus and cells were pre-incubated at 4 degrees C to coordinate virus entry initiated afterwards at 37 degrees C, reconciling controversy in the literature. As an explanation, CMV-induced phosphorylation of p36 was prevented by the 4 degrees C treatment. Supporting these immuno-inhibition data, purified A2t or p11 increased CMV infectious-progeny generation and CMV gene expression. A specific role for A2t was indicated by purified p36 having no effect. Unlike other steps, primary plaque formation was not enhanced by purified A2t or p11, possibly because of undetectable phosphorylation. As annexins 1 (A1) and 5 (A5) interact with A2, their effect on CMV was also tested. Both purified proteins inhibited CMV infection. In each experiment, the concentration of A1 required for half-maximal inhibition was five- to 10-fold lower than that of A5. Addition of A2 opposed A1- or A5-mediated inhibition of CMV, as did certain A2-specific antibodies that had no effect in the absence of added A1 or A5. Transfection of the p36-deficient cell line HepG2 increased CMV infection and was required for inhibition by the other annexins. These data suggest that CMV exploits A2t at physiological temperature to oppose the protection of cells conferred by A1 or A5.

Annexin A1↗

PubMed1993

Identification and immunolocalisation of annexins V and VI, the major cardiac annexins, in rat heart.

OBJECTIVE: The heart contains proteins of the annexin family, a unique group of calcium binding proteins. This study was aimed at identifying the major cardiac annexins and determining their distribution in the rat heart. METHODS: Annexins were isolated by affinity chromatography and purified by ion exchange high pressure liquid chromatography. Identification of isolated proteins by immunoblotting was confirmed by partial amino acid sequence determination. Antisera raised against the isolated proteins were used for immunohistochemistry by the avidin-biotin-peroxidase technique. RESULTS: Two annexins were isolated and purified. Amino acid sequencing confirmed their identities as annexin V and VI. Immunohistochemistry showed that both annexins were present in cardiac myocytes and non-myocytes, but a distinct pattern of distribution was seen for each annexin. Annexin V immunoreactivity was enhanced in the atria compared with the ventricles, whereas annexin VI was more uniformly distributed. In individual cardiac myocytes annexin V was distributed throughout the cell by contrast with annexin VI, which localised to the sarcolemma. Intercalated discs displayed immunoreactivity for both annexins, most prominently for annexin VI. The most striking immunoreactivity for annexin V occurred in vascular endothelial cells, both in the microcirculation and in the major coronary vessels. Immunoreactivity for annexin VI in vascular structures was localised to the nuclei of endothelial and smooth muscle cells. CONCLUSIONS: Annexins V and VI are the major cardiac annexins. The localisation of these annexins to different components of cardiac myocytes will serve to direct the search for their functions in the heart. The striking immunoreactivity for annexins, particularly annexin V, in the coronary vessels indicates that the functions of cardiac annexins include a role in the coronary circulatory system.

Amino Acid Sequence↗

PubMed2003

Nuclear annexin II negatively regulates growth of LNCaP cells and substitution of ser 11 and 25 to glu prevents nucleo-cytoplasmic shuttling of annexin II.

BACKGROUND: Annexin II heavy chain (also called p36, calpactin I) is lost in prostate cancers and in a majority of prostate intraepithelial neoplasia (PIN). Loss of annexin II heavy chain appears to be specific for prostate cancer since overexpression of annexin II is observed in a majority of human cancers, including pancreatic cancer, breast cancer and brain tumors. Annexin II exists as a heterotetramer in complex with a protein ligand p11 (S100A10), and as a monomer. Diverse cellular functions are proposed for the two forms of annexin II. The monomer is involved in DNA synthesis. A leucine-rich nuclear export signal (NES) in the N-terminus of annexin II regulates its nuclear export by the CRM1-mediated nuclear export pathway. Mutation of the NES sequence results in nuclear retention of annexin II. RESULTS: Annexin II localized in the nucleus is phosphorylated, and the appearance of nuclear phosphorylated annexin II is cell cycle dependent, indicating that phosphorylation may play a role in nuclear entry, retention or export of annexin II. By exogenous expression of annexin II in the annexin II-null LNCaP cells, we show that wild-type annexin II is excluded from the nucleus, whereas the NES mutant annexin II localizes in both the nucleus and cytoplasm. Nuclear retention of annexin II results in reduced cell proliferation and increased doubling time of cells. Expression of annexin II, both wild type and NES mutant, causes morphological changes of the cells. By site-specific substitution of glutamic acid in the place of serines 11 and 25 in the N-terminus, we show that simultaneous phosphorylation of both serines 11 and 25, but not either one alone, prevents nuclear localization of annexin II. CONCLUSION: Our data show that nuclear annexin II is phosphorylated in a cell cycle-dependent manner and that substitution of serines 11 and 25 inhibit nuclear entry of annexin II. Aberrant accumulation of nuclear annexin II retards proliferation of LNCaP cells.

Active Transport, Cell Nucleus↗

PubMed1993

Expression of annexin I, II, V, and VI by rat osteoblasts in primary culture: stimulation of annexin I expression by dexamethasone.

To determine whether rat osteoblasts synthesize proteins of the annexin family and to evaluate the extent to which glucocorticoids modulate the expression of annexins by these cells, osteoblasts were grown in primary cultures in the absence or presence of dexamethasone, and the expression of annexins was evaluated by immunoblotting using polyclonal antibodies against human annexins. Four different annexins (I, II, V, and VI) were found to be expressed by rat osteoblasts. The expression of annexin I, but not the other annexins studied, was increased in osteoblasts cultured in the presence of dexamethasone (173 +/- 33% increase comparing untreated cells and cells treated for 10 days with 5 x 10(-7) M dexamethasone). Increased expression of annexin I was observed after the third day of exposure to dexamethasone and rose thereafter until day 10; annexin I expression increased with dexamethasone concentrations above 10(-10) M throughout the range of concentrations studied. The increase in annexin I protein was associated with an increase in annexin I mRNA and was completely blocked by the concomitant addition of the glucocorticoid receptor antagonist RU 38486. The increase in annexin I content following dexamethasone treatment was associated with an increase in alkaline phosphatase activity and PTH-induced cAMP stimulation, whereas phospholipase A2 activity in the culture medium was reduced to undetectable levels. The finding that four annexins are expressed in rat osteoblasts in primary culture raises the possibility that these proteins could play an important role in bone formation by virtue of their ability to bind calcium and phospholipids, serve as Ca2+ channels, interact with cytoskeletal elements, and/or regulate phospholipase A2 activity. In addition, the dexamethasone-induced increase in annexin I may represent a mechanism by which glucocorticoids modify osteoblast function.

Alkaline Phosphatase↗

PubMed1992

Identification of a novel annexin in Hydra vulgaris. Characterization, cDNA cloning, and protein kinase C phosphorylation of annexin XII.

As a first step toward the elucidation of a simple animal model in which to investigate annexin function, we identified, isolated, and characterized a novel annexin from Hydra vulgaris, annexin XII. A hydra cDNA library was screened using a probe generated by polymerase chain reaction from primers based on the partial amino acid sequence of annexin XII. Annexin XII cDNA was cloned and the functional protein was expressed in high yields in Escherichia coli. The annexin XII cDNA sequence predicted a 316-amino acid protein that had between 44 and 54% sequence identity with the Ca2+-binding core domains of previously characterized vertebrate and Drosophila annexins. The amino-terminal domain of annexin XII did not have sequence similarity with other known annexins except at and around a site that resembled known protein kinase C (PKC) phosphorylation sites in other annexins. As anticipated from its sequence, annexin XII was a high affinity substrate for purified rat brain PKC; half-maximal phosphorylation occurred below 0.1 microM annexin XII, and incorporation of up to 0.8 mol of phosphate/mol of annexin XII was observed. A PKC-like activity in hydra extracts also phosphorylated annexin XII. In summary, hydra promises to be a valuable model system for investigating the biological function of annexins and for determining how this function is modulated by PKC phosphorylation.

Amino Acid Sequence↗

PubMed1994

Alterations of annexin expression in pathological neuronal and glial reactions. Immunohistochemical localization of annexins I, II (p36 and p11 subunits), IV, and VI in the human hippocampus.

Annexins are Ca(2+)-dependent membrane-binding proteins that are potentially important in Ca(2+)-induced neurotoxicity or neuroprotection. To address the possible involvement of annexins in cellular reactions to brain injury and neurodegenerative disease, we studied the immunohistochemical localization of annexins I, II (p36 and p11), IV, and VI in the adult human hippocampus. Formalin-fixed, paraffin-embedded tissue from autopsy cases representing hypoxic-ischemic injury, seizure disorders, Alzheimer's disease, and age-related controls were examined. Neurons showed cytoplasmic immunoreactivity for annexin I, whereas annexin VI was distributed in patterns suggesting plasma membrane and perisynaptic locations. The cytoarchitectural distribution of annexin VI within neurons was altered in pathological states and annexin VI was strongly associated with neuronal granulovacuolar bodies in Alzheimer's disease. Reactive astrocytes expressed annexins I, II (p36 and p11), and IV, whereas quiescent astrocytes were minimally immunoreactive. Significant annexin immunoreactivity was also detected in oligodendrocytes (annexin IV), ependymocytes (I, II, and IV), choroid plexus (I, IV, and VI), meningothelium (I, II, IV, and VI), and vascular endothelium (II and IV) and smooth muscle (I, IV, and VI). This is the first comparative study of immunoreactivities for multiple annexins in human brain. Neurons and glia display selective and different profiles of annexin protein expression and show immunohistochemical changes in pathological conditions, which suggest involvement of annexins in neuronal and glial reactions to injury.

Adult↗

PubMed1993

Interaction of annexins with membranes: the N-terminus as a governing parameter as revealed with a chimeric annexin.

The modulating effect of the variable N-terminus of annexins on the properties of these Ca(2+)-binding proteins was investigated. To this end, the interaction of annexin V and a mutant annexin, INVC, consisting of the N-terminus of annexin I (amino acids 1-45) and the core of annexin V (19-320), with large unilamellar phosphatidylserine (PS) vesicles was examined. In contrast to annexin V, the mutant annexin mediated Ca(2+)-dependent aggregation of the lipid vesicles at neutral pH. However, annexin V induces Ca(2+)-dependent aggregation at mild acidic pH. Moreover, both proteins can engage in hydrophobic interactions with PS vesicles, which results in release of the vesicle contents. These membrane-perturbing properties are expressed by both annexins in the absence of Ca2+ and occur at neutral and mild acidic pH. Interestingly, addition of Ca2+ inhibits annexin V-induced release, but sustains the release induced by the mutant annexin INVC. The Ca(2+)-dependent effects on the release of vesicle contents are reversed upon EDTA addition. Conformational changes revealed by binding of the hydrophobic probe, 4,4'-bis(1-anilino-8-naphthalenesulfonate), underly the observed Ca(2+)-modulated effects on leakage. However, low-pH-mediated aggregation by annexin V does not seem to be related to macroscopic conformational changes. Annexin INVC also affects Ca(2+)-induced fusion of PS vesicles, displaying synergistic properties in conjunction with Ca2+ at neutral pH. By contrast, annexin V does not display similar properties at mild acidic pH, in spite of its ability to aggregate vesicles under such conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Annexins↗

PubMed1997

Identification and characterization of a novel type of annexin-membrane interaction: Ca2+ is not required for the association of annexin II with early endosomes.

Annexin II, a member of a family of Ca2+ and membrane binding proteins, has been implicated in regulating membrane organization and membrane transport during endocytosis and Ca2+ regulated secretion. To characterize the mechanistic aspects of the annexin. II action we studied parameters which determine the endosomal association of annexin II. Immunoblot analysis of subcellular membrane fractions prepared from BHK cells in the presence of a Ca2+ chelating agent reveals that annexin II remains associated with endosomal membranes under such conditions. This annexin II behaviour is atypical for the Ca2+ regulated annexins and is corroborated by the finding that ectopically expressed annexin II mutants with inactivated Ca2+ binding sites continue to co-fractionate with endosomal membranes. The Ca(2+)-independent membrane association of annexin II is also not affected by introducing mutations interfering with the complex formation of annexin II with its intracellular protein ligand p11. However, a deletion of the unique N-terminal domain of annexin II, in particular the sequence spanning residues 15 to 24, abolishes the Ca(2+)-independent association of the protein with endosomes. These results describe a novel, Ca(2+)-independent type of annexin-membrane interaction and provide a first explanation for the observed preference of different annexins for different cellular membranes. In the case of annexin II this specificity could be mediated through specific membrane receptors interacting with a unique sequence in the annexin II molecule.

Amino Acid Sequence↗

PubMed1994

Annexin VI-binding proteins in brain. Interaction of annexin VI with a membrane skeletal protein, calspectin (brain spectrin or fodrin).

Identification of annexin VI-binding proteins is essential to elucidate the physiological functions of annexin VI. Here, we developed the methods to identify an annexin VI-binding protein and characterized the binding. Annexin VI bound to about 14 species of proteins in the whole homogenate of rat forebrain, when examined with 125I-annexin VI using blots of SDS-polyacrylamide gels. The binding was Ca(2+)-dependent and specific for phosphatidylserine (PS) and phosphatidic acid. A line of evidence indicates that the binding of annexin VI to its target proteins is a protein-protein interaction. One of annexin VI-binding proteins with M(r) 240,000 was enriched in the cytoskeletal fraction and was identified as calspectin (brain spectrin or fodrin). When the binding was examined with purified calspectin in the native state, the Ca2+ affinity (KCa) was 7.6 microM, and the affinity for annexin VI (Kd) was 68 nM. Annexin VI bound to beta subunit of calspectin, but not to alpha subunit. The binding site was localized to the NH2-terminal domain of beta subunit, which contains an actin-binding site and exhibits striking homology with the NH2-terminal regions of dystrophin and alpha-actinin. When the effect of annexin VI on the interaction between F-actin and calspectin was examined by low shear viscometry, annexin VI inhibited the F-actin cross-linking activity of calspectin in a Ca2+/PS-dependent manner. Cosedimentation assay showed that annexin VI dissociates calspectin from F-actin in the presence of Ca2+ and PS. These results suggest that annexin VI can dissociate and redistribute calspectin in a Ca2+/phospholipid-dependent manner under the plasma membrane and that annexin VI may be involved in the regulation of the membrane skeleton of neuronal cells in response to Ca2+.

Actins↗

PubMed1993

Aggregation of phospholipid vesicles by a chimeric protein with the N-terminus of annexin I and the core of annexin V.

A chimeric protein was produced with the N-terminal domain (amino acids 1-45) of annexin I and the core of annexin V (amino acids 19-320). This protein, annexin IN-VC, has a similar Ca2+ requirement for binding to phospholipid bilayers of 20% phosphatidylserine (PS)/80% phosphatidylcholine (PC) as annexin V. In contrast to annexin V, this protein has a strong potency to aggregate phospholipid vesicles as is shown by turbidimetric measurements and cryo-electron microscopy. Ellipsometry was employed to study quantitatively the phenomenon of phospholipid vesicle adhesion to annexin IN-VC bound to a planar phospholipid bilayer. The amount of phospholipid vesicles bound by annexin IN-VC on the planar bilayer is proportional to its surface coverage and can be inhibited by coadsorption of annexin V on the planar bilayer or by shielding the phospholipid surface of the vesicles with blood coagulation factor Va. Annexin IN-VC, like annexin V, does not bind to pure PC bilayers, but its adsorption on anionic phospholipid bilayers brings about the capacity to bind pure PC vesicles. This suggests that annexin IN-VC generates or exposes after binding to anionic phospholipids another phospholipid binding site, that differs from the annexin V phospholipid binding site. Collectively, the data suggest that two-dimensional cluster formation of annexin IN-VC on a bilayer with anionic phospholipids is involved in vesicle adherence.

Adsorption↗

PubMed1996

Annexins in the human neuroblastoma SH-SY5Y: demonstration of relocation of annexins II and V to membranes in response to elevation of intracellular calcium by membrane depolarisation and by the calcium ionophore A23187.

The human neuroblastoma SH-SY5Y was found to express annexins I, II, IV, V, and VI by western blot analysis. Calcium-dependent membrane-binding proteins were isolated from SH-SY5Y and analysed by 2-dimensional gel electrophoresis. Proteins with Mr and Pi values similar to those of annexins I, II, III, IV, V, and VI were observed. The identity of annexins II and V was confirmed by western blotting. The membrane association of annexins II and V was studied in cells that had been stimulated to release noradrenaline by K+ depolarisation or by treatment with the ionophore A23187. Annexins II and V were both found to associate with membranes in a manner that was resistant to elution with EGTA and required Triton X-100 for their solubilisation. Homogenisation of cells in calcium-containing buffers also resulted in the formation of EGTA-resistant membrane-associated annexins II and V. The results demonstrate calcium-dependent relocation of annexins II and V to membranes in intact cells and suggest that these annexins bind in a calcium-dependent manner to non-phospholipid components of SH-SY5Y membranes. Examination of cells by immunofluorescence microscopy demonstrated that annexin II was homogeneously associated with the plasma membrane before treatment with ionophore and relocated to discrete patches of staining after treatment. Annexin V was found by immunofluorescence to be present in the cytoplasm and in the nucleus, Stimulation of the cells produced no change in the cytoplasmic staining pattern but resulted in a partial relocation of nuclear annexin V to the periphery of the nucleus. The results argue for a general role for both annexins in calcium signalling at discrete intracellular locations. The results are not consistent with the specific involvement proposed previously for annexin II in membrane fusion at sites of vesicle exocytosis.

Annexin A2↗

PubMed2005

Expression of annexin A3 in primary cultured parenchymal rat hepatocytes and inhibition of DNA synthesis by suppression of annexin A3 expression using RNA interference.

Annexin A3 is a member of the lipocortin/annexin family, which binds to phospholipids and membranes in a Ca(2+)-dependent manner. Although annexin A3 has various functions in vitro, its cellular significance is completely unknown. Annexin A3 is not found in rat liver in vivo. In the present study, we investigated the expression of annexin A3 in primary cultured parenchymal rat hepatocytes. Annexin A3 protein was detected in 48-h, but not 2.5-h, cultured hepatocytes using Western blot analysis. The annexin A3 level further increased after an additional 24 h of culture. Annexin A3 mRNA was not detected in 2.5-h cultured hepatocytes but was detected 22 h after the start of culture by RT-PCR analysis, reaching a maximum value after 48 h of culture. To define the role of Annexin A3 in DNA synthesis, RNA interference was used to reduce annexin III gene expression in hepatocytes. The transfection of small interfering RNAs targeting annexin A3 in the hepatocytes reduced the corresponding mRNA and protein expression by approximately 80% and more than 90%, respectively, at 24 h after transfection. In the annexin A3 small interfering RNAs-transfected cells, DNA synthesis, as assessed by [3H]thymidine incorporation, decreased by approximately 70% not only in the control cultures, but also in the hepatocyte growth factor- or epidermal growth factor-treated cells. These findings show that annexin A3 is expressed in primary cultured parenchymal rat hepatocytes and that the suppression of annexin A3 expression using RNA interference inhibits DNA synthesis.

Animals↗

PubMed1997

Distinct annexin subfamilies in plants and protists diverged prior to animal annexins and from a common ancestor.

Annexin homologues in the kingdoms of Planta and Protista were characterized by molecular sequence analysis to determine their phylogenetic and structural relationship with annexins of Animalia. Sequence fragments from 19 plant annexins were identified in sequence databases and composite sequences were also assembled from expressed sequence tags for Arabidopsis thaliana. Length differences in protein aminotermini and evidence for unique exon splice sites indicated that plant annexins were distinct from those of animals. A third annexin gene of Giardia lamblia (Anx21-Gla) was identified as a distant relative to other protist annexins and to those of higher eukaryotes, thus providing a suitable outgroup for evolutionary reconstruction of the family tree. Rooted evolutionary trees portrayed protist, plant, and Dictyostelium annexins as early, monophyletic ramifications prior to the appearance of closely related animal annexin XIII. Molecular phylogenetic analyses of DNA and protein sequence alignments revealed at least seven separate plant subfamilies, represented by Anx18 (alfalfa, previously classified), Anx22 (thale cress), Anx23 (thale cress, cotton, rape and cabbage), Anx24 (bell pepper and tomato p34), Anx25 (strawberry, horseradish, pea, soybean, and castor bean), Anx26-Zma, and Anx27-Zma (maize). Other unique subfamilies may exist for rice, tomato p35, apple, and celery annexins. Consensus sequences compiled for each eukaryotic kingdom showed some breakdown of the "annexin-fold" motif in repeats 2 and 3 of protist and plant annexins and a conserved codon deletion in repeat 3 of plants. The characterization of distinct annexin genes in plants and protists reflects their comparable diversity among animal species and offers alternative models for the comparative study of structure-function relationships within this important gene family.

Amino Acid Sequence↗