Molecular characterization and expression analysis of annexin B3 and B38 as secretory proteins in Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis is a parasitic zoonotic disease, which poses a threat to public health and animal husbandry, and causes significant economic losses. Annexins are a family of phospholipid-binding proteins with calcium ion-binding activity, which have many functions. METHODS: Two annexin protein family genes [Echinococcus granulosus annexin B3 (EgAnxB3) and EgAnxB38] were cloned and molecularly characterized using bioinformatic analysis. The immunoreactivity of recombinant EgAnxB3 (rEgAnxB3) and rEgAnxB38 was investigated using western blotting. The distribution of EgAnxB3 and EgAnxB38 in protoscoleces (PSCs), the germinal layer, 18-day strobilated worms and 45-day adult worms was analyzed by immunofluorescence localization, and their secretory characteristics were analyzed preliminarily; in addition, quantitative real-time reverse transcription polymerase chain reaction was used to analyze their transcript levels in PSCs and 28-day strobilated worms stages. The phospholipid-binding activities of rEgAnxB3 and rEgAnxB38 were also analyzed. RESULTS: EgAnxB3 and EgAnxB38 are conserved and contain calcium-binding sites. Both rEgAnxB3 and rEgAnxB38 could be specifically recognized by the serum samples from E. granulosus-infected sheep, indicating that they had strong immunoreactivity. EgAnxB3 and EgAnxB38 were distributed in all stages of E. granulosus, and their transcript levels were high in the 28-day strobilated worms. They were found in liver tissues near the cysts. In addition, rEgAnxB3 has Ca2+-dependent phospholipid-binding properties. CONCLUSIONS: EgAnxB3 and EgAnxB38 contain calcium-binding sites, and rEgAnxB3 has Ca2+-dependent phospholipid-binding properties. EgAnxB3 and EgAnxB38 were transcribed in PSCs and 28-day strobilated worms. They were expressed in all stages of E. granulosus, and distributed in the liver tissues near the hydatid cyst, indicating that they are secreted proteins that play a crucial role in the development of E. granulosus.

Overexpression of annexin gene AnnSp2, enhances drought and salt tolerance through modulation of ABA synthesis and scavenging ROS in tomato.

Drought and high salinity are two major abiotic stresses that significantly affect agricultural crop productivity worldwide. Annexins are a multigene family that plays an essential role in plant stress responses and various cellular processes. Here, the AnnSp2 gene was cloned from drought-resistant wild tomato (Solanum pennellii) and functionally characterized in cultivated tomato. AnnSp2 protein was localized in the nucleus and had higher expression in leave, flower and fruit. It was induced by several phytohormones and some abiotic stresses. Tomato plants overexpressing AnnSp2 had increased tolerance to drought and salt stress, as determined by analysis of various physiological parameters. AnnSp2-transgenic plants were less sensitive to ABA during the seed germination and seedling stages. However, under drought stress, the ABA content significantly increased in the AnnSp2-overexpressing plants, inducing stomatal closure and reducing water loss, which underlay the plants' enhanced stress tolerance. Furthermore, scavenging reactive oxygen species (ROS), higher total chlorophyll content, lower lipid peroxidation levels, increased peroxidase activities (including APX, CAT and SOD) and higher levels of proline were observed in AnnSp2-overexpressing plants. These results indicate that overexpression of AnnSp2 in transgenic tomato improves salt and drought tolerance through ABA synthesis and the elimination of ROS.

Evolutionary adaptation of plant annexins has diversified their molecular structures, interactions and functional roles.

Annexins are an homologous, structurally related superfamily of proteins known to associate with membrane lipid and cytoskeletal components. Their involvement in membrane organization, vesicle trafficking and signaling is fundamental to cellular processes such as growth, differentiation, secretion and repair. Annexins exist in some prokaryotes and all eukaryotic phyla within which plant annexins represent a monophyletic clade of homologs descended from green algae. Genomic, proteomic and transcriptomic approaches have provided data on the diversity, cellular localization and expression patterns of different plant annexins. The availability of 35 complete plant genomes has enabled systematic comparative analysis to determine phylogenetic relationships, characterize structures and observe functional specificity between and within individual subfamilies. Short amino termini and selective erosion of the canonical type 2 calcium coordinating sites in domains 2 and 3 are typical of plant annexins. The convergent evolution of alternate functional motifs such as 'KGD', redox-sensitive Cys and hydrophobic Trp/Phe residues argues for their functional relevance and contribution to mechanistic diversity in plant annexins. This review examines recent findings and advances in plant annexin research with special focus on their structural diversity, cellular and molecular interactions and their potential integrated functions in the broader context of physiological responses.

Identification, phylogenetic relationships, characterization and gene expression patterns of six different annexins of channel catfish (Ictalurus punctatus Rafinesque, 1818).

Annexins are Ca(2+)-dependent phospholipid-binding proteins. They are ubiquitous in living organisms and are involved in many cellular processes. In the course of studying Edwardsiella ictaluri pathogenesis in channel catfish, we identified that six annexin expressed sequence tags (A1, A2, A4, A5, A6 and A11) were up-regulated at the early stage of infection. In this study, we cloned and characterized these transcripts. The full-length nucleic acid sequences of channel catfish annexins ranges from 1231 (annexin A1) to 2476 (annexin A6). Each transcript has one open reading, which appears to encode peptides ranges from 317 to 662 amino acid residues with the calculated molecular masses from 35.0 (annexin A5) to 74.5kDa (annexin A6). Phylogenetic and sequence analyses demonstrate that each channel catfish annexin had a diversified amino terminus, and had four structurally conserved 70-amino acid repeats. In addition, several important features for annexin functions were conserved in channel catfish. For expression profile, channel catfish annexin A1, A4 and A6 transcripts were detected in spleen, anterior kidney, liver, intestine, skin and gill of fish examined. However, annexin A2, A5 and A11 cDNAs were variously detected in tissues of fish sampled. This result provides important information for further elucidating channel catfish annexin functions in vivo.

Constitutive expression of mustard annexin, AnnBj1 enhances abiotic stress tolerance and fiber quality in cotton under stress.

Annexins belong to a multigene family of Ca(2+) dependent, phospholipid and cytoskeleton binding proteins. They have been shown to be upregulated under various stress conditions. We generated transgenic cotton plants expressing mustard annexin (AnnBj1), which showed enhanced tolerance towards different abiotic stress treatments like sodium chloride, mannitol, polyethylene glycol and hydrogen peroxide. The tolerance to these treatments was associated with decreased hydrogen peroxide levels and enhanced total peroxidase activity, enhanced content of osmoprotectants- proline and sucrose in transgenic plants. They showed higher retention of total chlorophyll and reduced TBARS in leaf disc assays with stress treatments, and decreased hydrogen peroxide accumulation in the stomatal guard cells when compared to their wild type counterparts. They also showed significantly enhanced fresh weight, relative water content, dry weight under stress. Treatment with sodium chloride resulted in enhanced expression of genes for Delta-pyrroline-5-carboxylase synthetase in leaves, and sucrose phosphate synthase, sucrose synthase and cellulose synthase A in the leaves and fibers of transgenic plants. The transgenic plants maintained normal seed development, fiber quality and cellulose content under stress.

Immunolocalization and histochemical evidence for the association of two different Arabidopsis annexins with secretion during early seedling growth and development.

Annexins are a multigene, multifunctional family of calcium-dependent, membrane-binding proteins found in animal and plant cells. In plants, annexins have been localized in the cytoplasm and at the cell periphery of highly secretory cell types, and in the tip region of polarly growing cells. Consequently, one proposed function for annexins in plant cells is participation in the Golgi-mediated secretion of new wall materials. In Arabidopsis, there are eight different annexin cDNAs, which share between 30% and 81% deduced amino acid sequence identity. We have used two monospecific Arabidopsis anti-annexin antibodies, raised against divergent 31-mer peptides from AnnAt1 and AnnAt2 and a previously characterized pea anti-annexin p35 antibody, for Western blot and immunolocalization studies in Arabidopsis. Western blot analyses of various Arabidopsis protein fractions showed that the two Arabidopsis antibodies are able to specifically recognize annexins in both soluble and membrane fractions. Immunofluorescence results with the three annexin antibodies show staining of secretory cells, especially at the cell periphery in developing sieve tubes, outer root cap cells, and in root hairs, consistent with previous results. In developmentally different stages some staining was also seen near the apical meristem, in some leaf cells, and in phloem-associated cells. Autoradiography following 3H-galactose incorporation was used to more clearly correlate active secretion of wall materials with the localization patterns of a specific individual annexin protein in the same cells at the same developmental stage. The results obtained in this study provide further support for the hypothesis that these two Arabidopsis annexins function in Golgi-mediated secretion during early seedling growth and development.

Evolutionary perspective on annexin calcium-binding domains.

Molecular systematic analysis of the annexin gene superfamily characterized the evolutionary origin, frequency and range of structural variation in calcium interaction domains that are considered intrinsic for membrane targeting and ion channel function. Approximately 36% of annexin repeat domains in an estimated 100 distinct subfamilies contained amino acid changes consistent with the functional loss of type two calcium-binding sites. At least 11% of annexin domains contained a novel K/H/RGD motif conserved in particular subfamilies and manifest in all phyla, apparently via convergent evolution. The first yeast annexin from Yarrowia lipolytica was classified in the ANXC1 subfamily with fungal and mycetozoan representatives. This clade had intact calcium-binding sites but disruption of the normally well-conserved, mid-repeat 4 region implicated in calcium channel regulation. Conversely, a tandem pair of novel annexins from the amphioxus Branchiostoma floridae resembled annexin A13 in gene structure and conserved the charged amino acids associated with the internal hydrophilic pore, but were devoid of external type 2 calcium-binding sites and incorporated K/RGD motifs instead, like annexin A9. The selective erosion of calcium-binding sites in annexin domains and the occurrence of alternate ligands in the same exposed, interhelical loops are pervasive features of the superfamily. This suggests greater complexity than previously appreciated in the mechanisms controlling annexin membrane interaction and calcium channel operation.

Selective vulnerability in amyotrophic lateral sclerosis: no evidence for a contribution of annexins, a family of calcium binding proteins.

Clinically, amyotrophic lateral sclerosis (ALS) usually presents as a pure motor system disorder, whereas oculomotor and sphincter muscle control of the anus and the bladder appear to be spared. Previously, a lacking expression of calcium binding proteins (CBPs) was demonstrated in vulnerable motor neurons in contrast to spared neuronal populations, e.g., the motor neurons of the cranial nerve III (NO) and the Onufrowicz nucleus (ON), suggesting a potential role of CBPs in the selective motoneuronal vulnerability in ALS. The annexins comprise a multigene family of CBPs, constituting a significant amount of total cellular protein and presumably involved in calcium-homeostasis and intracellular calcium-regulated pathways. We immunohistochemically investigated the expression patterns of annexins A1, A2, A4, A5, A6, and A7 in spinal cord and midbrain tissues from 24 ALS patients and 5 age-matched controls to test the hypothesis that annexins also contribute to the selective vulnerability in ALS. There was no difference in the expression patterns of ALS cases and normal controls. Annexin A1 was expressed in ependymal cells and motor neurons. Annexin A2 could be detected in ependymal and endothelial cells and motor neurons. Annexins A4 and A5 were found in both ependymal and glial cells, whereas annexin A6 was strongly expressed in motor neurons. Annexin A7 was totally absent from central nervous system tissue. A contribution of annexins to the selective vulnerability in ALS could not be derived from our results.

Identification of a novel class of annexin genes.

The annexins are a family of calcium- and phospholipid-binding proteins that have been widely studied in animals. Investigation of annexins in the fungus Aspergillus fumigatus identified a novel annexin-like gene (ANXC4) as well as two conventional annexins (ANXC3.1 and ANXC3.2). The genes were initially identified by bioinformatics, and sequences were then determined experimentally. Reverse transcription polymerase chain reaction indicated that all three genes were expressed. ANXC4 lacked calcium-binding consensus sequences and had a 553 residue N-terminal tail. However, bioinformatics indicated that ANXC4 is an annexin and homologues were identified in other filamentous fungi. ANXC4 therefore represents a new grouping within the annexin family.

Three-dimensional structure analysis of PROSITE patterns.

Pattern matches for each of the sequence patterns in PROSITE, a database of sequence patterns, were searched in all protein sequences in the Brookhaven Protein Data Bank (PDB). The three-dimensional structures of the pattern matches for the 20 patterns with the largest numbers of hits were analysed. We found that the true positives have a common three-dimensional structure for each pattern; the structures of false positives, found for six of the 20 patterns, were clearly different from those of the true positives. The results suggest that the true pattern matches each have a characteristic common three-dimensional structure, which could be used to create a template to define a three-dimensional functional pattern.

Human annexin 31 genetic mapping and origin.

The cDNA encoding novel human annexin 31 was utilized for chromosomal mapping, structural comparison, and phylogenetic analysis to clarify its genetic relationship to other annexins. The ANX31 gene locus was mapped by fluorescence in situ hybridization to human chromosome 1q21, remote from ten other paralogous human annexins on different chromosomes but near the epidermal differentiation gene complex, the S100A gene cluster and a breast-cancer translocation region. Protein homology testing and characterization of incompletely processed expressed sequence tags identified annexin 2 as the closest extant homologue. Maximum likelihood analysis confirmed its most recent common ancestor with vertebrate annexin 2 and validated its classification, in order of discovery, as annexin 31. This subfamily was formed approx. 500-600millionyears ago, subsequent to the gene duplication that produced annexin 1. It has diverged relatively rapidly and extensively, and specifically in the well-conserved, functionally critical type II calcium-binding sites.