2D crystal forms of annexin IV on lipid monolayers.

Two-dimensional crystalline arrays of annexin IV were generated by interaction of the purified protein with a phospholipid monolayer. Image analysis of electron micrographs of the protein crystals, which diffracted to 3.5 nm respectively, revealed p6 and p3 symmetry. Annexin IV gave two crystal forms with unit cells of 18 x 18 nm and 28 x 28 nm. The former unit cell was similar to a previously described form of annexin VI. The implications of these observations are discussed.     

Inhibition of EGF-dependent calcium influx by annexin VI is splice form-specific.

Annexin VI is a widely expressed calcium- and phospholipid-binding protein that lacks a clear physiological role. We now report that A431 cells expressing annexin VI are defective in their ability to sustain elevated levels of cytosolic Ca(2+) following stimulation with EGF. Other aspects of EGF receptor signaling, such as protein tyrosine phosphorylation and induction of c-fos are normal in these cells. However, EGF-mediated membrane hyperpolarization is attenuated and Ca(2+) entry abolished in cells expressing annexin VI. This effect of annexin VI was only observed for the larger of the two annexin VI splice forms, the smaller splice variant had no discernable effect on either cellular phenotype or growth rate. Inhibition of Ca(2+) influx was specific for the EGF-induced pathway; capacitative Ca(2+) influx initiated by emptying of intracellular stores was unaffected. These results provide the first evidence that the two splice forms of annexin VI have different functions.     

The Structure of Recombinant Human Annexin VI in Crystals and Membrane-bound

The crystal structure of calcium-free recombinant human annexin VI was solved at a resolution of 3.2 Å by using the annexin I model for Patterson search and refined to anR-factor of 19.0%. The molecule consists of two similar halves closely resembling annexin I connected by an α-helical segment and arranged perpendicular to each other. The calcium and membrane binding sites assigned by structural homology are therefore not located in the same plane. Analysis of the membrane-bound form of annexin VI by electron microscopy shows the two halves of the molecule coplanar with the membrane, but oriented differently to the crystal structure and suggesting a flexible arrangement. Ion channel activity has been found for annexin VI and the half molecules by electrophysiological experiments.     

Structural and dynamic changes in human annexin VI induced by a phosphorylation-mimicking mutation,T356D

Phosphorylation of some members of the annexin family of proteins may play a significant role in controlling their calcium-dependent interactions with membranes. Recent electron microscopic studies of annexin VI revealed that the protein's two core domains exhibit a great degree of flexibility and are able to undergo a relative conformational change that could potentially initiate contacts between membranes [Avila-Sakar, A. J., et al. (2000) J. Struct. Biol. 130, 54-62]. To assess the possibility of a regulatory role of phosphorylation in this behavior, the crystal structure of a phosphorylation-mimicking mutant (T356D in the flexible connector region of human annexin VI) was determined to 2.65 A resolution. When the mutant is compared to the wild-type annexin VI, subtle differences are seen at the site of the mutation, while larger changes are evident in one of the calcium-binding loops and in the presence of five calcium ions. Furthermore, biochemical studies provide evidence for additional conformational differences between the T356D and wild-type solution structures. Fluorescence emission and acrylamide quenching suggest a higher level of solvent exposure of Trp-343 in the connector region of T356D in the presence of calcium. Comparisons of retardation coefficients in native gel electrophoresis reveal that T356D has a more extended shape, while proteolytic studies show a greater accessibility of a trypsin cleavage site inside the linker region, indicating a conformation more open than the wild-type form. These data provide insights into a possible regulatory mechanism leading to a higher degree of flexibility and possibly a higher calcium binding affinity of annexin VI upon phosphorylation.     

Human apolipoprotein H may have various orientations when attached to lipid layer

Apolipoprotein H (ApoH), also known as beta(2)-glycoprotein I, is a plasma glycoprotein with its in vivo physiological and pathogenic roles being closely related to its interaction with negatively charged membranes. Although the three-dimensional crystal structure of ApoH has been recently solved, direct evidence about the spatial state of ApoH on the membrane is still lacking. In this work, the interactions of ApoH with the lipid layer are studied by a combination of lipid monolayer approach and surface concentration determination. The spatial state of the orientation of ApoH on the lipid layer is investigated by analyzing the process of membrane-attached ApoH molecules being extruded out from the phospholipid monolayer by compression. The results show that on neutral lipid layer ApoH has an upright orientation, which is not sensitive to the phase state of the lipid layer. However, on acidic lipid layer, ApoH may have two forms of orientation. One is an upright orientation in the liquid phase region, and the other is flat orientation on the condensed domain region. The variation of the spatial state of ApoH on the lipid layer may relate to a variety of its physiological functions.     

The roles of annexins and types II and X collagen in matrix vesicle-mediated mineralization of growth plate cartilage

Annexins II, V, and VI are major components of matrix vesicles (MV), i.e. particles that have the critical role of initiating the mineralization process in skeletal tissues. Furthermore, types II and X collagen are associated with MV, and these interactions mediated by annexin V stimulate Ca(2+) uptake and mineralization of MV. However, the exact roles of annexin II, V, and VI and the interaction between annexin V and types II and X collagen in MV function and initiation of mineralization are not well understood. In this study, we demonstrate that annexin II, V, or VI mediate Ca(2+) influx into phosphatidylserine (PS)-enriched liposomes, liposomes containing lipids extracted from authentic MV, and intact authentic MV. The annexin Ca(2+) channel blocker, K-201, not only inhibited Ca(2+) influx into fura-2-loaded PS-enriched liposomes mediated by annexin II, V, or VI, but also inhibited Ca(2+) uptake by authentic MV. Types II and X collagen only bound to liposomes in the presence of annexin V but not in the presence of annexin II or VI. Binding of these collagens to annexin V stimulated its Ca(2+) channel activities, leading to an increased Ca(2+) influx into the liposomes. These findings indicate that the formation of annexin II, V, and VI Ca(2+) channels in MV together with stimulation of annexin V channel activity by collagen (types II and X) binding can explain how MV are able to rapidly take up Ca(2+) and initiate the formation of the first crystal phase.     

Annexin VI Binds to a Synaptic Vesicle Protein, Synapsin I

Abstract: Annexin VI bound to >14 species of proteins in the whole homogenate of rat forebrain in a Ca²⁺/phosphatidylserine- or phosphatidic acid-dependent manner. When the subcellular fractions of rat forebrain were examined with a blot from a sodium dodecyl sulfate-polyacrylamide gel, each annexin VI-binding protein showed a different distribution, suggesting that annexin VI is a multifunctional protein. Of these proteins, the doublets of M_r 80,000 were enriched in the purified synaptic vesicles and were identified as synapsin I. Annexin VI bound to the head domain of synapsin I. When the binding of annexin VI to synapsin I was characterized in the native state, the affinity of the binding for Ca²⁺ ( K _Ca) was 12.6 µ M , and the affinity for annexin VI ( K _D) was ∼270 n M . Phosphorylation of synapsin I by cyclic AMP-dependent protein kinase and by Ca²⁺/calmodulin-dependent protein kinase II inhibited the annexin VI binding. The mode of the inhibition was different between the two kinases. These results indicate that annexin VI may modulate the function of synapsin I in a Ca²⁺- and phospholipid-dependent manner.     

Trimers, dimers of trimers, and trimers of trimers are common building blocks of annexin a5 two-dimensional crystals

Annexin A5 is a member of a family of homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca(2+)-dependent manner. Annexin A5, as well as other annexins, self-assembles into two-dimensional (2D) ordered arrays upon binding to membranes, a property that has been proposed to have functional implications. Electron microscopy and atomic force microscopy experiments have revealed that annexin A5 forms two types of 2D crystals-with either p6 or p3 symmetry-that are both based on annexin trimers. In this study, we describe three other crystal forms that coexist with the p6 crystals. All crystal forms are made of the same building blocks, namely, dimers of trimers and trimers of trimers. A mechanistic model of the formation of the annexin A5 2D crystals is proposed.     

Investigating the role played by protein-lipid and protein-protein interactions in the membrane association of the p120GAP CaLB domain.

The GTPase activating protein, p120GAP, contains an amino acid sequence motif called the Ca2+-dependent lipid binding domain (CaLB) which mediates a protein-protein interaction between p120GAP and annexin VI and also binds to negatively charged phospholipids. Because membrane association of p120GAP is important for the regulation of p21 Ras activity, we have studied the roles played by Ca2+, phospholipids and annexin VI in the membrane association of p120GAP. Here we demonstrate that a truncated CaLB domain GST fusion protein (GSTGAP618-632), lacking the ability to bind to phospholipids, is able to bind to rat fibroblast membranes in a Ca2+- and concentration-dependent manner. In addition, this fusion protein also binds to annexin VI in an amino acid sequence specific but Ca2+ independent manner. Also, when bound to annexin VI in the presence of Ca2+, this fusion protein has the ability to co-bind to phosphatidylserine vesicles. Thus, annexin VI may simultaneously mediate an interaction with p120GAP and also an interaction with membrane phospholipids. This may in part explain the mechanism by which p120GAP associates with membranes in response to Ca2+ elevation and suggests the potential importance of annexin VI in the regulation of p21 Ras and the role CaLB domains may play in the specific recognition of cellular membranes.     

LOCALIZATION OF ANNEXIN VI IN THE ADULT AND NEONATAL HEART

Annexins are a major family of intracellular Ca²⁺-binding proteins which have been implicated in a variety of cellular functions. In this paper the authors have used confocal microscopy to compare the distribution of annexin VI in vibratome sections of the rat adult left ventricle and striated muscle of the rat oesophagus. It is shown that in rat cardiac myocytes annexin VI is associated with only the sarcolemma and intercalated discs. In contrast, it is demonstrated that in rat skeletal muscle annexin VI is associated with the sarcoplasmic reticulum, in addition to the plasma membrane, suggesting that annexin VI is regulating different processes in these tissues. Also shown is that in vibratome sections of the neonatal rat left ventricle, annexin VI has a different subcellular location to that observed in the terminally differentiated adult myocyte. In these differentiating neonatal cells annexins VI is also associated with specific subcellular structures. Furthermore, using confocal microscopy of isolated myocytes the authors demonstrate that the association of annexin VI with the sarcolemma is stable even after cells are treated with the intracellular calcium chelator BAPTA-AM, to greatly deplete cytosolic calcium levels. This demonstrates that annexin VI associates tightly with the sarcolemma, and suggests that components in addition to phospholipid are involved in binding annexin VI to the membrane. These results demonstrate that the subcellular location of annexin VI is differentially regulated, and suggest that annexin VI is required for a process or processes characteristic of the sarcolemma, and of the sarcoplasmic reticulum of skeletal but not of heart muscle.     

S100A1 and S100B interactions with annexins

Members of the annexin protein family interact with members of the S100 protein family thereby forming heterotetramers in which an S100 homodimer crossbridges two copies of the pertinent annexin. Previous work has shown that S100A1 and S100B bind annexin VI in a Ca(2+)-dependent manner and that annexin VI, but not annexin V, blocks the inhibitory effect of S100A1 and S100B on intermediate filament assembly. We show here that both halves of annexin VI (i.e., the N-terminal half or annexin VI-a and the C-terminal half or annexin VI-b) bind individual S100s on unique sites and that annexin VI-b, but not annexin VI-a, blocks the ability of S100A1 and S100B to inhibit intermediate filament assembly. We also show that the C-terminal extension of S100A1 (and, by analogy, S100B), that was previously demonstrated to be critical for S100A1 and S100B binding to several target proteins including intermediate filament subunits, is not part of the S100 surface implicated in the recognition of annexin VI, annexin VI-a, or annexin VI-b. Evaluation of functional properties with a liposome stability and a calcium influx assay reveals the ability of both S100 proteins to permeabilize the membrane bilayer in a similar fashion like annexins. When tested in combinations with different annexin proteins both S100 proteins mostly lead to a decrease in the calcium influx activity although not all annexin/S100 combinations behave in the same manner. Latter observation supports the hypothesis that the S100-annexin interactions differ mechanistically depending on the particular protein partners.     

Modulation of Ca2+ release channel activity from sarcoplasmic reticulum by annexin VI (67-kDa calcimedin)

The effect of annexin VI (67-kDa calcimedin) on the activity of the Ca2+ release channel was studied using heavy sarcoplasmic reticulum membranes reconstituted into planar bilayers. Annexin VI, in a range of 5-40 nM, modified the gating behavior of the Ca2+ release channel by increasing the probability of opening by 2.7-fold and the mean open time by 82-fold relative to controls. Annexin VI caused no change in the slope conductance of the channel. The modulatory effect of annexin VI on the activity of Ca2+ release channels was Ca2+ dependent, and the annexin VI-modified channel was sensitive to both ruthenium red and ryanodine. The effect of annexin VI was observed when this protein was added specifically to the trans chamber, which corresponds to the luminal side of sarcoplasmic reticulum as determined by the ATP activation of the channel. In addition, differential extraction studies demonstrated that some annexin VI is localized within the lumen of the isolated heavy sarcoplasmic reticulum vesicles prepared by several different procedures. Annexin VI did not modify, from either the cis or trans chambers, the activity of K+ or Cl- channels from sarcoplasmic reticulum or the dihydropyridine sensitive Ca2+ channel from transverse tubules. In addition, the 38-kDa core proteolytic fragments of annexin VI had no effect on the Ca2+ release channel activity. Annexin VI is therefore a candidate for a physiological modulator of the Ca2+ release channel and as such, may play an important role in the excitation-contraction coupling.     

Endocytosis occurs independently of annexin VI in human A431 cells

Annexin VI is one of a family of calcium-dependent phospholipid-binding proteins. Although the function of this protein is not known, various physiological roles have been proposed, including a role in the budding of clathrin-coated pits (Lin et al., 1992. Cell. 70:283-291.). In this study we have investigated a possible endocytotic role for annexin VI in intact cells, using the human squamous carcinoma cell line A431, and report that these cells do not express endogenous annexin VI, as judged by Western and Northern blotting and PCR/Southern blotting. To examine whether endocytosis might in some way be either facilitated or inhibited by the presence of annexin VI, a series of A431 clones were isolated in which annexin VI expression was achieved by stable transfection. These cells expressed annexin VI at similar levels to other human cell types. Using assays for endocytosis and recycling of the transferrin receptor, we report that each of these cellular processes occurs with identical kinetics in both transfected and wild- type A431 cells. In addition, purified annexin VI failed to support the scission of coated pits in permeabilized A431 cells. We conclude that annexin VI is not an essential component of the endocytic pathway, and that in A431 cells, annexin VI fails to exert any influence on internalization and recycling of the transferrin receptor.     

Interaction of annexin VI with actin

Actin polymerization was investigated using fluorescence probe N-(1-pyrenyl)iodoacetamide, which was bound covalently to reactive sulfhydryl group, Cys-373. Labeled actin in the bulk was 0.5 to 1% of total actin concentration. Actin polymerization at concentration 12 mM was started by addition of 20 mM KCl and 2 mM MgCl2. The label fluorescence was excited at 365 nm and registered at 386 nm. Under actin polymerization the label fluorescence increased almost 10 times. Two main phases may be distinguished in the process of actin polymerization: 1) monomer activation and nucleus (trimer) formation, 2) growth of actin filaments on the nuclei. In our experimental conditions, both for pure actin and for that with added annexin VI, the 1st phase continued for about 3 min and after that the 2nd phase was perfectly approximated by exponential dependence. An analysis of the exponential curves showed that actin monomer lifetime increased from 327 s, at annexin absence, to about 373 s at 0.7 microM annexin and more. Calculation of rate constants at two ends of growing actin filament suggests that annexin VI binds with pointed ("slow") end so that at sufficient annexin concentration the filament grows only on barbed ("fast") end. Our results, together with data of other researchers showing that annexin VI binds with the inner membrane surface of smooth muscle cell through Ca2+, may indicate that, at Ca2+ entering the cell, this annexin binds actin filament pointed ends to cell surface making it ready for the act of contraction.     

Regulation of the sarcoplasmic reticulum Ca(2+)-release channel requires intact annexin VI.

Annexin VI has eight highly conserved repeated domains; all other annexins have four. Díaz-Mu?oz et al. (J Biol Chem 265:15894, 1990) reported that annexin VI alters the gating properties of the ryanodine-sensitive Ca(2+)-release channel isolated from sarcoplasmic reticulum. The investigate the domain structure of rat annexin VI (67 kDa calcimedin) required for this channel regulation, various proteolytic digestions were performed. In each case, protease-resistant core polypeptides were produced. Annexin VI was digested with V8 protease and two core polypeptides were purified by Ca(2+)-dependent phospholipid binding followed by HPLC. The purified fragments were shown to be derived from the N- and C-terminal halves of annexin VI, and demonstrated differential immunoreactivity with monoclonal antibodies to rat annexin VI. While both core polypeptides retained their ability to bind phospholipids in a Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-release channel as did intact annexin VI.     

Immunological development and cardiovascular function are normal in annexin VI null mutant mice

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.     

Annexin VI stimulates endocytosis and is involved in the trafficking of low density lipoprotein to the prelysosomal compartment

Annexins are calcium-binding proteins with a wide distribution in most polarized and nonpolarized cells that participate in a variety of membrane-membrane interactions. At the cell surface, annexin VI is thought to remodel the spectrin cytoskeleton to facilitate budding of coated pits. However, annexin VI is also found in late endocytic compartments in a number of cell types, indicating an additional important role at later stages of the endocytic pathway. Therefore overexpression of annexin VI in Chinese hamster ovary cells was used to investigate its possible role in endocytosis and intracellular trafficking of low density lipoprotein (LDL) and transferrin. While overexpression of annexin VI alone did not alter endocytosis and degradation of LDL, coexpression of annexin VI and LDL receptor resulted in an increase in LDL uptake with a concomitant increase of its degradation. Whereas annexin VI showed a wide intracellular distribution in resting Chinese hamster ovary cells, it was mainly found in the endocytic compartment and remained associated with LDL-containing vesicles even at later stages of the endocytic pathway. Thus, data presented in this study suggest that after stimulating endocytosis at the cell surface, annexin VI remains bound to endocytic vesicles to regulate entry of ligands into the prelysosomal compartment.     

Late endocytic compartments are major sites of annexin VI localization in NRK fibroblasts and polarized WIF-B hepatoma cells

Annexin VI is an abundant calcium- and phospholipid-binding protein whose intracellular distribution and function are still controversial. Using a highly specific antibody, we have studied the distribution of annexin VI in NRK fibroblasts and the polarized hepatic cell line WIF-B by confocal microscopy. In NRK cells, annexin VI was almost exclusively found associated with endocytic compartments, which were defined by their ability to receive fluid-phase marker internalized from the cell surface. However, extensive colocalization of annexin VI and the endocytic marker was only observed after about 45 min, indicating that annexin VI was primarily in late endocytic compartments or (pre)lysosomes. Consistent with this, annexin VI was predominantly seen on structures that contained the lysosomal protein lgp120, although not on dense core lysosomes by electron microscopy. Two major populations of annexin VI-containing structures were present in polarized WIF-B hepatocytes. One correlated to lgp120-positive (pre)lysosomes and was still observed after treatment with brefeldin A (BFA), while the other appeared to be partially associated with Golgi membranes and was BFA-sensitive. The striking association with prelysosomal compartments in NRK and WIF-B cells suggests that annexin VI could play a role in fusion events in the late endocytic pathway, possibly by acting as a tether between membranes.     

Annexin VI is required for budding of clathrin-coated pits.

Isolated plasma membranes attached to a solid substratum at 4 degrees C have numerous clathrin-coated pits. These pits initially are flat but become deeply invaginated after warming to 37 degrees C. The pits remain tethered to the membrane in this rounded condition unless supplied with ATP, Ca2+, and cytosol. We now show that when cytosol is treated to remove the Ca(2+)-dependent, phospholipid-binding protein annexin VI, coated pit budding no longer takes place. Addition of purified annexin VI back to the annexin VI-depleted cytosol restores budding activity to normal. Purified annexin VI alone shows only a modest budding activity, suggesting that the cytosol contains a factor(s) in addition to annexin VI that is required for full activity. Cytosol-dependent activation of annexin VI requires both ATP and Ca2+. Annexin VI appears to be not only an active component in the detachment of coated pits from the membrane but also a site for regulating the formation of coated vesicles.     

Expression of annexin VI (p68, 67 kDa-calelectrin) in normal human tissues: evidence for developmental regulation in B- and T-lymphocytes.

This paper describes the tissue distribution of annexin VI, a Ca(2+)-dependent phospholipid binding protein, and a member of the annexin super-gene family. In order to determine whether annexin VI expression correlated with a particular functional phenotype, an extensive series of non-pathological human tissues were examined, in which annexin VI was detected either immunohistochemically or by immunofluorescence, using a rabbit polyclonal anti(human annexin VI)-IgG of known specificity. Although most tissues investigated were found to express annexin VI, the protein was usually confined to highly specific cell types within each tissue, the staining generally appearing cytoplasmic and diffuse. There was particularly good correlation between annexin VI expression and hormone secreting cells, with positive staining in the islet cells of the pancreas, the Leydig cells of the testis and the cells of the adrenal cortex. A notable exception was the parathyroid gland, which lacked detectable annexin VI. Although the protein was absent in most epithelia, it was expressed strongly in certain secretory epithelia; e.g. the ductal epithelial cells of the salivary glands and non-lactating breast, and the sweat glands and their ducts. The observation that the epithelial cells of lactating breast failed to stain for annexin VI suggests functional regulation of protein expression in this tissue. However, the most interesting finding was that annexin VI expression appeared to be developmentally regulated in B- and T-lymphocyte differentiation, with negative staining in the proliferating B cells of the germinal centre of the lymph nodes, but strong staining in the mature small lymphocytes of the cortex, mantle zone and paracortex.