ATP hydrolyzing salivary enzymes of caterpillars suppress plant defenses

The oral secretions of herbivores are important recognition cues that can be used by plants to mediate induced defenses. In this study, a degradation of adenosine-5'-triphosphate (ATP) in tomato leaves was detected after treatment with Helicoverpa zea saliva. Correspondingly, a high level of ATPase activity in saliva was detected and three ATP hydrolyzing enzymes: apyrase, ATP synthase and ATPase 13A1 were identified in salivary glands. To determine the functions of these proteins in mediating defenses, they were cloned from H. zea and expressed in Escherichia coli. By applying the purified expressed apyrase, ATP synthase or ATPase 13A1 to wounded tomato leaves, it was determined that these ATP hydrolyzing enzymes suppressed the defensive genes regulated by the jasmonic acid and ethylene pathways in tomato plant. Suppression of glandular trichome production was also observed after treatment. Blood-feeding arthropods employ 5'-nucleotidase family of apyrases to circumvent host responses and the H. zea apyrase, is also a member of this family. The comparatively high degree of sequence similarity of the H. zea salivary apyrase with mosquito apyrases suggests a broader evolutionary role for salivary apyrases than previously envisioned.     

Evolution of the salivary apyrases of blood-feeding arthropods

Phylogenetic analyses of three families of arthropod apyrases were used to reconstruct the evolutionary relationships of salivary-expressed apyrases, which have an anti-coagulant function in blood-feeding arthropods. Members of the 5′nucleotidase family were recruited for salivary expression in blood-feeding species at least five separate times in the history of arthropods, while members of the Cimex-type apyrase family have been recruited at least twice. In spite of these independent events of recruitment for salivary function, neither of these families showed evidence of convergent amino acid sequence evolution in salivary-expressed members. On the contrary, in the 5′-nucleotide family, salivary-expressed proteins conserved ancestral amino acid residues to a significantly greater extent than related proteins without salivary function, implying parallel evolution by conservation of ancestral characters. This unusual pattern of sequence evolution suggests the hypothesis that purifying selection favoring conservation of ancestral residues is particularly strong in salivary-expressed members of the 5′-nucleotidase family of arthropods because of constraints arising from expression within the vertebrate host.     

Functions of plant apyrases

This publication presents results of the recent studies on plant NTPDases (apyrases). The structure and major physicochemical properties of this enzymes are reviewed. The attention has been paid to metabolic functions of apyrases from Solanum tuberosum and Arabidopsis thaliana. Apyrases constitute a family of proteins hydrolyzing phosphoanhydride bonds of nucleoside tri- and di-phosphates. They share common features like a similar structure, broad nucleotide substrate specificity and divalent cation requirement for their catalytic activity. The presence of plant NTPDases was detected in various cellular compartments. They are soluble or membrane-bound proteins. In hydrolytic processes catalyzed by activity of apoplastic apyrases and other ectoenzymes, adenine, ribose and orthophosphate are produced. These compounds are transported to the cell. Apyrases have been speculated to be involved in the regulation of starch synthesis and signal transmission. Their activity is necessary for development and growth of tubers and roots. Enzymes from leguminous plants activate the symbiosis with root nodule bacteria. Considering the fact, that NTPDases change the nucleotide concentration in cells and tissues, most of described functions may be related to the regulation of the energy charge of cell.     

Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation

Apyrase activity is present in the saliva of haematophagous arthropods. It is related to blood-feeding because of the apyrase ability to hydrolyse ADP, a key component of platelet aggregation. Five apyrases with apparent molecular masses of 88, 82, 79, 68 and 67 kDa were identified in the saliva of the vector of Chagas disease, Triatoma infestans. The large size observed during purification of these enzymes suggested oligomerization. In the present study, we confirmed, using gel-filtration and analytical ultracentrifugation, the presence of apyrase oligomers with molecular masses of 200 kDa in the saliva. Electrophoretic analyses showed that disulphide bonds were involved in homo-oligomerization. In addition, heterogeneity in disulphide bonds and in pI was detected, with the pI ranging from 4.9 to 5.4. The present study gives the first insights into the quaternary structure of soluble apyrases.     

Shared weapons of blood- and plant-feeding insects: Surprising commonalities for manipulating hosts

Insects that reprogram host plants during colonization remind us that the insect side of plant–insect story is just as interesting as the plant side. Insect effectors secreted by the salivary glands play an important role in plant reprogramming. Recent discoveries point to large numbers of salivary effectors being produced by a single herbivore species. Since genetic and functional characterization of effectors is an arduous task, narrowing the field of candidates is useful. We present ideas about types and functions of effectors from research on blood-feeding parasites and their mammalian hosts. Because of their importance for human health, blood-feeding parasites have more tools from genomics and other – omics than plant-feeding parasites. Four themes have emerged: (1) mechanical damage resulting from attack by blood-feeding parasites triggers “early danger signals” in mammalian hosts, which are mediated by eATP, calcium, and hydrogen peroxide, (2) mammalian hosts need to modulate their immune responses to the three “early danger signals” and use apyrases, calreticulins, and peroxiredoxins, respectively, to achieve this, (3) blood-feeding parasites, like their mammalian hosts, rely on some of the same “early danger signals” and modulate their immune responses using the same proteins, and (4) blood-feeding parasites deploy apyrases, calreticulins, and peroxiredoxins in their saliva to manipulate the “danger signals” of their mammalian hosts. We review emerging evidence that plant-feeding insects also interfere with “early danger signals” of their hosts by deploying apyrases, calreticulins and peroxiredoxins in saliva. Given emerging links between these molecules, and plant growth and defense, we propose that these effectors interfere with phytohormone signaling, and therefore have a special importance for gall-inducing and leaf-mining insects, which manipulate host-plants to create better food and shelter.     

Multiple variability in the sequence of a family of maize endosperm proteins

A collection of cDNA clones, corresponding to a group of maize endosperm proteins classified in the glutelin-2 (or reduced soluble proteins) and in the zein-2 subfractions, has been identified and characterized. The nucleotide sequence of three of these clones has been obtained and the amino acid sequence deduced. They appear to correspond to a small family of genes that are specifically expressed in immature endosperm simultaneously to zeins, the best characterized proteins from this tissue. Unlike zeins, the proteins of the glutelin-2 and zein-2 family contain sequences homologous to storage proteins from other cereals such as gliadins or hordeins. The cDNA clones encoding for the two types of proteins have been compared, and a high degree of homology has been observed for both the nucleotide and amino acid sequences. The differences existing in both the coding and non-coding regions allow the definition of multiple types of variability in their sequence. An hypothesis is proposed on how sequence diversity may have been generated in this particular class of plant proteins.     

Phylogenetic analyses of proton-translocating transhydrogenases

The proton-translocating nicotinamide nucleotide transhydrogenases (TH) provide a simple model for understanding chemically coupled transmembrane proton translocation. To further our understanding of TH structure-function relationships, we have identified all sequenced homologous of these vectorial enzymes and have conducted sequence comparison studies. The NAD-binding domains of TH are homologous to bacterial alanine dehydrogenases (ADH) and eukaryotic saccharopine dehydrogenases (SDH) as well as N5(carboxyethyl)-L-ornithine synthase of Lactococcus lactis and dipicolinate synthase of Bacillus subtilis. A multiple alignment, a phylogenetic tree, and two signature sequences for this family, designated the TH-ADH-SDH or TAS superfamily, have been derived. Additionally, the TH family has been characterized. Phylogenetic analyses suggested that these proteins have evolved without inter-system shuffling. However, interdomain splicing-fusion events have occurred during the evolution of several of these systems. Analyses of the multiple alignment for the TH family revealed that domain conservation occurs in the order: NADP-binding domain (domain III) > NAD-binding domain (domain I) > proton-translocating transmembrane domain (domain II). A topologic model for the proton-translocating transmembrane domain consistent with published data is presented, and a possible involvement of specific transmembrane alpha-helical segments in channel formation is suggested.     

节肢动物ABC转运蛋白及其介导的杀虫剂抗性

腺苷三磷酸结合盒转运蛋白（ATP-binding cassette transporter),简称ABC转运蛋白（ABC transporter）,是继细胞色素P450单加氧酶、羧酸酯酶、谷胱甘肽S-转移酶之后又一类参与解毒作用的重要蛋白家族,因其在杀虫剂解毒等方面起着非常重要的作用,近年来逐渐受到广泛关注。ABC转运蛋白是一大类跨膜蛋白,其核心结构通常由4个结构域组成,包括2个高度疏水的跨膜结构域（transmembrane domains , TMD）和2个核苷酸结合域（nucleotide binding domains, NBD）。根据序列相似性和保守结构域,可以把ABC转运蛋白家族分为8个亚家族,每个亚家族的成员数及功能不同。这类蛋白在各种生物体内均有分布,其主要功能包括转运物质、信号传导、细胞表面受体及参与细胞内DNA修复,转录及调节基因的表达过程等。此外,近年来的研究表明,ABC转运蛋白的突变或过表达不仅与节肢动物对化学农药的抗药性密切相关,而且在抗Bt毒素方面也起着非常重要的作用,对转Bt作物造成严重威胁。本文综述了节肢动物ABC转运蛋白的结构,ATP水解介导的作用机制,亚家族的分类、结构及生理功能,以及由ABC转运蛋白介导的抗药性研究进展,旨在深入了解ABC转运蛋白的研究现状及其在节肢动物抗药性方面的作用,为阐明节肢动物抗药性机制提供新的理论依据,对改进农业害虫的抗性监测和治理策略也具有一定的指导意义。     

Function and evolution of a mosquito salivary protein family

Saliva of blood-sucking arthropods contains a complex and diverse mixture of antihemostatic, antiinflammatory, and immunomodulatory compounds. The D7 salivary family of proteins is abundantly expressed in blood-feeding Diptera and is distantly related to the odorant-binding protein superfamily. In mosquitoes, two subfamilies exist, the long and short D7 proteins. Ticks and kissing bugs evolved salivary lipocalins that act as efficient scavengers of biogenic amines, and a similar function was postulated for the D7 proteins. Accordingly, we expressed the five members of the small D7 family of the African malaria vector Anopheles gambiae and a D7 long form from Aedes aegypti and showed by isothermal microcalorimetry, a modified and very sensitive non-equilibrium chromatography/spectrum distortion method, and by smooth muscle bioassay that four of these five short D7 proteins and the D7 long form bind serotonin with high affinity, as well as histamine and norepinephrine. The nonbinding D7 protein is poorly expressed in the salivary glands and appears to be on the path to becoming a pseudogene. Scavenging of host amines would antagonize their vasoconstrictor, platelet-aggregating, and pain-inducing properties. It appears that counteracting biogenic amines is of strong adaptive value in the convergent evolution of arthropods to hematophagy. This adaptation has been solved independently in ticks, bugs, and mosquitoes by co-option of either member of the lipocalin or, as shown here, by the odorant-binding protein families.     

Bioinformatics perspective on rhomboid intramembrane protease evolution and function

Endopeptidase classification based on catalytic mechanism and evolutionary history has proven to be invaluable to the study of proteolytic enzymes. Such general mechanistic- and evolutionary- based groupings have launched experimental investigations, because knowledge gained for one family member tends to apply to the other closely related enzymes. The serine endopeptidases represent one of the most abundant and diverse groups, with their apparently successful proteolytic mechanism having arisen independently many times throughout evolution, giving rise to the well-studied soluble chemotrypsins and subtilisins, among many others. A large and diverse family of polytopic transmembrane proteins known as rhomboids has also evolved the serine protease mechanism. While the spatial structure, mechanism, and biochemical function of this family as intramembrane proteases has been established, the cellular roles of these enzymes as well as their natural substrates remain largely undetermined. While the evolutionary history of rhomboid proteases has been debated, sorting out the relationships among current day representatives should provide a solid basis for narrowing the knowledge gap between their biochemical and cellular functions. Indeed, some functional characteristics of rhomboid proteases can be gleaned from their evolutionary relationships. Finally, a specific case where phylogenetic profile analysis has identified proteins that contain a C-terminal processing motif (GlyGly-Cterm) as co-occurring with a set of bacterial rhomboid proteases provides an example of potential target identification through bioinformatics. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Characterisation of the secreted apyrase family of Heligmosomoides polygyrus

Apyrases are a recurrent feature of secretomes from numerous species of parasitic nematodes. Here we characterise the five apyrases secreted by Heligmosomoides polygyrus, a natural parasite of mice and a widely used laboratory model for intestinal nematode infection. All five enzymes are closely related to soluble calcium-activated nucleotidases described in a variety of organisms, and distinct from the CD39 family of ecto-nucleotidases. Expression is maximal in adult worms and restricted to adults and L4s. Recombinant apyrases were produced and purified from Pichia pastoris. The five enzymes showed very similar biochemical properties, with strict calcium dependence and a broad substrate specificity, catalysing the hydrolysis of all nucleoside tri- and diphosphates, with no activity against nucleoside monophosphates. Natural infection of mice provoked very low antibodies to any enzyme, but immunisation with an apyrase cocktail showed partial protection against reinfection, with reduced egg output and parasite recovery. The most likely role for nematode secreted apyrases is hydrolysis of extracellular ATP, which acts as an alarmin for cellular release of IL-33 and initiation of type 2 immunity.     

Sequences related to the ox pancreatic ribonuclease coding region in the genomic DNA of mammalian species

Mammalian pancreatic ribonucleases form a family of homologous proteins that has been extensively investigated. The primary structures of these enzymes were used to derive phylogenetic trees. These analyses indicate that the presence of three strictly homologous enzymes in the bovine species (the pancreatic, seminal, and cerebral ribonucleases) is due to gene duplication events which occurred during the evolution of ancestral ruminants.In this paper we present evidence that confirms this finding and that suggests an overall structural conservation of the putative ribonuclease genes in ruminant species.We could also demonstrate that the sequences related to ox ribonuclease coding regions present in genomic DNA of the giraffe species are the orthologues of the bovine genes encoding the three ribonucleases mentioned above.Correspondence to: A. Furia     

A CTP-dependent archaeal riboflavin kinase forms a bridge in the evolution of cradle-loop barrels

Proteins of the cradle-loop barrel metafold are formed by duplication of a conserved betaalphabeta-element, suggesting a common evolutionary origin from an ancestral group of nucleic acid-binding proteins. The basal fold within this metafold, the RIFT barrel, is also found in a wide range of enzymes, whose homologous relationship with the nucleic acid-binding group is unclear. We have characterized a protein family that is intermediate in sequence and structure between the basal group of cradle-loop barrels and one family of RIFT-barrel enzymes, the riboflavin kinases. We report the structure, substrate-binding mode, and catalytic activity for one of these proteins, Methanocaldococcus jannaschii Mj0056, which is an archaeal riboflavin kinase. Mj0056 is unusual in utilizing CTP rather than ATP as the donor nucleotide, and sequence conservation in the relevant residues suggests that this is a general feature of archaeal riboflavin kinases.     

Molecular genetics of nucleotide sugar interconversion pathways in plants

Nucleotide sugar interconversion pathways represent a series of enzymatic reactions by which plants synthesize activated monosaccharides for the incorporation into cell wall material. Although biochemical aspects of these metabolic pathways are reasonably well understood, the identification and characterization of genes encoding nucleotide sugar interconversion enzymes is still in its infancy. Arabidopsis mutants defective in the activation and interconversion of specific monosaccharides have recently become available, and several genes in these pathways have been cloned and characterized. The sequence determination of the entire Arabidopsis genome offers a unique opportunity to identify candidate genes encoding nucleotide sugar interconversion enzymes via sequence comparisons to bacterial homologues. An evaluation of the Arabidopsis databases suggests that the majority of these enzymes are encoded by small gene families, and that most of these coding regions are transcribed. Although most of the putative proteins are predicted to be soluble, others contain N-terminal extensions encompassing a transmembrane domain. This suggests that some nucleotide sugar interconversion enzymes are targeted to an endomembrane system, such as the Golgi apparatus, where they may co-localize with glycosyltransferases in cell wall synthesis. The functions of the predicted coding regions can most likely be established via reverse genetic approaches and the expression of proteins in heterologous systems. The genetic characterization of nucleotide sugar interconversion enzymes has the potential to understand the regulation of these complex metabolic pathways and to permit the modification of cell wall material by changing the availability of monosaccharide precursors.     

Differential expression of two soybean apyrases, one of which is an early nodulin

Two cDNA clones were isolated from soybean (Glycine soja) by polymerase chain reaction with primers designed to conserved motifs found in apyrases (nucleotide phosphohydrolase). The two cDNAs are predicted to encode for two, distinct, apyrase proteins of approximately 50 kDa (i.e., GS50) and 52 kDa (i.e., GS52). Phylogenetic analysis indicated that GS52 is orthologous to a family of apyrases recently suggested to play a role in legume nodulation. GS50 is paralogous to this family and, therefore, likely plays a different physiological role. Consistent with this analysis, GS50 mRNA was detected in root, hypocotyls, flowers, and stems, while GS52 mRNA was found in root and flowers. Neither gene was expressed in leaves or cotyledons. Inoculation of roots with Bradyrhizobium japonicum, nitrogen-fixing symbiont of soybean, resulted in the rapid (<6 h) induction of GS52 mRNA expression. The level of GS50 mRNA expression was not affected by bacterial inoculation. Western blot (immunoblot) analysis of GS50 expression mirrored the results obtained by mRNA analysis. However, in contrast to the mRNA results, GS52 protein was found in stems. Interestingly, anti-GS52 antibody recognized a 50-kDa protein found only in nodule extracts. Treatment of roots with anti-GS52 antibody, but not anti-GS50 antibody or preimmune serum, blocked nodulation by B. japonicum. Fractionation of cellular membranes in sucrose density gradients and subsequent Western analysis of the fractions revealed that GS50 colocalized with marker enzymes for the Golgi, while GS52 colocalized with marker enzymes for the plasma membrane. Restriction fragment length polymorphism (RFLP)-based mapping placed the gs52 gene on major linkage group J of the integrated genetic map of soybean. These data suggest that GS50 is likely an endo-apyrase involved in Golgi function, while GS52 is localized on the root surface and appears to play an important role in nodulation.