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.     

Chromosomal mapping of the human annexin IV (ANX4) gene.

Annexin IV (placental anticoagulant protein II) is a member of the annexin or lipocortin family of calcium-dependent phospholipid-binding proteins. A cDNA for human annexin IV was isolated from a placental library that is 675 bases longer in the 3' untranslated region than previously reported, indicating the existence of alternative mRNA processing for this gene. Genomic Southern blotting with a cDNA probe indicated a gene size of 18-56 kb. Primers developed for polymerase chain reaction (PCR) allowed amplification of a 1.6-kb portion of the ANX4 gene. DNA sequence analysis showed that this PCR product contained a single intron with exon-intron boundaries in exactly the same position as in the mouse annexin I and annexin II genes. PCR analysis of a somatic cell hybrid panel mapped the ANX4 gene to chromosome 2, and in situ hybridization with a cDNA probe showed a unique locus for ANX4 at 2p13. This study provides further evidence that genes for the annexins are dispersed throughout the genome but are similar in size and exon-intron organization.     

Binding of annexins to lung lamellar bodies and the PMA-stimulated secretion of annexin V from alveolar type II cells

To identify lung lamellar body (LB)-binding proteins, the fractions binding to LB-Sepharose 4B in a Ca(2+)-dependent manner from the lung soluble fractions were analyzed with Mono Q column. Four annexins (annexins III, IV, V, and VIII) were identified by partial amino acid sequence analyses as the LB-binding proteins in the lung soluble fractions. A control experiment using phospholipid (phosphatidylserine/phosphatidylglycerol/phosphtidylcholine) liposome-Sepharose 4B revealed that annexins III, IV and V were the Ca(2+)-dependent proteins binding to the column in the lung soluble fractions, while annexin VIII was not detected. Thus, annexin VIII might preferentially bind to LB. On the other hand, the only Ca(2+)-dependent LB-binding protein identified in the bronchoalveolar lavage fluids was annexin V. It was further demonstrated that annexin V was secreted by isolated alveolar type II cells from rats and that the secretion was stimulated by the addition of phorbol ester (PMA), a potent stimulator of surfactant secretion. The PMA-dependent stimulation of annexin V was attenuated by preincubation with surfactant protein-A (SP-A), a potent inhibitor of surfactant secretion. As LB is thought to be an intracellular store of pulmonary surfactant, which is secreted by alveolar type II cells, annexin V is likely to be secreted together with the lamellar body.     

Calcium-dependent self-association of annexin II: a possible implication in exocytosis.

Alveolar type II cells secrete lung surfactant through exocytosis of lamellar bodies. We previously showed that the annexin II tetramer (Anx IIt) mediates the fusion of lamellar bodies with liposomes. The present study examined the possible involvement of membrane proteins in this process. Pre-treatment of lamellar bodies with trypsin and alpha-chymotrypsin reduced Anx IIt-mediated membrane fusion. With the use of an Anx IIt-conjugated Sepharose column, three Anx IIt-binding proteins with molecular weights of 67,000, 36,000 and 34,000 were isolated froM the Triton X-100 extract of bovine lung tissue membranes. These proteins were identified as annexins VI, II and IV by Western blot. The interaction of Anx IIt with annexins II and IV was confirmed by ligand blot assay. An EGTA-resistant membrane-bound annexin II was present in lung type II cells. Anx IIt preferentially hound to membranous annexin II compared with cytosolic annexin II of type II cells. With the use of immunofluorescence, annexin II was found to translocate from cytoplasm to plasma membranes in type II cells upon stimulation with phorbol 12-myristate 13-acetate. These results suggest that cytosolic annexin II may bind to membranous annexin II and form a protein-protein bridge to bring two membranes together.     

The genetic origin of mouse annexin VIII

Mouse annexin VIII cDNA was characterized by DNA sequencing of expressed sequence tag clones, molecular systematic analysis, and genetic linkage mapping to investigate its evolutionary origin. Its subfamily identity, divergence pattern, and nucleotide substitution rate were established by comparison with other annexin cDNA and deduced protein sequences. The known phylogenetic association of annexin VIII in an evolutionary clade with annexins XI, IV, V, and VIa identified these close homologs as potential progenitors or duplication products. Cladistic analysis confirmed the base position of annexin XI and its relationship to annexin IV as a direct duplication product. Although annexin VIII also derived from annexin XI, the evolutionary branching order, gene separation times, and mapping results indicated that it was probably a subsequent duplication product of annexin IV about 300 million years ago. Dates were calibrated against the assumed separation time of 75 Mya for rodents from other mammals, divergence rates were based on comparisons of all available annexin species, and relative rate tests implied individually stable gene clocks for most annexins. Linkage mapping of mouse Anx8 to the centromeric region of Chromosome (Chr) 14 placed it in a more distal homology group from previously mapped Anx7 and Anx11. Despite their synteny, the combined proximity and segregation of these three annexins diminished the likelihood that they were mutual gene duplication products. Received: 25 May 1997 / Accepted: 13 September 1997     

Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions

The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell–cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell–cell and/or cell–ECM contacts and anchorage-independent cell proliferation.     

Atypical properties displayed by annexin A9, a novel member of the annexin family of Ca(2+) and lipid binding proteins

Annexin A9 is a novel member of the annexin family of Ca(2+) and phospholipid binding proteins which has so far only been identified in EST data bases and whose deduced protein sequence shows mutations in residues considered crucial for Ca(2+) coordination in other annexins. To elucidate whether the annexin A9 protein is expressed as such and to characterize its biochemical properties we probed cell extracts with specific anti-annexin A9 antibodies and developed a recombinant expression system. We show that the protein is found in HepG2 hepatoma cell lysates and that a green fluorescent protein-tagged form is abundantly expressed in the cytosol of HeLa cells. Recombinant expression in bacteria yields a soluble protein that can be enriched by conventional chromatographic procedures. The protein is capable of binding phosphatidylserine containing liposomes albeit only at Ca(2+) concentrations exceeding 2 mM. Moreover and in contrast to other annexins this binding appears to be irreversible as the liposome-bound annexin A9 cannot be released by Ca(2+) chelation. These results indicate that annexin A9 is a unique member of the annexin family whose intracellular activity is not subject to Ca(2+) regulation.     

Membrane-bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins.

The distribution of annexin V isoforms (CaBP33 and CaBP37) and of annexin VI in bovine lung, heart, and brain subfractions was investigated with special reference to the fractions of these proteins which are membrane-bound. In addition to EGTA-extractable pools of the above proteins, membranes from lung, heart, and brain contain EGTA-resistant annexins V and VI which can be solubilized with detergents (Triton X-100 or Triton X-114). A strong base like Na2CO3, which is usually effective in extracting membrane proteins, only partially solubilizes the membrane-bound, EGTA-resistant annexins analyzed here. Also, only 50-60% of the Triton X-114-soluble annexins partition in the aqueous phase, the remaining fractions being recovered in the detergent-rich phase. Altogether, these findings suggest that, by an as yet unknown mechanism, following Ca(2+)-dependent association of annexin V isoforms and annexin VI with membranes, substantial fractions of these proteins remain bound to membranes in a Ca(2+)-independent way and behave like integral membrane proteins. These results further support the possibility that the above annexins might play a role in membrane trafficking and/or in the regulation of the structural organization of membranes.     

Structural determinant of the vesicle aggregation activity of annexin I.

Some annexins, including annexins I, II, IV, and VII, can promote membrane aggregation. To identify amino acids involved in annexin I-mediated membrane aggregation, we generated truncated mutants of human annexin I lacking various parts of the amino terminus. The in vitro vesicle binding and aggregation activities of these mutants indicated that both the amino-terminal region of annexin I spanning residues 26-29 and the carboxy-terminal core are involved in membrane aggregation. This notion was further supported by the finding that a chimera composed of residues 24-35 of annexin I and the core of annexin V has vesicle aggregation activity that is significantly higher than that of annexin V but lower than that of annexin I. Further site-specific mutations in the amino-terminal region of annexin I indicated that Lys-26 and Lys-29 are essential for its membrane aggregation activity. The comparison of tryptic digest patterns of free and vesicle-bound wild type and K29E mutant suggests that a primary role of Lys-26 and Lys-29 is to induce and stabilize an active conformation of annexin I for vesicle aggregation.     

Differential expression of annexins I, II and IV in human tissues: an immunohistochemical study

 Annexins constitute a family of Ca²⁺- and phospholipid-binding proteins. Although their functions are still not clearly defined, several members of the annexin family have been implicated in membrane-related events along exocytotic and endocytotic pathways. To elucidate a possible correlation of those functional proposals with the tissue distribution of annexins, we analysed immunohistochemically the expression of annexins I, II and IV in a broad variety of human tissues. Annexins I and II were chosen for this study since their functionally relevant N-terminal domains are structurally closely related, whilst annexin IV is structurally less related to the former two proteins. The study revealed distinct expression patterns of annexins I, II and IV throughout the body. Annexin I was found in leucocytes of peripheral blood, tissue macrophages and T-lymphocytes and in certain epithelial cells (respiratory and urinary system, superficial cells of non-keratinised squamous epithelium), annexin II in endothelial cells, myoepithelial cells and certain epithelial cells (mainly respiratory and urinary system), whereas annexin IV was almost exclusively found in epithelial cells. Epithelia of the upper respiratory system, Bowman’s capsule, urothelial cells, mesothelial cells, peripheral nerves, the choroid plexus, ependymal cells and pia mater and arachnoid of meninges generally strongly expressed all three annexins investigated. The characteristic expression in different tissues and the intracellular distribution indicates that the three annexins investigated are involved in aspects of differentiation and/or physiological functions specific to these tissues. Accepted: 15 January 1998     

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.     

Characterization of mammalian heart annexins with special reference to CaBP33 (annexin V)

Porcine heart was observed to express annexins V (CaBP33) and VI in large amounts, and annexins III and IV in much smaller amounts. Annexin V (CaBP33) in porcine heart was examined in detail by immunochemistry. Homogenization and further processing of heart in the presence of EGTA resulted in the recovery of annexin V (CaBP33) in the cytosolic fraction and in an EGTA-resistant, Triton X-100-soluble fraction from cardiac membranes. Including Ca2+ in the homogenization medium resulted in a significant decrease in the annexin V (CaBP33) content of the cytosolic fraction with concomitant increase in the content of this protein in myofibrils, mitochrondria, the sarcoplasmic reticulum and the sarcolemma. The amount of annexin V (CaBP33) in each of these subfractions depended on the free Ca2+ concentration in the homogenizing medium. At the lowest free Ca2+ concentration tested, 0.8 microM, only the sarcolemma appeared to contain bound annexin V (CaBP33). Membrane-bound annexins V (CaBP33) and VI partitioned in two fractions, one EGTA-resistant and Triton X-100-extractable, and one Triton X-100-resistant and EGTA-extractable. Altogether, these data suggest that annexins V and VI are involved in the regulation of membrane-related processes.     

Crystallization and preliminary X-ray crystallographic studies of human placental annexin IV

Human placental annexin IV, a member of the annexin family of calcium and phospholipid-binding proteins, has been crystallized by the vapour diffusion method in the presence of calcium, using polyethylene glycol 8000. The crystals are orthorhombic, space C222(1), cell dimensions a = 105.4 A, b = 115.7 A, c = 80.7 A and diffract to at least 2.5 A resolution on a synchrotron source.     

A comparison of the energetics of annexin I and annexin V.

The annexins comprise a family of soluble Ca2+- and phospholipid-binding proteins. Although highly similar in three-dimensional structure, different annexins are likely to exhibit different biochemical and functional properties and to play different roles in various membrane related events. Since it must be expected that these functional differences arise from differences in the characteristic thermodynamic parameters of these proteins, we performed high-sensitivity differential scanning microcalorimetry (DSC) and isothermal guanidinium hydrochloride (GdnHCl)-induced unfolding studies on annexin I and compared its thermodynamic parameters with those of annexin V published previously. The DSC data were analyzed using a model that permits quantitative treatment of the irreversible reaction. It turned out, however, that provided a heating rate of 2 K min-1 is used, unfolding of annexin I can be described satisfactorily in terms of a simple two-state reaction. At pH 6.0 annexin I is characterized by the following thermodynamic parameters: t1/2=61.8 degrees C, DeltaHcal=824 kJ mol-1 and DeltaCp=19 kJ mol-1 K-1. These parameters result in a stability value of DeltaG0D (20 degrees C)=51 kJ mol-1. The GdnHCl induced isothermal unfolding of annexin I in Mes buffer (pH 6.0), yielded DeltaG0D (buffer) values of 48, 60 and 36 kJ mol-1 at 20, 12 and 5 degrees C, respectively. These DeltaG0D values are in reasonable agreement with the values obtained from the DSC studies. The comparison of annexin I and annexin V under identical conditions (pH 8.0 or pH 6.0) shows that despite the pronounced structural homology of these two members of the annexin familiy, the stability parameters are remarkably different. This difference in stability is consistent with and provides a thermodynamic basis for the potential different in vivo functions proposed for these two annexins.     

Kidney proximal tubule cells: epithelial cells without EGTA-extractable annexins?

The expression and the subcellular localizations of annexins I, II, IV, VI, and XIII in renal epithelial cells were investigated, using immunological techniques with specific monoclonal antibodies. Upon performing Western blotting experiments, no annexins VI and XIII were detected in kidney, whereas annexins I, II, and IV were. Immunofluorescence labelling procedure performed on thin frozen renal sections showed the presence of these three annexins along the plasma membrane of the collecting duct cells with a restricted expression of annexin I at principal cells. Annexin I was also found present in some glomerular cells. None of these annexins, however, were detected in the proximal tubular cells upon performing immunofluorescence labelling and electrophoretic analysis on an EGTA (ethylenebis(oxyethylenenitrilo)tetraacetic acid)-extractable annexin fraction prepared from freshly isolated cells. This is the first time a mammalian epithelial cell has been found to express non-typical annexin (at least partly solubilized with EGTA). However, when these cells were grown in primary culture, they were found to express annexins I, II, IV, and V. As well as being located along the basolateral membrane, annexins I and II are also present on vesicles, which suggests that these annexins may be involved in vesicular traffic under cell culture conditions.     

A potential endogenous ligand of annexin IV in the exocrine pancreas. Carbohydrate structure of GP-2, a glycosylphosphatidylinositol-anchored glycoprotein of zymogen granule membranes

We demonstrated previously that annexins IV, V, and VI, proteins of the calcium/phospholipid-binding annexin family, have glycosaminoglycan binding properties (Ishitsuka, R., Kojima, K., Utsumi, H., Ogawa, H., and Matsumoto, I. (1998) J. Biol. Chem. 273, 9935-9941). In this study, we investigated the endogenous ligands of annexin IV in the exocrine pancreas. Immunohistochemical study of bovine pancreas showed that annexin IV localized in the apical cytoplasmic region of pancreatic acinar cells where zymogen granules are concentrated. Because it is the major component of the zymogen granule membrane, the glycosylphosphatidylinositol-anchored glycoprotein GP-2 was suggested to play a role in apical sorting and secretion of zymogens. We isolated GP-2 from porcine pancreas extract and determined the structure of its N-linked oligosaccharides by two-dimensional mapping. The major carbohydrate structures of porcine GP-2 were trisialo-triantennary and tetrasialo-tetra-antennary complex-type oligosaccharides. Dot-blot assay showed that annexin IV interacts with GP-2 in the presence of calcium and that it recognizes the terminal sialic acid residues linked through alpha2-3 linkages to the carbohydrate of GP-2. Lectin blot assay showed that Maackia amurensis mitogen, a plant lectin specific for the trisaccharide sequence Sia(alpha)2-3Galbeta1-4GlcNAc of N-linked oligosaccharides, has strong affinity for GP-2. Thus, M. amurensis mitogen was used as a specific probe for GP-2 in the histochemical staining of the bovine pancreas. GP-2 was found to localize exclusively in the same apical cytoplasmic region of pancreatic acinar cells as annexin IV does. These results suggest that GP-2 is an endogenous ligand of annexin IV in the exocrine pancreas.     

Engineering, biophysical characterisation and binding properties of a soluble mutant form of annexin A2 domain IV that adopts a partially folded conformation

The four approximately 75-residue domains (repeats) that constitute the annexin core structure all possess an identical five-alpha-helix bundle topology, but the physico-chemical properties of the isolated domains are different. Domain IV of the annexins has previously been expressed only as inclusion bodies, resistant to solubilisation. Analysis of the conserved, exposed hydrophobic residues of the four annexin domains reveals that domain IV contains the largest number of hydrophobic residues involved in interfacial contacts with the other domains. We designed five constructs of domain IV of annexin A2 in which several interfacial hydrophobic residues were substituted by hydrophilic residues. The mutant domain, in which all fully exposed hydrophobic interfacial residues were substituted, was isolated as a soluble protein. Circular dichroism measurements indicate that it harbours a high content of alpha-helical secondary structure and some tertiary structure. The CD-monitored (lambda=222 nm) thermal melting profile suggests a weak cooperative transition. Nuclear magnetic resonance (1H-15N) correlation spectroscopy reveals heterogeneous line broadening and an intermediate spectral dispersion. These properties are indicative of a partially folded protein in which some residues are in a fairly structured conformation, whereas others are in an unfolded state. This conclusion is corroborated by 1-anilinonaphthalene-8-sulfonate fluorescence (ANS) analyses. Surface plasmon resonance measurements also indicate that this domain binds heparin, a known ligand of domain IV in the full-length annexin A2, although with lower affinity.