Characterization of caged compounds binding to proteins by NMR spectroscopy.

Photolysable caged ligands are used to investigate protein function and activity. Here, we investigate the binding properties of caged nucleotides and their photo released products to well established but evolutionary and structurally unrelated nucleotide-binding proteins, rabbit muscle creatine kinase (RMCK) and human annexin A6 (hAnxA6), using saturation transfer difference NMR spectroscopy. We detect the binding of the caged nucleotides and discuss the general implications on interpreting data collected with photolysable caged ligands using different techniques. Strategies to avoid non-specific binding of caged compound to certain proteins are also suggested.

Characterization of a recombinant form of annexin VI for detection of apoptosis.

We developed a recombinant form of human annexin VI called annexin VI-601 (M(r) 76,224) with the N-terminal extension of Ala-Gly-Gly-Cys-Gly-His to allow ready attachment of fluorescent or radioactive labels. The protein was produced by expression in E. coli and was purified by calcium-dependent membrane binding, anion-exchange chromatography, and heparin-Sepharose affinity chromatography. The protein could be readily labeled with iodoacetamidofluorescein and with (99m)Tc. The protein bound with high affinity to PS-containing phospholipid vesicles and to erythrocytes with exposed phosphatidylserine. Fluorescent annexin VI-601 readily detected apoptosis of Jurkat cells by flow cytometry at much lower calcium concentrations than those required for equivalent detection by annexin V. In vivo administration of radiolabeled protein showed that blood clearance was much slower than annexin V. In conclusion, annexin VI may have advantages over annexin V in certain situations for both in vitro and in vivo detection of apoptosis and therapeutic targeting of PS due to its lower calcium requirement for membrane binding and its higher molecular weight.

Annexin-A6 in Membrane Repair of Human Skeletal Muscle Cell: A Role in the Cap Subdomain.

Defects in membrane repair contribute to the development of some muscular dystrophies, highlighting the importance to decipher the membrane repair mechanisms in human skeletal muscle. In murine myofibers, the formation of a cap subdomain composed notably by annexins (Anx) is critical for membrane repair. We applied membrane damage by laser ablation to human skeletal muscle cells and assessed the behavior of annexin-A6 (AnxA6) tagged with GFP by correlative light and electron microscopy (CLEM). We show that AnxA6 was recruited to the site of membrane injury within a few seconds after membrane injury. In addition, we show that the deficiency in AnxA6 compromises human sarcolemma repair, demonstrating the crucial role played by AnxA6 in this process. An AnxA6-containing cap-subdomain was formed in damaged human myotubes in about one minute. Through transmission electron microscopy (TEM), we observed that extension of the sarcolemma occurred during membrane resealing, which participated in forming a dense lipid structure in order to plug the hole. By properties of membrane folding and curvature, AnxA6 helped in the formation of this tight structure. The compaction of intracellular membranes-which are used for membrane resealing and engulfed in extensions of the sarcolemma-may also facilitate elimination of the excess of lipid and protein material once cell membrane has been repaired. These data reinforce the role played by AnxA6 and the cap subdomain in membrane repair of skeletal muscle cells.

Annexins sense changes in intracellular pH during hypoxia.

The pH(i) (intracellular pH) is an important physiological parameter which is altered during hypoxia and ischaemia, pathological conditions accompanied by a dramatic decrease in pH(i). Sensors of pH(i) include ion transport systems which control intracellular Ca2+ gradients and link changes in pH(i) to functions as diverse as proliferation and apoptosis. The annexins are a protein family characterized by Ca2+-dependent interactions with cellular membranes. Additionally, in vitro evidence points to the existence of pH-dependent, Ca(2+)-independent membrane association of several annexins. We show that hypoxia promotes the interaction of the recombinant annexin A2-S100A10 (p11) and annexin A6 with the plasma membrane. We have investigated in vivo the influence of the pH(i) on the membrane association of human annexins A1, A2, A4, A5 and A6 tagged with fluorescent proteins, and characterized this interaction for endogenous annexins present in smooth muscle and HEK (human embryonic kidney)-293 cells biochemically and by immunofluorescence microscopy. Our results show that annexin A6 and the heterotetramer A2-S100A10 (but not annexins A1, A4 and A5) interact independently of Ca2+ with the plasma membrane at pH 6.2 and 6.6. The dimerization of annexin A2 within the annexin A2-S100A10 complex is essential for the pH-dependent membrane interaction at this pH range. The pH-induced membrane binding of annexins A6 and A2-S100A10 might have consequences for their functions as membrane organizers and channel modulators.

Annexin A6 at the cardiac myocyte sarcolemma--evidence for self-association and binding to actin.

The plasma membrane of the heart muscle cell and its underlying cytoskeleton are vitally important to the function of the heart. Annexin A6 is a major cellular calcium and phospholipid binding protein. Here we show that annexin A6 copurifies with sarcolemma isolated from pig heart. Two pools of annexin A6 are present in the sarcolemma fraction, one dependent on calcium and one that resists extraction by the calcium chelator EGTA. Potential annexin A6 binding proteins in the sarcolemma fraction were identified using Far Western blotting. Two major annexin A6 binding proteins were identified as actin and annexin A6 itself. Annexin A6 bound to itself both in the presence and in the absence of calcium ions. Sites for self association were mapped by performing Western blots on proteolytic fragments of recombinant annexin A6. Annexin A6 bound preferentially not only to the N terminal fragment (domains I-IV, residues 1-352) but also to C-terminal fragments corresponding to domains V+VI and domains VII+VIII. Actin binding to annexin A6 was calcium-dependent and exclusively to the N-terminal fragment of annexin A6. A calcium-dependent complex of annexin A6 and actin may stabilize the cardiomyocyte sarcolemma during cell stimulation.

Clustering of protein structures using hydrophobic free energy and solvent accessibility of proteins.

The hydrophobic free energy and solvent accessibility of amino acids are used to study the relationship between the primary structure and structural classification of large proteins. A measure representation and a Z curve representation of protein sequences are proposed. Fractal analysis of the measure and Z curve representations of proteins and multifractal analysis of their hydrophobic free energy and solvent accessibility sequences indicate that the protein sequences possess correlations and multifractal scaling. The parameters from the fractal and multifractal analyses on these sequences are used to construct some parameter spaces. Each protein is represented by a point in these spaces. A method is proposed to distinguish and cluster proteins from the alpha, beta, alpha + beta, and alpha/beta structural classes in these parameter spaces. Fisher's linear discriminant algorithm is used to give a quantitative assessment of our clustering on the selected proteins. Numerical results indicate that the discriminant accuracies are satisfactory. In particular, they reach 94.12% and 88.89% in separating proteins from {alpha, alpha + beta, alpha/beta} proteins in a three-dimensional space.

Mechanism of Fibronectin Binding to Human Trabecular Meshwork Exosomes and Its Modulation by Dexamethasone.

Exosomes are emerging as important mediators of cell-matrix interactions by means of specific adhesion proteins. Changes in the tissue-specific exosomal protein expression may underlie pathological conditions whereby extracellular matrix turnover and homeostasis is disrupted. Ocular hypertension due to extracellular matrix accumulation in the trabecular meshwork is a hallmark of glucocorticoid-induced glaucoma. In the trabecular meshwork, exosomal fibronectin mediates cell matrix interactions at cellular structures called "invadosomes". Trabecular meshwork cells use invadosomes to turn over their surrounding matrix and maintain passageways for flow of aqueous humor. In this study, we observed that human trabecular meshwork explants treated with dexamethasone released exosomes with significantly reduced amounts of fibronectin bound per exosome. Further, we found that exosome-fibronectin binding is heparan sulfate-dependent, consistent with our observation that trabecular meshwork exosomes are enriched in the heparin/heparan sulfate binding annexins A2 and A6. In this way, dexamethasone-treated explants released exosomes with a significant reduction in annexin A2 and A6 per exosome. Interestingly, we did not detect exosomal matrix metalloproteinases, but we identified abundant dipeptidyl peptidase 4, a serine protease whose activity was reduced on exosomes isolated from dexamethasone-treated explants. Together, our findings demonstrate mechanistically how corticosteroid-induced alterations in exosomal adhesion cargo and properties can account for the pathological matrix accumulation seen in many glaucoma patients.

The crystal structure of annexin VI indicates relative rotation of the two lobes upon membrane binding.

The crystal structure of bovine liver annexin VI has been determined to low resolution by molecular replacement. The first lobe (domains 1-4) is rotated about 90 degrees relative to the second lobe (domains 5-8). Since the same crystal form (P4(3), 68 X 68 X 205 A) grew from (NH4)2SO4, polyethylene glycol, and sodium acetate with and without added calcium, this probably reflects the structure in solution. When bound to a lipid monolayer both lobes of annexin VI are coplanar. This implies a significant change in conformation upon binding to membranes.

Annexin VI isoforms are differentially expressed in mammalian tissues.

Purified annexin VI migrates as a closely spaced doublet when separated by SDS-PAGE. Immunolocalization of annexin VI in heart demonstrates staining at different defined subcellular compartments. Moss et al. identified two cDNAs, one having an insert of 18 bases encoding VAAEIL at the beginning of repeat domain seven. We have identified the splicing site of the murine annexin VI gene. It contains a single small exon of 18 bases. PCR amplification of reverse transcribed (RT) mRNA demonstrates that, in all tissues tested, the mRNA isoform containing the insert is predominant. Site-directed antibody was produced and affinity purified against peptides reflecting the insert and deletion sequences. The steady-state isoform ratio of the annexin VI protein is consistent with the RT-PCR data. Chromatographic experiments demonstrate that the annexin VI protein isoforms have biochemical differences. These differences may target the individual isoforms to unique cellular compartments or alter functional properties.

Characterization and immunolocalization of rat liver annexin VI.

Annexin VI has been purified to homogeneity from rat liver and monospecific antibodies have been produced. The antibodies have been used for immunoblot analysis of rat tissues. Annexin VI is present in most tissues, with particularly high concentrations in liver, spleen, muscle, and intestine. In liver, annexin VI constitutes approximately 0.25% of total cellular protein. Immunohistochemical studies have located annexin VI on plasma membranes of hepatocytes with enhanced concentration on bile canaliculi. Annexin VI binds in a Ca(2+)-dependent manner to a sub-cellular fraction containing membranes. In the presence of physiological concentrations of ATP, the free Ca2+ concentration required for half-maximal binding of annexin VI to membranes is significantly reduced. While annexin VI binds in vitro to membranes in the presence of Ca2+, in rat liver about 31% of the annexin VI is associated with membranes in a Ca(2+)-independent manner and its solubilization requires the presence of Triton X-100. However, studies using Triton X-114 showed no increase in the hydrophobicity of this fraction of the protein compared to the purified EGTA-soluble annexin VI.

Crystal structure of bovine annexin VI in a calcium-bound state.

The crystal structure of a calcium-bound form of bovine annexin VI has been determined with X-ray diffraction data to 2.9 A by molecular replacement. Six Ca2+ ions were found, five in AB loops, one in a DE loop. Two loops (II-AB, which binds calcium, and V-AB, which does not) have conformations that differ significantly from those in calcium-free, human recombinant annexin VI. There are only small differences between the calci- and the apo-annexin VI in the rest of the molecule. Calcium by itself does not promote a major conformational change.

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.

In adrenocortical tissue, annexins II and VI are attached to clathrin coated vesicles in a calcium-independent manner.

We have previously characterized three populations of clathrin coated vesicles (CCVs) isolated from bovine adrenocortical tissue and designated them as large, medium and small coated vesicles, i.e., LCV, MCV and SCV, respectively. Here, we show that annexins II and VI, two of the annexins involved in membrane traffic, are present in the three populations of CCVs but with different distributions between coat proteins (CP) and lipidic vesicle membrane. Annexin VI is only associated with the membrane, whatever the CCV population. In contrast, annexin II is differently distributed between coat and membrane, depending on the CCV population. Both annexins are bound to membranes in a calcium-independent manner and solubilization studies in Triton X114 (TX114) suggest that they interact poorly with lipids by hydrophobic interactions. Ligand blotting experiments show that both annexins bind to CCV proteins: annexin II to a 200-kDa component in all CCVs and annexin VI to a 100-kDa component in LCV and SCV identified as dynamin, a GTPase essential for endocytic CCV pinching off. Dynamin is tightly associated to annexin VI only in LCVs, the endocytic [transferrin (Tf) positive] vesicles. Our data suggest that annexins II and VI could define specific protein-lipid interaction microdomains that could play a role in the different functions of the CCVs.

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 A by using the annexin I model for Patterson search and refined to an R-factor of 19.0%. The molecule consists of two similar halves closely resembling annexin I connected by an alpha-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.

The effect of annexin IV and VI on the fluidity of phosphatidylserine/phosphatidylcholine bilayers studied with the use of 5-deoxylstearate spin label.

An effect of annexin IV and VI on the fluidity of phosphatidylserine/phosphatidylcholine (PS/PC) membranes was studied by spin labeling technique with the use of 5-doxylstearic acid. It was found that calcium ions at micromolar concentrations induced a marked decrease in the order parameter of PS/PC membranes. This effect was completely abolished by both annexins. The influence of annexins on the mobility of fatty acid chains in the studied region in PE/PC membranes was insignificant.

Conformational states of annexin VI in solution induced by acidic pH.

Acidic pH-induced folding of annexin (Anx)VI in solution was investigated in order to study the mechanism of formation of ion channels by the protein in membranes. Using 2-(p-toluidino)naphthalene-6-sulfonic acid as a hydrophobic probe, it was demonstrated that AnxVI exerts a large change in hydrophobicity at acidic pH. Moreover, circular dichroism spectra indicated that the native state of AnxVI changes at acidic pH towards a state characterized by a significant loss of alpha-helix content and appearance of new beta-structures. These changes are reversible upon an increase of pH. It is postulated that the structural folding of AnxVI could explain how a soluble protein may undergo transition into a molecule able to penetrate the membrane hydrophobic region. The physiological significance of these observations is discussed.

Mapping the site of interaction between annexin VI and the p120GAP C2 domain.

Annexin VI is a Ca(2+)-dependent membrane and phospholipid binding protein. It mediates a protein-protein interaction with the Ras p21 regulatory protein p120GAP. In this study we have mapped the binding site of GAP within the annexin VI protein. Using Far Western overlay binding assays and cell lysate competition studies we have mapped the site of interaction to the inter-lobe linker region; amino acids 325-363. Finally, using a GST fusion protein corresponding to this linker region we have demonstrated that cellular loading of the fusion protein into Rat-1 fibroblasts by electroporation blocks the interaction and co-immunoprecipitation of annexin VI and GAP.

Annexin VI interacts with adenine nucleotides and their analogs.

Annexin VI (AnxVI), a member of the annexin family of Ca2+- and membrane-binding proteins, has been shown to interact in vitro with adenine nucleotides. Furthermore, it has been proposed that within the AnxVI molecule a nucleotidde-binding domain exists, which is located in the C-terminal half of the protein, in the vicinity of Trp343. By comparison of exposure of tryptophan and multiple tyrosine residues upon nucleotide binding, as revealed by quenching of intrinsic fluorescence of AnxVI by ATP, ADP or cAMP, it can be concluded that the binding of nucleotides evokes changes in the protein tertiary structure. Moreover, in the course of present study we have found that AnxVI binds to a non-hydrolysable analog of ATP, the triazine dye Cibacron blue 3GA (CB3GA), immobilized on agarose. Binding reveals negative cooperativity with respect to protein concentration and is Ca2+-dependent. Binding is prevented by ATP. CB3GA binds to AnxVI also in solution, evoking the formation of annexin multimers. On the basis of this observation it can be suggested that interaction of CB3GA with AnxVI is useful to examine, with some limitations, the self-association of annexin molecules implying to play a role in interacting of AnxVI with biological membranes.

Affinity labeling of annexin VI with a triazine dye, Cibacron blue 3GA. Probable interaction of the dye with C-terminal nucleotide-binding site within the annexin molecule.

Annexin VI (AnxVI) from porcine liver, a member of the annexin family of Ca(2+)- and membrane-binding proteins, has been shown to bind ATP in vitro with a K(d) in the low micromolar concentration range. However, this protein does not contain within its primary structure any ATP-binding consensus motifs found in other nucleotide-binding proteins. In addition, binding of ATP to AnxVI resulted in modulation of AnxVI function, which was accompanied by changes in AnxVI affinity to Ca2+ in the presence of ATP. Using limited proteolytic digestion, purification of protein fragments by affinity chromatography on ATP-agarose, and direct sequencing, the ATP-binding site of AnxVI was located in a C-terminal half of the AnxVI molecule. To further study AnxVI-nucleotide interaction we have employed a functional nucleotide analog, Cibacron blue 3GA (CB3GA), a triazine dye which is commonly used to purify multiple ATP-binding proteins and has been described to modulate their activities. We have observed that AnxVI binds to CB3GA immobilized on agarose in a Ca(2+)-dependent manner. Binding is reversed by EGTA and by ATP and, to a lower extent, by other adenine nucleotides. CB3GA binds to AnxVI also in solution, evoking reversible aggregation of protein molecules, which resembles self-association of AnxVI molecules either in solution or on a membrane surface. Our observations support earlier findings that AnxVI is an ATP-binding protein.

Annexin VI: an intracellular target for ATP.

Annexin VI (AnxVI), an Ca2+- and phospholipid-binding protein, interacts in vitro with ATP in a calcium-dependent manner. Experimental evidence indicates that its nucleotide-binding domain which is localized in the C-terminal half of the protein differs structurally from ATP/GTP-binding motifs found in other nucleotide-binding proteins. The amino-acid residues of AnxVI directly involved in ATP binding have not been yet defined. Binding of ATP to AnxVI induces changes in the secondary and tertiary structures of protein, affecting the affinity of AnxVI for Ca2+ and, in consequence, influencing the Ca2+-dependent activities of AnxVI: binding to F-actin and to membranous phospholipids, and self-association of the annexin molecules. These observations suggest that ATP is a functional ligand for AnxVI in vivo, and ATP-sensitive AnxVI may play the role of a factor coupling vesicular transport and calcium homeostasis to cellular metabolism.