Localization and quantitation of cardiac annexins II, V, and VI in hypertensive guinea pigs.
Annexins are characterized by Ca2+-dependent binding to phospholipids. Annexin II mainly participates in cell-cell adhesion and signal transduction, whereas annexins V and VI also seem to regulate intracellular calcium cycling. Their abundance and localization were determined in left ventricle (LV) and right ventricle (RV) from hypertensive guinea pigs, during the transition from compensatory hypertrophy to heart failure. Immunoblot analysis of annexins II, V, and VI revealed an increased accumulation (2.6-, 1.45-, and 2.3-fold, respectively) in LV from hypertensive guinea pigs and no modification in RV. Immunofluorescent labeling of annexins II, V, and VI; of Na+-K+-ATPase; and of sarcomeric alpha-actinin showed that in control LV and RV, 1) annexin II is present in nonmuscle cells; 2) annexins V and VI are mainly observed in the sarcolemma and intercalated disks of myocytes; 3) annexins II, V, and VI strongly label endothelial cells and adventitia of coronary arteries; and 4) annexin VI is present in the media. At the onset of heart failure, the most striking changes are the increased protein accumulation in LV and the very strong labeling of annexins II, V, and VI in interstitial tissue, suggesting a role in fibrosis development and cardiac remodeling. (+info)
A conserved nuclear element with a role in mammalian gene regulation.
Mammalian genomes contain numerous fragments of DNA that are derived from inactivated transposable elements. The accumulation and persistence of these elements is generally attributed to transposase activity rather than through possession or acquisition of a function of value to the host genome. Here we describe such a repetitive element, named ALF (forannexin VILINE-2fragment), comprising 130 bp of DNA derived from a LINE-2 sequence, which functions as a potent T-cell-specific silencer. The expansion of the DNA database arising as a result of the human genome sequencing project enabled us to identify ALF in, or close to, several well characterized genes including those for annexin VI, interleukin-4 and protein kinase C-beta. A systematic analysis of the entire LINE-2 sequence revealed that ALF, and not other regions of the LINE-2 sequence, was especially highly represented in the human genome. Acquisition of a function by this repetitive element may explain its abundance. These data show that a conserved fragment of an interspersed nuclear element has the potential to modulate gene expression, a discovery that has broad implications for the way in which we view so-called 'junk' DNA and our understanding of eukaryotic gene regulation. (+info)
Differential expression of Ca(2+)-binding proteins on follicular dendritic cells in non-neoplastic and neoplastic lymphoid follicles.
We studied the Ca(2+)-capture ability of follicular dendritic cells (FDCs) in tonsillar secondary lymphoid follicles (LFs) and the expression of six Ca(2+)-binding proteins (CBPs), caldesmon, S-100 protein, calcineurin, calbindin-D, calmodulin, and annexin VI in LFs of various lymphoid tissues and caldesmon and S-100 protein in neoplastic follicles of follicular lymphomas. First, Ca(2+)-capture cytochemistry revealed extensive Ca(2+) capture in the nuclei and cytoplasm of FDCs, but little or none in follicular lymphocytes. All six CBPs were localized immunohistochemically in the LFs and were always present in the basal light zone. Immunoelectron microscopic staining of FDCs was classified into two patterns: caldesmon was distributed in the peripheral cytoplasm like a belt; S-100 protein, calcineurin, calbindin-D, and calmodulin were distributed diffusely in the cytosol. Annexin VI was, however, negative on FDCs. Immunocytochemistry also demonstrated CBP-positive FDCs within FDC-associated clusters isolated from germinal centers. In situ hybridization revealed diffuse calmodulin mRNA expression throughout the secondary LFs. These data indicate that the CBPs examined may regulate Ca(2+) in the different subcellular sites of FDCs, and the roles of CBPs may be heterogeneous. We also investigated the distribution of caldesmon and S-100 protein in follicular lymphomas on paraffin-embedded tissue sections. FDCs within grades I and II neoplastic follicles clearly expressed caldesmon, but not S-100 protein, except a part of grade II neoplastic follicles. FDCs within grade III follicles showed no caldesmon, but frequently expressed S-100 protein. These results demonstrate that the caldesmon and S-100 protein staining patterns of grade I follicular lymphomas are different from those of grade III follicular lymphomas and suggest that FDC networks in grade I neoplastic follicles may be similar to those in the light zone within non-neoplastic follicles, FDC networks in grade III neoplastic follicles may be similar to those in dark and basal light zones within non-neoplastic follicles, and grade II follicles may be intermediate between grade I and grade III follicles. (+info)
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. (+info)
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. (+info)
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. (+info)
Annexin VI participates in the formation of a reversible, membrane-cytoskeleton complex in smooth muscle cells.
The plasmalemma of smooth muscle cells is periodically banded. This arrangement ensures efficient transmission of contractile activity, via the firm, actin-anchoring regions, while the more elastic caveolae-containing "hinge" regions facilitate rapid cellular adaptation to changes in cell length. Since cellular mechanics are undoubtedly regulated by components of the membrane and cytoskeleton, we have investigated the potential role played by annexins (a family of phospholipid- and actin-binding, Ca(2+)-regulated proteins) in regulating sarcolemmal organization. Stimulation of smooth muscle cells elicited a relocation of annexin VI from the cytoplasm to the plasmalemma. In smooth, but not in striated muscle extracts, annexins II and VI coprecipitated with actomyosin and the caveolar fraction of the sarcolemma at elevated Ca(2+) concentrations. Recombination of actomyosin, annexins, and caveolar lipids in the presence of Ca(2+) led to formation of a structured precipitate. Participation of all 3 components was required, indicating that a Ca(2+)-dependent, cytoskeleton-membrane complex had been generated. This association, which occurred at physiological Ca(2+) concentrations, corroborates our biochemical fractionation and immunohistochemical findings and suggests that annexins play a role in regulating sarcolemmal organization during smooth muscle contraction. (+info)
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. (+info)