Chitinases as Food Allergens.

Food allergies originate from adverse immune reactions to some food components. Ingestion of food allergens can cause effects of varying severity, from mild itching to severe anaphylaxis reactions. Currently there are no clues to predict the allergenic potency of a molecule, nor are cures for food allergies available. Cutting-edge research on allergens is aimed at increasing information on their diffusion and understanding structure-allergenicity relationships. In this context, purified recombinant allergens are valuable tools for advances in the diagnostic and immunotherapeutic fields. Chitinases are a group of allergens often found in plant fruits, but also identified in edible insects. They are classified into different families and classes for which structural analyses and identification of epitopes have been only partially carried out. Moreover, also their presence in common allergen databases is not complete. In this review we provide a summary of the identified food allergenic chitinases, their main structural characteristics, and a clear division in the different classes.

Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase.

A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/ß-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/ß-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.

Very rapid phosphorylation kinetics suggest a unique role for Lhcb2 during state transitions in Arabidopsis.

Light-harvesting complex II (LHCII) contains three highly homologous chlorophyll-a/b-binding proteins (Lhcb1, Lhcb2 and Lhcb3), which can be assembled into both homo- and heterotrimers. Lhcb1 and Lhcb2 are reversibly phosphorylated by the action of STN7 kinase and PPH1/TAP38 phosphatase in the so-called state-transition process. We have developed antibodies that are specific for the phosphorylated forms of Lhcb1 and Lhcb2. We found that Lhcb2 is more rapidly phosphorylated than Lhcb1: 10 sec of 'state 2 light' results in Lhcb2 phosphorylation to 30% of the maximum level. Phosphorylated and non-phosphorylated forms of the proteins showed no difference in electrophoretic mobility and dephosphorylation kinetics did not differ between the two proteins. In state 2, most of the phosphorylated forms of Lhcb1 and Lhcb2 were present in super- and mega-complexes that comprised both photosystem (PS)I and PSII, and the state 2-specific PSI-LHCII complex was highly enriched in the phosphorylated forms of Lhcb2. Our results imply distinct and specific roles for Lhcb1 and Lhcb2 in the regulation of photosynthetic light harvesting.

Chitinolytic Enzymes of the Hyperparasite Fungus Aphanocladium album: Genome-Wide Survey and Characterization of A Selected Enzyme.

The filamentous fungus Aphanocladium album is known as a hyperparasite of plant pathogenic fungi; hence, it has been studied as a possible agent for plant protection. Chitinases secreted by A. album have proven to be essential for its fungicidal activity. However, no complete analysis of the A. album chitinase assortment has been carried out, nor have any of its chitinases been characterized yet. In this study, we report the first draft assembly of the genome sequence of A. album (strain MX-95). The in silico functional annotation of the genome allowed the identification of 46 genes encoding chitinolytic enzymes of the GH18 (26 genes), GH20 (8 genes), GH75 (8 genes), and GH3 (4 genes) families. The encoded proteins were investigated by comparative and phylogenetic analysis, allowing clustering in different subgroups. A. album chitinases were also characterized according to the presence of different functional protein domains (carbohydrate-binding modules and catalytic domains) providing the first complete description of the chitinase repertoire of A. album. A single chitinase gene was then selected for complete functional characterization. The encoded protein was expressed in the yeast Pichia pastoris, and its activity was assayed under different conditions of temperature and pH and with different substrates. It was found that the enzyme acts mainly as a chitobiosidase, with higher activity in the 37-50 °C range.

Genome walking by Klenow polymerase.

Genome walking procedures are all based on a final polymerase chain reaction amplification, regardless of the strategy employed for the synthesis of the substrate molecule. Here we report a modification of an already established genome walking strategy in which a single-strand DNA substrate is obtained by primer extension driven by Klenow polymerase and which results suitable for the direct sequencing of complex eukaryotic genomes. The efficacy of the method is demonstrated by the identification of nucleotide sequences in the case of two gene families (chiA and P1) in the genomes of several maize species.

Changes in morphology, cell wall composition and soluble proteome in Rhodobacter sphaeroides cells exposed to chromate.

The response of the carotenoidless Rhodobacter sphaeroides mutant R26 to chromate stress under photosynthetic conditions is investigated by biochemical and spectroscopic measurements, proteomic analysis and cell imaging. Cell cultures were found able to reduce chromate within 3-4 days. Chromate induces marked changes in the cellular dimension and morphology, as revealed by atomic force microscopy, along with compositional changes in the cell wall revealed by infrared spectroscopy. These effects are accompanied by significant changes in the level of several proteins: 15 proteins were found up-regulated and 15 down-regulated. The protein content found in chromate exposed cells is in good agreement with the biochemical, spectroscopic and microscopic results. Moreover at the present stage no specific chromate-reductase could be found in the soluble proteome, indicating that detoxification of the pollutant proceeds via aspecific reductants.

Identification of an Amylomaltase from the Halophilic Archaeon Haloquadratum walsbyi by Functional Metagenomics: Structural and Functional Insights.

Amylomaltases are prokaryotic 4-α-glucanotransferases of the GH77 family. Thanks to the ability to modify starch, they constitute a group of enzymes of great interest for biotechnological applications. In this work we report the identification, by means of a functional metagenomics screening of the crystallization waters of the saltern of Margherita di Savoia (Italy), of an amylomaltase gene from the halophilic archaeon Haloquadratum walsbyi, and its expression in Escherichia coli cells. Sequence analysis indicated that the gene has specific insertions yet unknown in homologous genes in prokaryotes, and present only in amylomaltase genes identified in the genomes of other H. walsbyi strains. The gene is not part of any operon involved in the metabolism of maltooligosaccharides or glycogen, as it has been found in bacteria, making it impossible currently to assign a precise role to the encoded enzyme. Sequence analysis of the H. walsbyi amylomaltase and 3D modelling showed a common structure with homologous enzymes characterized in mesophilic and thermophilic bacteria. The recombinant H. walsbyi enzyme showed starch transglycosylation activity over a wide range of NaCl concentrations, with maltotriose as the best acceptor substrate compared to other maltooligosaccharides. This is the first study of an amylomaltase from a halophilic microorganism.

Amylomaltases in Extremophilic Microorganisms.

Amylomaltases (4-α-glucanotransferases, E.C. 2.4.1.25) are enzymes which can perform a double-step catalytic process, resulting in a transglycosylation reaction. They hydrolyse glucosidic bonds of α-1,4'-d-glucans and transfer the glucan portion with the newly available anomeric carbon to the 4'-position of an α-1,4'-d-glucan acceptor. The intramolecular reaction produces a cyclic α-1,4'-glucan. Amylomaltases can be found only in prokaryotes, where they are involved in glycogen degradation and maltose metabolism. These enzymes are being studied for possible biotechnological applications, such as the production of (i) sugar substitutes; (ii) cycloamyloses (molecules larger than cyclodextrins), which could potentially be useful as carriers and encapsulating agents for hydrophobic molecules and also as effective protein chaperons; and (iii) thermoreversible starch gels, which could be used as non-animal gelatin substitutes. Extremophilic prokaryotes have been investigated for the identification of amylomaltases to be used in the starch modifying processes, which require high temperatures or extreme conditions. The aim of this article is to present an updated overview of studies on amylomaltases from extremophilic Bacteria and Archaea, including data about their distribution, activity, potential industrial application and structure.

Plant Health and Rhizosphere Microbiome: Effects of the Bionematicide Aphanocladium album in Tomato Plants Infested by Meloidogyne javanica.

The artificial introduction in the soil of antagonistic microorganisms can be a successful strategy, alternative to agrochemicals, for the control of the root-knot nematodes (Meloidogyne spp.) and for preserving plant health. On the other hand, plant roots and the associated rhizosphere constitute a complex system in which the contribution of microbial community is fundamental to plant health and development, since microbes may convert organic and inorganic substances into available plant nutrients. In the present study, the potential nematicidal activity of the biopesticide Aphanocladium album (A. album strain MX-95) against the root-knot nematode Meloidogyne javanica in infected tomato plants was investigated. Specifically, the effect of the A. album treatment on plant fitness was evaluated observing the plant morphological traits and also considering the nematode propagation parameters, the A. album MX-95 vitality and population density. In addition, the treatment effects on the rhizosphere microbiome were analysed by a metabarcoding procedure. Treatments with A. album isolate MX-95 significantly decreased root gall severity index and soil nematode population. The treatment also resulted in increased rhizosphere microbial populations. A. album MX-95 can be favourably considered as a new bionematicide to control M. javanica infestation.

A Differential Metabarcoding Approach to Describe Taxonomy Profiles of Bacteria and Archaea in the Saltern of Margherita di Savoia (Italy).

Microorganisms inhabiting saline environments are an interesting ecological model for the study of the adaptation of organisms to extreme living conditions and constitute a precious resource of enzymes and bioproducts for biotechnological applications. We analyzed the microbial communities in nine ponds with increasing salt concentrations (salinity range 4.9-36.0%) of the Saltern of Margherita di Savoia (Italy), the largest thalassohaline saltern in Europe. A deep-metabarcoding NGS procedure addressing separately the V5-V6 and V3-V4 hypervariable regions of the 16S rRNA gene of Bacteria and Archaea, respectively, and a CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) analysis allowed us to profile the dynamics of microbial populations at the different salt concentrations. Both the domains were detected throughout the saltern, even if the low relative abundance of Archaea in the three ponds with the lowest salinities prevented the construction of the relative amplicon libraries. The highest cell counts were recorded at 14.5% salinity for Bacteria and at 24.1% salinity for Archaea. While Bacteria showed the greatest number of genera in the first ponds (salinity range 4.9-14.5%), archaeal genera were more numerous in the last ponds of the saltern (salinity 24.1-36.0%). Among prokaryotes, Salinibacter was the genus with the maximum abundance (~49% at 34.6% salinity). Other genera detected at high abundance were the archaeal Haloquadratum (~43% at 36.0% salinity) and Natronomonas (~18% at 13.1% salinity) and the bacterial "Candidatus Aquiluna" (~19% at 14.5% salinity). Interestingly, "Candidatus Aquiluna" had not been identified before in thalassohaline waters.

Identification and characterization of protease inhibitors in Diplotaxis species.

PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.

Progress in the Analysis of Food Allergens through Molecular Biology Approaches.

Food allergies associated with class E immunoglobulins (IgE) are a serious health problem that affects between 1% and 10% of the population of developing countries, with a variability that depends on the geographical area and age range considered. These allergies are caused by a cross-link reaction between a specific food protein (the allergen) and the host IgE. Allergic reactions can range from mild itching to anaphylactic shock and there are no clues to predict the effects of an allergen. Strict avoidance of allergenic food is the only way to avoid possible serious allergic reactions. In the last 30 years a growing number of molecular studies have been conducted to obtain information on the diffusion of food allergens and to establish the structural basis of their allergenicity. At the same time, these studies have also allowed the development of molecular tools (mainly based on synthetic peptides and recombinant allergens) that can be of great help for diagnostic and therapeutic approaches of food allergies. Accordingly, this review focuses on advances in the study of food allergens made possible by molecular technologies and how results and technologies can be integrated for the development of a systematic food molecular allergology. The review may be of interest both to scientists approaching this field of investigation and to physicians who wish to have an update on the progress of research in diagnosis and therapy of food allergies.

RNA editing in plants: A comprehensive survey of bioinformatics tools and databases.

RNA editing is a widespread epitranscriptomic mechanism by which primary RNAs are specifically modified through insertions/deletions or nucleotide substitutions. In plants, RNA editing occurs in organelles (plastids and mitochondria), involves the cytosine to uridine modification (rarely uridine to cytosine) within protein-coding and non-protein-coding regions of RNAs and affects organelle biogenesis, adaptation to environmental changes and signal transduction. High-throughput sequencing technologies have dramatically improved the detection of RNA editing sites at genomic scale. Consequently, different bioinformatics resources have been released to discovery and/or collect novel events. Here, we review and describe the state-of-the-art bioinformatics tools devoted to the characterization of RNA editing in plant organelles with the aim to improve our knowledge about this fascinating but yet under investigated process.

A genome walking strategy for the identification of eukaryotic nucleotide sequences adjacent to known regions.

Determination of nucleotide sequences adjacent to a known region is a recurring need in many genome scale studies. Various methods have been developed based on PCR techniques in order to fulfill this aim and overcome the time-consuming approach of screening genomic libraries. Usually these protocols rely on specific requirements and strategies, such as the presence of suitable nucleotide restriction sites and ligation of specific single- or double-strand linkers, thus limiting their application to a certain extent. In this paper we present an alternative PCR-based protocol, consisting of four main steps: (i) extension of a sequence-specific primer; (ii) 3'-tailing of extended single-strand DNA; (iii) PCR; and (iv) nested PCR amplifications. This method, which appears to be a valid alternative to the other PCR-based protocols, was used for the identification of sequences flanking the cDNA encoding region of the Lhcb 1.1 gene (one member of the multigene family coding for the light harvesting protein Lhcbl) in the spinach genome.

One of the three proteinase inhibitor genes newly identified in the Brassica napus genome codes for an inhibitor of glutamyl endopeptidase.

Three proteinase inhibitor genes have been identified in the rapeseed (Brassica napus) genome. They are highly homologous to other genes of the mustard inhibitor (MSI) family of proteinase inhibitors characteristic of Cruciferae. In germinating seeds, only the transcript of one gene, coding for a trypsin inhibitor, is detectable by Northern analysis. The other two genes are transcribed at basal levels detectable only by reverse transcription PCR. One of the other two genes (rti-2) encodes a polypeptide with a glutamic residue in the P1 position, characteristic of glutamyl proteinase inhibitors. The recombinant RTI-2 protein strongly inhibits (Ki=44 nM) a glutamyl proteinase from Streptomyces griseus.

Identification and characterization of digestive serine proteases from inhibitor-resistant Helicoverpa zea larval midgut.

Protease inhibitors mediate a natural form of plant defence against insects, by interfering with the digestive system of the insect. In this paper, affinity chromatography was used to isolate trypsins and chymotrypsins from Helicoverpa zea larvae, which had been raised on inhibitor-containing diet. Sensitivity of the fractions to inhibition by plant proteinase inhibitors was tested, and compared to the sensitivity of proteinases found in insects raised on diet to which no inhibitor had been added. The isolated chymotrypsin activity was found to be less sensitive to plant protease inhibitors. The sensitivity of the isolated trypsin activity was found to be intermediate between completely sensitive trypsins and completely insensitive forms that have been previously described. Mass spectrometry was used to identify one trypsin and two chymotrypsins in the partially purified protease fraction. The sequence features of these proteases are discussed in relation to their sensitivity to inhibitors. The results provide insight in the enzymes deployed by Helicoverpa larvae to overcome plant defence.

PLMItRNA, a database on the heterogeneous genetic origin of mitochondrial tRNA genes and tRNAs in photosynthetic eukaryotes.

The updated version of PLMItRNA reports information and multialignments on 609 genes and 34 tRNA molecules active in the mitochondria of Viridiplantae (27 Embryophyta and 10 Chlorophyta), and photosynthetic algae (one Cryptophyta, four Rhodophyta and two Stramenopiles). Colour-code based tables reporting the different genetic origin of identified genes allow hyper-textual link to single entries. Promoter sequences identified for tRNA genes in the mitochondrial genomes of Angiospermae are also reported. The PLMItRNA database is accessible at http://bighost.area.ba.cnr.it/PLMItRNA/.

Identification and Characterization of the Sucrose Synthase 2 Gene (Sus2) in Durum Wheat.

Sucrose transport is the central system for the allocation of carbon resources in vascular plants. Sucrose synthase (SUS), which reversibly catalyzes sucrose synthesis and cleavage, represents a key enzyme in the control of the flow of carbon into starch biosynthesis. In the present study the genomic identification and characterization of the Sus2-2A and Sus2-2B genes coding for SUS in durum wheat (cultivars Ciccio and Svevo) is reported. The genes were analyzed for their expression in different tissues and at different seed maturation stages, in four tetraploid wheat genotypes (Svevo, Ciccio, Primadur, and 5-BIL42). The activity of the encoded proteins was evaluated by specific activity assays on endosperm extracts and their structure established by modeling approaches. The combined results of sucrose synthase 2 expression and activity levels were then considered in the light of their possible involvement in starch yield.

Targeted quantitative phosphoproteomic analysis of erythrocyte membranes during blood bank storage.

One of the hallmarks of blood bank stored red blood cells (RBCs) is the irreversible transition from a discoid to a spherocyte-like morphology with membrane perturbation and cytoskeleton disorders. Therefore, identification of the storage-associated modifications in the protein-protein interactions between the cytoskeleton and the lipid bilayer may contribute to enlighten the molecular mechanisms involved in the alterations of mechanical properties of stored RBCs. Here we report the results obtained analyzing RBCs after 0, 21 and 35 days of storage under standard blood banking conditions by label free mass spectrometry (MS)-based experiments. We could quantitatively measure changes in the phosphorylation level of crucial phosphopeptides belonging to ß-spectrin, ankyrin-1, α-adducin, dematin, glycophorin A and glycophorin C proteins. Data have been validated by both western blotting and pseudo-Multiple Reaction Monitoring (MRM). Although each phosphopeptide showed a distinctive trend, a sharp increase in the phosphorylation level during the storage duration was observed. Phosphopeptide mapping and structural modeling analysis indicated that the phosphorylated residues localize in protein functional domains fundamental for the maintenance of membrane structural integrity. Along with previous morphological evidence acquired by electron microscopy, our results seem to indicate that 21-day storage may represent a key point for the molecular processes leading to the erythrocyte deformability reduction observed during blood storage. These findings could therefore be helpful in understanding and preventing the morphology-linked mechanisms responsible for the post-transfusion survival of preserved RBCs.

Expression and characterization of a new isoform of the 9 kDa allergenic lipid transfer protein from tomato (variety San Marzano).

Lipid transfer proteins (LTPs) are food allergens found first in fruits of the Rosaceae family and later identified in other food plants. Their high structural stability causes them to behave as allergens in cooked and processed foods. Allergenic LTPs have been identified in tomato fruits as well, but studies of their thermal stability and structural characteristics are limited. In this article we report the identification of the coding region for a novel 9 kDa LTP isoform in the tomato variety San Marzano, together with the expression of the recombinant mature protein. The purified recombinant protein was further characterized for its thermal stability and was found to bind 1-palmitoil-2-lysophosphatidylcholine (Lyso-C16) after thermal treatments up to 105 °C. Analysis of a modeling derived structure of the protein allowed the identification of possible epitope regions on the molecular surface.