Bioinformatics Analysis of Bacterial Annexins – Putative Ancestral Relatives of Eukaryotic Annexins

Annexins are Ca²⁺-binding, membrane-interacting proteins, widespread among eukaryotes, consisting usually of four structurally similar repeated domains. It is accepted that vertebrate annexins derive from a double genome duplication event. It has been postulated that a single domain annexin, if found, might represent a molecule related to the hypothetical ancestral annexin. The recent discovery of a single-domain annexin in a bacterium, Cytophaga hutchinsonii, apparently confirmed this hypothesis. Here, we present a more complex picture. Using remote sequence similarity detection tools, a survey of bacterial genomes was performed in search of annexin-like proteins. In total, we identified about thirty annexin homologues, including single-domain and multi-domain annexins, in seventeen bacterial species. The thorough search yielded, besides the known annexin homologue from C. hutchinsonii, homologues from the Bacteroidetes/Chlorobi phylum, from Gemmatimonadetes, from beta- and delta-Proteobacteria, and from Actinobacteria. The sequences of bacterial annexins exhibited remote but statistically significant similarity to sequence profiles built of the eukaryotic ones. Some bacterial annexins are equipped with additional, different domains, for example those characteristic for toxins. The variation in bacterial annexin sequences, much wider than that observed in eukaryotes, and different domain architectures suggest that annexins found in bacteria may actually descend from an ancestral bacterial annexin, from which eukaryotic annexins also originate. The hypothesis of an ancient origin of bacterial annexins has to be reconciled with the fact that remarkably few bacterial strains possess annexin genes compared to the thousands of known bacterial genomes and with the patchy, anomalous phylogenetic distribution of bacterial annexins. Thus, a massive annexin gene loss in several bacterial lineages or very divergent evolution would appear a likely explanation. Alternative evolutionary scenarios, involving horizontal gene transfer between bacteria and protozoan eukaryotes, in either direction, appear much less likely. Altogether, current evidence does not allow unequivocal judgement as to the origin of bacterial annexins.     

Induction of annexin by heavy metals and jasmonic acid in Zea mays

Plant annexins are Ca²⁺- and phospholipid-binding proteins forming an evolutionary conserved multi-gene family. They are implicated in the regulation of plant growth, development, and stress responses. With the availability of the maize genome sequence information, we identified 12 members of the maize annexin genes. Analysis of protein sequence and gene structure of maize annexins led to their classification into five different orthologous groups. Expression analysis by RT-PCR revealed that these genes are responsive to heavy metals (Ni, Zn, and Cd). The maize annexin genes were also found to be regulated by Ustilago maydis and jasmonic acid. Additionally, the promoter of the maize annexin gene was analyzed for the presence of different stress-responsive cis-elements, such as ABRE, W-box, GCC-box, and G-box. RT-PCR and microarray data show that all 12 maize annexin genes present differential, organ-specific expression patterns in the maize developmental steps. These results indicate that maize annexin genes may play important roles in the adaptation of plants to various environmental stresses.     

Sequence and Chromosomal Localization of Mouse Annexin XI

Mouse annexin XI (anx11)²was cloned from a macrophage cDNA library and characterized by genetic linkage mapping, DNA sequencing, and structural comparison with other annexins. TheAnx11gene localized to mouse chromosome 14 in close linkage with theRarb, Plau,andWnt5agenes near the centromere and 1.8 cM distal from theAnx7gene. The open reading frame was flanked by long, untranslated regions and encoded a 503-amino-acid protein with 93.1% identity to its human orthologue. Its 189-aa amino terminus corresponded to the widely expressed variant 1 of two possible, alternatively spliced forms. A previously described peptide fromAplysia brasilianawas identified as a closely related invertebrate homologue. Since annexin XI is known to be localized in the nucleus at certain stages of development, the identification of a region in tetrad repeats 3 and 4 resembling the “chromo box” domain may be relevant to a nuclear regulatory function of annexin XI. Knowledge of the mouse cDNA sequence and genetic map location will assist in the analysis of genomic organization and expression and provide a useful animal model to investigate gene function and hereditary phenotype for annexin XI.     

Plasma Membrane-associated Annexin A6 Reduces Ca2+ Entry by Stabilizing the Cortical Actin Cytoskeleton

The annexins are a family of Ca²⁺- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca²⁺]_i or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca²⁺ entry (SOCE), but did not influence the rates of Ca²⁺ extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca²⁺ entry as long as [Ca²⁺]_i was below the threshold of annexin A6-membrane translocation. However, when [Ca²⁺]_i reached the levels necessary for the Ca²⁺-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca²⁺ entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca²⁺ entry, with consequences for the rates of cell proliferation.Calcium entry into cells either through voltage- or receptor-operated channels, or following the depletion of intracellular stores is a major factor in maintaining intracellular Ca²⁺ homeostasis. Resting [Ca²⁺]_i is low (∼100 nm compared with extracellular [Ca²⁺]_ex of 1.2 mm) and can be rapidly increased by inositol triphosphate-mediated release from the intracellular Ca²⁺ stores (mostly endoplasmic reticulum (ER)³), or by channel-mediated influx across the plasma membrane (PM). Store-operated calcium entry (SOCE) has been proposed as the main process controlling Ca²⁺ entry in non-excitable cells (1), and the recent discovery of Orai1 and STIM provided the missing link between the Ca²⁺-release activated current (I_CRAC) and the ER Ca²⁺ sensor (2–4). Translocation of STIM within the ER, accumulation in punctae at the sites of contact with PM and activation of Ca²⁺ channels have been proposed as a model of its regulation of Orai1 activity (5, 6). However, many details of the functional STIM-Orai1 protein complex and its regulation remain to be elucidated. The actin cytoskeleton plays a major role in the regulation of SOCE, possibly by influencing the function of ion channels or by interfering with the interaction between STIM and Orai1 (7–9). However, the proteins connecting the actin cytoskeleton and SOCE activity at the PM have yet to be identified.The annexins are a multigene family of Ca²⁺- and phospholipid-binding proteins, which have been implicated in many Ca²⁺-regulated processes. Their C-terminal core is evolutionarily conserved and contains Ca²⁺-binding sites, their N-terminal tails are unique and enable the protein to interact with distinct cytoplasmic partners. At low [Ca²⁺]_i, annexins are diffusely distributed throughout the cytosol, however, after stimulation resulting in the increase of [Ca²⁺]_i, annexins are targeted to distinct subcellular membrane locations, such as the PM, endosomes, or secretory vesicles (10). Annexins are involved in the processes of vesicle trafficking, cell division, apoptosis, calcium signaling, and growth regulation (11), and frequent changes in expression levels of annexins are observed in disease (12, 13). Previously, using biochemical methods and imaging of fluorescent protein-tagged annexins in live cells, we demonstrated that annexins A1, A2, A4, and A6 interacted with the PM as well as with internal membrane systems in a highly coordinated manner (10, 14). In addition, there is evidence of Ca²⁺-independent membrane association of several annexins, including annexin A6 (15–19); some of which point to the existence of pH-dependent binding mechanisms (20–22). Given the fact that several annexins are present within any one cell, it is likely that they form a [Ca²⁺] and pH sensing system, with a regulatory influence on other signaling pathways.The role of annexins as regulators of ion channel activity has been addressed previously (23–25). In particular, annexin A6 has been implicated in regulation of the sarcoplasmic reticulum ryanodine-sensitive Ca²⁺ channel (25), the neuronal K⁺ and Ca²⁺ channels (26), and the cardiac Na⁺/Ca²⁺ exchanger (27). Cardiac-specific overexpression of annexin A6 resulted in lower basal [Ca²⁺], a depression of [Ca²⁺]_i transients and impaired cardiomyocyte contractility (28). In contrast, the cardiomyocytes from the annexin A6 null-mutant mice showed increased contractility and accelerated Ca²⁺ clearance (29). Consistent with its role in mediating the intracellular Ca²⁺ signals, especially Ca²⁺ influx, ectopic overexpression of annexin A6 in A431 cells, which lack endogenous annexin A6, resulted in inhibition of EGF-dependent Ca²⁺ entry (30).The difficulty of investigating the influence of annexins on signaling events occurring at the PM lies in the transient and reversible nature of their Ca²⁺ and pH-dependent lipid binding. Although the intracellular Ca²⁺ increase following receptor activation or Ca²⁺ influx promotes the association of the Ca²⁺-sensitive annexins A2 and A6 with the PM, the proteins quickly resume their cytoplasmic localization upon restoration of the basal [Ca²⁺]_i (14). Therefore, to investigate the effects of membrane-associated annexins on Ca²⁺ homeostasis and the cell signaling machinery, we aimed to develop a model system allowing for a constitutive membrane association of annexins. Here we used the PM-anchoring sequences of the H- and K-Ras proteins to target annexins A6 and A1 to the PM independently of [Ca²⁺]. The Ras GTPases are resident at the inner leaflet of the PM and function as molecular switches (31). The C-terminal 9 amino acids of H- and N-Ras and the C-terminal 14 amino acids of K-Ras comprise the signal sequences for membrane anchoring of Ras isoforms (32). Although the palmitoylation and farnesylation of the C terminus of H-Ras (tH) serves as a targeting signal for predominantly cholesterol-rich membrane microdomains at the PM (lipid rafts/caveolae) (33), the polybasic group and the lipid anchor of K-Ras (tK) ensures the association of K-Ras with cholesterol-poor PM membrane domains. Importantly, these minimal C-terminal amino acid sequences are sufficient to target heterologous proteins, for example GFP, to different microdomains at the PM and influence their trafficking (34).In the present study we fused annexins A6, A2, and A1 with fluorescent proteins and introduced the PM-anchoring sequences of either H-Ras (annexin-tH) or K-Ras (annexin-tK) at the C termini of the fusion constructs. We demonstrate that the constitutive PM localization of annexin A6 results in down-regulation of store-operated Ca²⁺ entry. Expression of membrane-anchored annexin A6 causes an accumulation of the cortical F-actin, and cytoskeletal destabilization with latrunculin A abolishes the inhibitory effect of PM-anchored annexin A6 on SOCE. Taken together, our results implicate annexin A6 in the maintenance of intracellular Ca²⁺ homeostasis via actin-dependent regulation of Ca²⁺ entry.     

Molecular cloning and characterization of five annexin genes from Indian mustard (Brassica juncea L. Czern and Coss)

Plant annexins constitute a multigene family having suggested roles in a variety of cellular processes including stress responses. We have isolated and characterized five different cDNAs of mustard, Brassica juncea (AnnBj1, AnnBj2, AnnBj3, AnnBj6 and AnnBj7) encoding annexin proteins using a RT-PCR/RACE-PCR based strategy. The predicted molecular masses of these annexins are ～36.0 kDa with acidic pIs. At the amino acid level, they share high sequence similarity with each other and with annexins from higher plants. Phylogenetic analysis revealed their evolutionary relationship with corresponding orthologous sequences in Arabidopsis and deduced proteins in various plant species. Expression analysis by semi-quantitative RT-PCR revealed that these genes are differentially expressed in various tissues. The expression patterns of these genes also showed regulation by various stress conditions such as exposure to signaling molecules, salinity and oxidative stress and wounding. Additionally, the in silico promoter analysis (of AnnBj1, AnnBj2 and AnnBj3) showed the presence of different cis-responsive elements that could respond to various stress conditions. These results indicate that AnnBj genes may play important roles in adaptation of plants to various environmental stresses.     

Genome-wide Comparative Analysis of Annexin Superfamily in Plants

Most annexins are calcium-dependent, phospholipid-binding proteins with suggested functions in response to environmental stresses and signaling during plant growth and development. They have previously been identified and characterized in Arabidopsis and rice, and constitute a multigene family in plants. In this study, we performed a comparative analysis of annexin gene families in the sequenced genomes of Viridiplantae ranging from unicellular green algae to multicellular plants, and identified 149 genes. Phylogenetic studies of these deduced annexins classified them into nine different arbitrary groups. The occurrence and distribution of bona fide type II calcium binding sites within the four annexin domains were found to be different in each of these groups. Analysis of chromosomal distribution of annexin genes in rice, Arabidopsis and poplar revealed their localization on various chromosomes with some members also found on duplicated chromosomal segments leading to gene family expansion. Analysis of gene structure suggests sequential or differential loss of introns during the evolution of land plant annexin genes. Intron positions and phases are well conserved in annexin genes from representative genomes ranging from Physcomitrella to higher plants. The occurrence of alternative motifs such as K/R/HGD was found to be overlapping or at the mutated regions of the type II calcium binding sites indicating potential functional divergence in certain plant annexins. This study provides a basis for further functional analysis and characterization of annexin multigene families in the plant lineage.     

Apo and Calcium-Bound Crystal Structures of Cytoskeletal Protein Alpha-14 Giardin (Annexin E1) from the Intestinal Protozoan Parasite Giardia lamblia

Alpha-14 giardin (annexin E1), a member of the alpha giardin family of annexins, has been shown to localize to the flagella of the intestinal protozoan parasite Giardia lamblia. Alpha giardins show a common ancestry with the annexins, a family of proteins most of which bind to phospholipids and cellular membranes in a Ca²⁺-dependent manner and are implicated in numerous membrane-related processes including cytoskeletal rearrangements and membrane organization. It has been proposed that alpha-14 giardin may play a significant role during the cytoskeletal rearrangement during differentiation of Giardia. To gain a better understanding of alpha-14 giardin's mode of action and its biological role, we have determined the three-dimensional structure of alpha-14 giardin and its phospholipid-binding properties. Here, we report the apo crystal structure of alpha-14 giardin determined in two different crystal forms as well as the Ca²⁺-bound crystal structure of alpha-14 giardin, refined to 1.9, 1.6 and 1.65 Å, respectively. Although the overall fold of alpha-14 giardin is similar to that of alpha-11 giardin, multiwavelength anomalous dispersion phasing was required to solve the alpha-14 giardin structure, indicating significant structural differences between these two members of the alpha giardin family. Unlike most annexin structures, which typically possess N-terminal domains, alpha-14 giardin is composed of only a core domain, followed by a C-terminal extension that may serve as a ligand for binding to cytoskeletal protein partners in Giardia. In the Ca²⁺-bound structure we detected five bound calcium ions, one of which is a novel, highly coordinated calcium-binding site not previously observed in annexin structures. This novel high-affinity calcium-binding site is composed of seven protein donor groups, a feature rarely observed in crystal structures. In addition, phospholipid-binding assays suggest that alpha-14 giardin exhibits calcium-dependent binding to phospholipids that coordinate cytoskeletal disassembly/assembly during differentiation of the parasite.     

Characterization of Annexin gene family and functional analysis of RsANN1a involved in heat tolerance in radish (Raphanus sativus L.)

Plant annexins are a kind of conserved Ca²⁺-dependent phospholipid-binding proteins which are involved in plant growth, development and stress tolerance. Radish is an economically important annual or biennial root vegetable crop worldwide. However, the genome-wide characterization of annexin (RsANN) gene family remain largely unexplored in radish. In this study, a comprehensive identification of annexin gene family was performed at the whole genome level in radish. In total, ten RsANN genes were identified, and these putative RsANN proteins shared typical characteristics of the annexin family proteins. Phylogenetic analysis showed that the RsANNs together with annexin from Arabidopsis and rice were clustered into five groups with shared similar motif patterns. Chromosomal localization showed that these ten RsANN genes were distributed on six chromosomes (R3-R8) of radish. Several cis-elements involved in abiotic stress response were identified in the promoter regions of RsANN genes. Expression profile analysis indicated that the RsANN genes exhibited tissue-specific patterns at different growth stages and tissues. The Real-time quantitative PCR (RT-qPCR) revealed that the expression of most RsANN genes was induced under various abiotic stresses including heat, drought, salinity, oxidization and ABA stress. In addition, stress assays showed that overexpression of RsANN1a improved plant’s growth and heat tolerance, while artificial microRNAs (amiRNA)-mediated knockdown of RsANN1a caused dramatically decreased survival ratio of Arabidopsis plants. These findings not only demonstrate that RsANN1a might play a critical role in the heat stress response of radish, but also facilitate clarifying the molecular mechanism of RsANN genes in regulating the biological process governing plant growth and development.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01056-5.     

Identification and characterization of annexin gene family in rice

Plant annexins are Ca²⁺-dependent phospholipid-binding proteins and are encoded by multigene families. They are implicated in the regulation of plant development as well as protection from drought and other stresses. They are well characterized in Arabidopsis, however no such characterization of rice annexin gene family has been reported thus far. With the availability of the rice genome sequence information, we have identified ten members of the rice annexin gene family. At the protein level, they share 16–64% identity with predicted molecular masses ranging from 32 to 40 kDa. Phylogenetic analysis of rice annexins together with annexins from other monocots led to their classification into five different orthologous groups and share similar motif patterns in their protein sequences. Expression analysis by real-time RT-PCR revealed differential temporal and spatial regulation of these genes. The rice annexin genes are also found to be regulated in seedling stage by various abiotic stressors including salinity, drought, heat and cold. Additionally, in silico analysis of the putative upstream sequences was analyzed for the presence of stress-responsive cis-elements. These results provide a basis for further functional characterization of specific rice annexin genes at the tissue/developmental level and in response to abiotic stresses.     

Stereochemistry of lepidopteran sex pheromone biosynthesis: a comparison of fatty acid-CoA Δ11-(Z)-desaturases in Bombyx mori and Manduca sexta female moths

The absolute stereochemistry of fatty acid (FA) desaturation in Bombyx mori and Manduca sexta female pheromone glands (PGs), catalysed by FA-CoA Δ11-(Z)-desaturases, was determined using chiral, specifically labelled palmitic acids {[2,2,3,4,5,5,6,6,7,8,9,9,11,12−²H₁₄]–(11R,12S)−1 and [2,2,3,4,5,5,6,6,7,8,9,9,11,12−²H₁₄]–(11S,12R)−1)} as metabolic probes. The (11R,12S)−1 acid was converted in PGs of treated virgin females to labelled methyl (11Z)-hexadecenoate ([²H₁₄]−2, Mw=282 Da). In incubations with the opposite enantiomer two deuterium atoms from (11S,12R)−1 were removed, yielding [²H₁₂]−2 of Mw=280 Da. These results were confirmed by methylthiolation of [²H₁₄]−2 and [²H₁₂]−2 with a dimethyl disulfide/iodine mixture. Mass spectra of the DMDS adducts directly showed the distribution of deuterium atoms in the labelled methyl esters of 2. The data consistently indicate, that the studied insects possess Δ11-(Z)-desaturases with pro-(R) C(11)-H and pro-(R) C(12)-H stereospecificity, catalysing a syn-elimination of two hydrogen atoms.     

Distinct Annexin Subfamilies in Plants and Protists Diverged Prior to Animal Annexins and from a Common Ancestor

Annexin homologues in the kingdoms of Planta and Protista were characterized by molecular sequence analysis to determine their phylogenetic and structural relationship with annexins of Animalia. Sequence fragments from 19 plant annexins were identified in sequence databases and composite sequences were also assembled from expressed sequence tags for Arabidopsis thaliana. Length differences in protein amino-termini and evidence for unique exon splice sites indicated that plant annexins were distinct from those of animals. A third annexin gene of Giardia lamblia (Anx21-Gla) was identified as a distant relative to other protist annexins and to those of higher eukaryotes, thus providing a suitable outgroup for evolutionary reconstruction of the family tree. Rooted evolutionary trees portrayed protist, plant, and Dictyostelium annexins as early, monophyletic ramifications prior to the appearance of closely related animal annexin XIII. Molecular phylogenetic analyses of DNA and protein sequence alignments revealed at least seven separate plant subfamilies, represented by Anx18 (alfalfa, previously classified), Anx22 (thale cress), Anx23 (thale cress, cotton, rape and cabbage), Anx24 (bell pepper and tomato p34), Anx25 (strawberry, horseradish, pea, soybean, and castor bean), Anx26-Zma, and Anx27-Zma (maize). Other unique subfamilies may exist for rice, tomato p35, apple, and celery annexins. Consensus sequences compiled for each eukaryotic kingdom showed some breakdown of the ``annexin-fold' motif in repeats 2 and 3 of protist and plant annexins and a conserved codon deletion in repeat 3 of plants. The characterization of distinct annexin genes in plants and protists reflects their comparable diversity among animal species and offers alternative models for the comparative study of structure–function relationships within this important gene family. Received: 30 May 1996 / Accepted: 20 August 1996     

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in Campylobacter lari

PCR amplifications using primers for the clustered regularly interspaced short palindromic repeats (CRISPRs)-associated gene 1 (cas1), cas2, putative (p)-cas and CRISPRs genes generated cas1, cas2, p-cas and CRISPRs genes segments with 9–28 of 28 urease-positive thermophilic Campylobacter (UPTC) isolates, respectively. The p-cas and CRISPRs genes segments were amplified with 10 of 11 and 0 of 11 urease-negative (UN) Campylobacter lari isolates, respectively. When the nucleotide sequences of the CRISPRs consensus sequence repeats of each 33–37 base pairs from the 18 Campylobacter jejuni isolates were aligned, as well as from the four C. jejuni reference and UPTC CF89-12 strains, the repeats were identified as being almost identical. Although a total of all 18 C. jejuni isolates examined gave PCR-positive signals for the CRISPRs genes, it was, interestingly, suggested that many numbers of C. lari and C. jejuni isolates may possibly carry cas but not CRISPRs genes within their CRISPRs loci. In addition, PCR amplification by using a novel primer pair of f-ClCRISPR-ladder and ClCRISPRs-R, which were novel to this study, with the UPTC CF89-12 strain was shown to be useful for the detection of the putative CRISPRs separated by the non-repetitive unique spacer regions, with the electrophoretic ladder DNA profile following 5.0 % polyacrylamide gel electrophoresis. Secondary structure models of the CRISPRs repeats were predicted with UPTC CF89-12 and two C. jejuni strains.     

Temperature and Development Impacts on Housekeeping Gene Expression in Cowpea Aphid,Aphis craccivora (Hemiptera: Aphidiae)

Quantitative real-time PCR (qRT-PCR) is a powerful technique to quantify gene expression. To standardize gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to consistently expressed housekeeping genes (HKGs) is required. In this study, ten candidate HKGs including elongation factor 1 α (EF1A), ribosomal protein L11 (RPL11), ribosomal protein L14 (RPL14), ribosomal protein S8 (RPS8), ribosomal protein S23 (RPS23), NADH-ubiquinone oxidoreductase (NADH), vacuolar-type H+-ATPase (ATPase), heat shock protein 70 (HSP70), 18S ribosomal RNA (18S), and 12S ribosomal RNA (12S) from the cowpea aphid, Aphis craccivora Koch were selected. Four algorithms, geNorm, Normfinder, BestKeeper, and the ΔC_t method were employed to evaluate the expression profiles of these HKGs as endogenous controls across different developmental stages and temperature regimes. Based on RefFinder, which integrates all four analytical algorithms to compare and rank the candidate HKGs, RPS8, RPL14, and RPL11 were the three most stable HKGs across different developmental stages and temperature conditions. This study is the first step to establish a standardized qRT-PCR analysis in A. craccivora following the MIQE guideline. Results from this study lay a foundation for the genomics and functional genomics research in this sap-sucking insect pest with substantial economic impact.