Phase Separation and Fibrillization of Human Annexin A7 Are Mediated by Its Proline-Rich Domain.

Human annexin A7, a calcium- and phospholipid-binding protein, governs calcium homeostasis, plasma membrane repair, apoptosis, and tumor progression. A7 contains an N-terminal proline-rich domain (PRD; 180 residues, ∼24% prolines) that determines its functional specificity. Using microscopy and dye-binding assays, we show that recombinant A7 and its isolated PRD spontaneously phase separate into spherical condensates, which subsequently transform into ß-sheet-rich fibrils. We demonstrate that fibrillization of A7-PRD proceeds via primary nucleation and fibril-catalyzed secondary nucleation processes, as determined by chemical kinetics, providing a mechanistic basis for its amyloid assembly. This study confirms and highlights a subclass of eukaryotic PRDs prone to forming aggregates with important physiological and pathological implications.

Annexin A7 trafficking to alveolar type II cell surface: possible roles for protein insertion into membranes and lamellar body secretion.

A role for annexin A7 (A7) is postulated in the obligatory fusion between lamellar bodies and the plasma membrane during surfactant secretion in alveolar type II cells. This study investigated if surfactant secretagogues increase cell surface A7, which could support A7 insertion into plasma membrane as annexin proteins reportedly lack membrane penetration ability. In vivo trafficking of A7 to cell surface was determined by immuno-staining after non-permeabilizing fixation of alveolar type II cells. Stimulation with various secretagogues increased protein kinase-dependent staining for A7 and ABCA3 in comparison to control cells. Biotin-labeling of surface proteins showed ~4% of total A7 in control cells, which increased ~3-4 folds in stimulated type II cells. Increased cell surface A7 was also observed by protein cross-linking studies showing ~70kDa A7-adduct in the membranes but not in the cytosol fraction of PMA- or A23187-stimulated cells. In vitro phosphorylation increased the Ca(2+)-dependent binding of recombinant A7 to lung plasma membranes; and subsequent cross-linking showed increased levels of ~70kDa A7-adduct. PMA-stimulation of type II cells increased A7 trafficking to lipid rafts suggesting that the latter are involved in A7 trafficking to the cell surface. However, in vitro membrane insertion of recombinant A7 and its tryptophan mutants as determined by fluorescence quenching with doxylPC suggested only shallow membrane insertion by A7. Together, our studies support in vivo association between surfactant secretion and cell surface A7 occurring by insertion into plasma membrane and by fusion of A7 containing lamellar bodies.

Echinophilic proteins stomatin, sorcin, and synexin locate outside gangliosideM1 (GM1) patches in the erythrocyte membrane.

The detergent (Triton X-100, 4°C)-resistant membrane (DRM)-associated membrane proteins stomatin, sorcin, and synexin (anexin VII) exposed on the cytoplasmic side of membrane were investigated for their lateral distribution in relation to induced ganglioside(M1) (GM1) raft patches in flat (discocytic) and curved (echinocytic) human erythrocyte membrane. In discocytes, no accumulation of stomatin, sorcin, and synexin in cholera toxin subunit B (CTB) plus anti-CTB-induced GM1 patches was detected by fluorescence microscopy. In echinocytes, stomatin, sorcin, and synexin showed a similar curvature-dependent lateral distribution as GM1 patches by accumulating to spiculae induced by ionophore A23187 plus calcium. Stomatin was partly and synexin and sorcin were fully recruited to the spiculae. However, the DRM-associated proteins only partially co-localized with GM1 and were frequently distributed into different spiculae than GM1. The study indicates that stomatin, sorcin, and synexin are echinophilic membrane components that mainly locate outside GM1 rafts in the human erythrocyte membrane. Echinophilicity is suggested to contribute to the DRM association of a membrane component in general.

Novel protein ligands of the annexin A7 N-terminal region suggest pro-beta helices engage one another with high specificity.

The N-terminal regions of annexins A7 (synexin) and A11 consist of an extended series of short sequence repeats rich in tyrosine, proline, and glycine that provide binding sites for other proteins that may be recruited to membranes by the annexins and that may modulate the calcium and membrane binding activities of the annexin core domains. In this study two new ligands for the annexin A7 N terminal region were identified by yeast two hybrid screening: the TNFalpha receptor regulatory protein SODD (Suppressor Of Death Domains) and KIAA0280, a protein of unknown function. Strikingly, the sites of interaction of these proteins with the annexin also contain sequence repeats similar to those present in the N-termini of annexins A7 and A11. It was also found that the annexin A7 N-terminal region interacts with itself in the two hybrid assay. These results suggest that sequence repeats of this nature form novel structures, called YP pro-beta helices, that are characterized by an ability to interact with one another. Specificity of interactions between the pro-beta helices in different proteins may be encoded by the variations of residues and lengths of the sequence repeats.

A ten-residue domain (Y11-A20) in the NH2-terminus modulates membrane association of annexin A7.

Annexin A7 (synexin, annexin VII) is postulated to promote membrane fusion during surfactant secretion in alveolar type II cells and catecholamine secretion in adrenal chromaffin cells. Recently, we demonstrated that the 1-29 residues in the NH(2)-terminus could, possibly by interaction with the COOH-terminus, influence the Ca(2+)-dependent membrane binding, aggregation, and fusion properties of annexin A7 (A7). In this study, we further investigated this 29-residue domain by evaluating several deletion and point mutations for membrane-associated functions of A7. In comparison to A7, the mutants lacking 1-29 residues (A7Delta(1-29)) or 1-21 residues (A7Delta(1-21)), but not those lacking 1-10 residues (A7Delta(1-10)) or 21-29 residues (A7Delta(21-29)), showed diminished membrane binding. Segmental deletion of 10-20 residues (A7Delta(10-20)) also decreased the protein binding to membranes. The Ca(2+)-dependent membrane aggregation of PLV with A7Delta(1-29) was maximally diminished but less so with A7Delta(10-20) or A7Delta(1-21) in comparison to that with A7. However, phospholipid vesicle (PVL) aggregation was unaffected with A7Delta(1-10) or A7Delta(21-29). The Ca(2+)-dependent membrane fusion of PLV was also diminished with A7Delta(10-20) and A7Delta(1-29), but not with A7Delta(1-10). Since the mode of annexin A7 association and function with biological membranes could be different, we also evaluated these proteins for functional changes with isolated lung lamellar bodies. In comparison to A7, the binding to lamellar bodies was diminished for A7Delta(1-29) and A7Delta(1-21) but not for A7Delta(1-10). The Ca(2+)-dependent fusion of isolated lamellar bodies with PLV was also diminished with A7Delta(1-29), but not with A7Delta(10-20) or A7Delta(1-21). Taken together, our studies suggest that the 10-residue domain (Y(11)-A(20)) in the NH(2)-terminus modifies the phospholipid binding and aggregation properties of annexin A7. For binding and fusion of biological membranes, the 10-29-residue domain may be required although the annexin A7 properties are primarily modulated through the Y(11)-A(20) domain.

Partial truncation of the NH2-terminus affects physical characteristics and membrane binding, aggregation, and fusion properties of annexin A7.

Annexin A7 (synexin, annexin VII), a member of the annexin family of proteins, causes aggregation of membranes in a Ca2+-dependent manner and has been suggested to promote membrane fusion during exocytosis of lung surfactant, catecholamines, and insulin. Although annexin A7 (A7) was one of the first annexin proteins described, limited studies of its physical characteristics or of structural domains affecting any of its proposed functions have been conducted. As postulated for other annexin proteins, the unique NH2-domain possibly determines the functional specificity of A7. Therefore, we evaluated the effects of segmental deletions in the NH2-terminus on several characteristics associated with the COOH-terminus of A7. The COOH-terminus contains the only tryptophan residue, and all potential trypsin sites, and the Ca2+ and phospholipid binding sites. Recombinant rat A7 and its deletion mutants were expressed using constructs based on the cDNA sequence obtained by screening a rat lung cDNA library. Ca2+ increased the tryptophan fluorescence of A7 and caused a small red shift in the emission maximum (lambdamax), which was further increased in presence of phospholipid vesicles (PLV). NH2-terminal deletions of 29, 51, and 109 residues affected the peak width of fluorescence and lambdamax, surface-exposure of tryptophan residue, and caused a smaller Ca2+-dependent red shift in lambdamax of membrane-bound protein in comparison to A7. Limited proteolysis with trypsin showed that Ca2+ increased the proteolysis of all proteins, but the deletions also affected the pattern of proteolysis. The presence of PLV protected against Ca2+-dependent increase in proteolysis of all proteins. The deletion of first 29 residues also caused decreased membrane binding, aggregation, and fusion, when compared with A7. Collectively, these results suggest that specific NH2-terminus domains can alter those properties of A7 that are normally associated with the COOH-terminus. We speculate that interactions between the NH2- and COOH-termini are required for membrane binding, and aggregation and fusion properties of annexin A7.

Prognostic impact of ANX7-GTPase in metastatic and HER2-negative breast cancer patients.

PURPOSE: ANX7-GTPase located on chromosome 10q21 is significantly altered and associated with hormone-refractory metastatic prostate cancers. Therefore, we investigated whether levels of ANX7 correlate with breast cancer progression and survival EXPERIMENTAL DESIGN: A diagnostic tumor tissue microarray containing 525 human breast tissue specimens at different stages of the disease was assayed for ANX7 using immunocytochemical methods with ANX7 monoclonal antibody. A separate prognostic tumor tissue microarray containing 553 human breast tissue specimens annotated with clinicopathological parameters was assayed for ANX7, HER2, estrogen receptor, progesterone receptor, and p53 protein. RESULTS: We report here for the first time that the expression of ANX7-GTPase is significantly enhanced and associated with the presence of metastatic disease (P < 0.0001) in the 525 human breast tissue specimens analyzed. Furthermore, using a separate 553 case retrospective prognostic tumor tissue microarray, we found that increased ANX7 expression is also significantly associated with poor overall patient survival (P < 0.014). This is particularly true when restricted to patients in whom the BRE clinical grade is 2 (P < 0.001) or for whom there is a lack of HER2 expression (P < 0.002). Finally, Cox regression analysis shows that as the expression of ANX7 rises, the probability of survival decreases by more than 10-fold for those patients with HER2-negative tumors. These latter patients represented 66% of the population affected with breast cancer in this study. CONCLUSIONS: High levels of ANX7 in tumor correlate strongly with poor survival of HER2-negative patients and the most aggressive forms of breast cancer. This is the first study to demonstrate that ANX7 antibody has the potential for development into an in vivo diagnostic and therapeutic tool. This simple and reliable immunohistochemical assay may therefore become an important biomarker for metastatic breast cancer diagnosis and management of HER2-negative breast tumor patients.

The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin VII and annexin XI in a Ca2+-dependent manner.

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca(2+)-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST-Anx7N and GST-Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2-DeltaN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants (K(d)) were estimated to be approximately 40-60 nM for the high-affinity site and 500-700 nM for the low-affinity site.

Crystal structure of the C-terminal tetrad repeat from synexin (annexin VII) of Dictyostelium discoideum.

Synexin (annexin VII) is a cytosolic Ca(2+)-binding protein that promotes membrane fusion and forms voltage-regulated ion channels in artificial and natural membranes. The crystal structure of the C-terminal tetrad repeat from recombinant synexin (annexin VII) of Dictyostelium discoideum was solved to 2.45 A resolution. The protein crystallized in a dimeric form with two molecules joined face-to-face by their convex sides. Mainly hydrogen bonds and van der Waals contacts are involved in dimer formation, while not Ca2+ is bound to the conserved Ca(2+)-binding sites. The truncated N terminus is folded into a short antiparallel beta-sheet, from which the side-chain of Tyr111 penetrates sideways into the central, hydrophilic pore and may directly affect the ion channel activity. In order to investigate the structure of the missing N-terminal domain, we synthesized a 37-membered peptide of the N-terminal tail, (GYPPQQ)6G. CD and NMR studies showed a random coil conformation of the peptide in solution, suggesting for the synexin N terminus the lack of a well-ordered, three-dimensional fold.

Novel isoforms of synexin in Xenopus laevis: multiple tandem PGQM repeats distinguish mRNAs in specific adult tissues and embryonic stages.

Synexin (annexin VII) is a calcium-dependent, phospholipid-binding and membrane fusion protein in the annexin gene family, which forms calcium channels and may play a role in exocytotic secretion. We report here the cloning and characterization of five novel isoforms of cDNAs encoding Xenopus synexin from brain, oocyte and stage 24 cDNA libraries. The most prevalent Xenopus synexin has 1976 bp of cDNA sequence, which contains a 1539 bp open reading frame of 512 amino acids encoding a 54 kDa protein. This Xenopus protein is 6 kDa larger than the previously reported human and mouse synexins with which it shares approx. 73% identity in the C-terminal region and approx. 44% identity in the N-terminal region. Further studies with PCR revealed the molecular basis of the substantial divergence in the Xenopus synexin's N-terminal domain. The domain equivalent to the mammalian tissue-specific cassette exon occurs at a different position and is variable in size and sequence. The most interesting observation relates to the occurrence of different forms of synexin due to the varying numbers of tandem PGQM repeats that are expressed differently in different adult tissues and embryonic stages. For these reasons we have labelled this set of unique isoforms annexin VIIb, referring to mammalian forms, which lack the PGQM tandem repeats, as annexin VIIa. In spite of these differences from annexin VIIa, the form of recombinant annexin VIIb with three PGQM repeats was found to be catalytically active. We interpret these results to indicate that the actual calcium and phospholipid binding sites are conserved in Xenopus, and that the variations observed between members of the synexin gene family in the regulatory domain clearly point towards the tissue- and stage-specific roles of individual members, possibly involving the exocytotic process.

Annexin VII relocalization as a result of dystrophin deficiency.

Annexin VII (synexin) is a member of the annexin family of proteins, which are characterized by Ca(2+)-dependent binding to phospholipids. In normal skeletal muscle annexin VII is located preferentially at the plasma membrane and the t-tubule system [Selbert et al. (1995) J. Cell. Sci. 108, 85-95]. Here we have analyzed the distribution of annexin VII in muscle disorders in which the Ca2+ regulation is affected. A remarkable difference was observed in muscle specimens from patients suffering from Duchenne muscular dystrophy and also in muscle from the MDX mouse where annexin VII was gradually released from the sarcolemmal membrane into the cytosol and into the extracellular space during progression of the disease. Hypercontracted muscle fibers positive in Ca2+ staining were devoid of cytosolic annexin VII. Annexins IV and VI were similarly released into the extracellular space. Whereas normal skeletal muscle showed specifically the 51-kDa annexin VII isoform, in dystrophic muscle different ratios of the 51-kDa and the muscle-atypic 47-kDa isoforms were observed. The potential of annexin VII to serve as a tool with which cellular Ca2+ levels can be studied and different muscular disorders classified is discussed.

Stilbene disulfonic acids inhibit synexin-mediated membrane aggregation and fusion.

Stilbene disulfonic acids inhibit surfactant secretion from lung epithelial type II cells by an undefined mechanism, and inhibit CD4 mediated cell-cell fusion. We have previously shown that lung synexin promotes in vitro fusion of lamellar bodies and plasma membranes, an obligatory process for surfactant secretion. This study investigates the effect of stilbene disulfonic acids, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMDS), on synexin-mediated liposome aggregation and fusion. Structurally, these three stilbene compounds differ in the number of isothiocyano groups present (DIDS = 2, SITS = 1, and AMDS = 0). At 10 micrograms synexin/ml, DIDS and SITS inhibited synexin-mediated liposome aggregation with an EC50 of 3.5 microM and 148 microM, respectively. In comparison, AMDS was least inhibitory (EC50 > 1 mM). Thus, the inhibitory potency (DIDS > SITS > AMDS) was partly dependent upon the number of isothiocyano groups. The EC50 was also dependent on synexin concentration. Stilbene disulfonic acids were also inhibitory for arachidonic acid-enhanced synexin-mediated liposome fusion. The EC50 for DIDS and SITS for fusion were similar to that for liposome aggregation. Ca(2+)-induced synexin polymerization, measured by 90 degrees light scattering, was increased by DIDS, suggesting binding of stilbene disulfonic acids to synexin. The binding of DIDS to synexin was dependent on the molar ratio of synexin to DIDS. These results indicate that stilbene disulfonic acids interact directly with synexin to inhibit membrane aggregation and fusion. Our results suggest that such inhibition of synexin activity may contribute towards inhibition of surfactant secretion by DIDS, and support a physiological role for synexin in lung surfactant secretion.

Genomic organization and chromosomal localization of the mouse synexin gene.

We have isolated and characterized the gene encoding mouse synexin, which consists of 14 exons and spans approximately 30 kbp of genomic DNA. The protein's unique N-terminal domain is encoded by six exons, and the C-terminal tetrad repeat, the site of the membrane-fusion and ion-channel domain, is encoded by seven exons. The first exon encodes the 5'-untranslated region. Analysis of synexin-gene expression in different mouse tissues shows that mRNA with exon 6 is only present in brain, heart and skeletal muscle. mRNA lacking exon 6 is expressed in all tissues we have examined. The initiation site for transcription was determined by primer-extension analysis and S1 nuclease mapping. Sequence analysis of the 1.3 kb 5'-flanking region revealed that the promoter has a TATA box located at position -25 and a number of potential promoter and regulatory elements. A CCAAT motif was not observed but CCATT is located in an appropriate position for the CCAAT motif upstream from the transcription-initiation start site. In addition, the 5'-flanking region contains two sets of palindromic sequences. Finally, we have determined that the functional synexin gene (Anx7) is located on mouse chromosome 14 and that a pseudogene (Anx7-ps1) is located on chromosome 10.

Structure-function correlations of calcium binding and calcium channel activities based on 3-dimensional models of human annexins I, II, III, V and VII.

The annexins are a family of calcium-dependent phospholipid-binding proteins which share a high degree of primary sequence similarity. Using a model of the crystal structure of annexin V as a template, 3-dimensional models of human annexins I, II, III and VII were constructed by homology modeling (J. Greer, J. Mol. Biol. 153, 1027-1042, 1981; J.M. Chen, G. Lee, R.B. Murphy, R.P. Carty, P.W. Brant-Rauf, E. Friedman and M.R. Pincus, J. Biomolec. Str. Dyn. 6, 859-87, 1989) for the 316 amino acid portions corresponding to the annexin V structure published by Huber et al. (J. Mol. Biol. 223, 683-704, 1992). These methods were used to study structure-function correlations for calcium ion binding and calcium channel activity. Published experimental data are specifically shown to be consistent with the annexin models. Possible intramolecular disulfide bridges were identified in annexin I (between Cys297 and Cys316) and in annexins II and VII (between Cys115 and Cys243). Each of the annexin models have 3 postulated calcium binding sites, usually via a Gly-Xxx-Gly-Thr loop with an acidic Glu or Asp residue 42 positions C-terminal to the first Gly. Despite a nonconserved binding site sequence, annexins I and II are able to coordinate calcium in domain 3 since the residue in the second loop position is directed toward the solvent away from the binding pocket. This finding also suggests a mechanism for a conformational change upon binding calcium. Highly conserved Arg and acidic sidechains stabilize the channel pore structure; annexin channels probably exist in a closed state normally. Arg271 may be involved in channel opening upon activation: basic residue 254 can stabilize Glu112, which allows Arg271 to interact with residue 95 instead of Glu112. Residue 267, found on the convex surface at the pore opening, may also be important in modifying channel activity.