Alison Lovegrove

Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses

Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by α-(1,2)– and α-(1,3)–linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown. Here we demonstrate that knocking-down glycosyltransferase (GT) 61 expression in wheat endosperm strongly decreases α-(1,3)–linked arabinosyl substitution of xylan. Moreover, heterologous expression of wheat and rice GT61s in Arabidopsis leads to arabinosylation of the xylan, and therefore provides gain-of-function evidence for α-(1,3)-arabinosyltransferase activity. Thus, GT61 proteins play a key role in arabinoxylan biosynthesis and therefore in the evolutionary divergence of grass cell walls.

Carbohydrate structural analysis of wheat flour arabinogalactan protein

The water-extractable arabinogalactan protein (AGP) was isolated from bread wheat flour (Triticum aestivum L. variety Cadenza) and the structure of the arabinogalactan (AG) carbohydrate component was studied. Oligosaccharides, released by hydrolysis of the AG with a range of AGP-specific enzymes, were characterised by Matrix Assisted Laser Desorption Ionisation (MALDI)-Time of Flight (ToF)-Mass Spectrometry (MS), MALDI-ToF/ToF high energy collision induced dissociation (CID) and Polysaccharide Analysis by Carbohydrate gel Electrophoresis (PACE). The AG is composed of a β-(1→3)-D-galactan backbone with β-(1→6)-D-galactan side chains. These side chains are highly variable in length, from one to at least 20 Gal residues and are highly substituted with α-L-Araf. Single GlcA residues are also present at the non-reducing termini of some short β-(1→6)-galactan side chains. In addition, the β-(1→6)-galactan side chains are also substituted with β-L-Arap. We propose a polysaccharide structure of the wheat flour AGP that is substantially revised from earlier models.

Cell Walls of Developing Wheat Starchy Endosperm: Comparison of Composition and RNA-Seq Transcriptome

The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3;1,4)-β-d-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.

Hazelnut allergy across Europe dissected molecularly: A EuroPrevall outpatient clinic survey.

BACKGROUND Hazelnut allergy is birch pollen-driven in Northern/Western Europe and lipid transfer protein-driven in Spain and Italy. Little is known about other regions and other allergens. OBJECTIVE Establishing a molecular map of hazelnut allergy across Europe. METHODS In 12 European cities, subjects reporting reactions to hazelnut (n = 731) were evaluated and sensitization to 24 foods, 12 respiratory allergen sources, and latex was tested by using skin prick test and ImmunoCAP. A subset (124 of 731) underwent a double-blind placebo-controlled food challenge to hazelnut. Sera of 423 of 731 subjects were analyzed for IgE against 7 hazelnut allergens and cross-reactive carbohydrate determinants by ImmunoCAP. RESULTS Hazelnut allergy was confirmed in 70% of those undergoing double-blind placebo-controlled food challenges. Birch pollen-driven hazelnut sensitization (Cor a 1) dominated in most cities, except in Reykjavik, Sofia, Athens, and Madrid, where reporting of hazelnut allergy was less frequent anyhow. In Athens, IgE against Cor a 8 dominated and strongly correlated with IgE against walnut, peach, and apple and against Chenopodium, plane tree, and mugwort pollen. Sensitization to seed storage proteins was observed in less than 10%, mainly in children, and correlated with IgE to nuts, seeds, and legumes. IgE to Cor a 12, observed in all cities (10% to 25%), correlated with IgE to nuts, seeds, and pollen. CONCLUSIONS In adulthood, the importance of hazelnut sensitization to storage proteins, oleosin (Cor a 12), and Cor a 8 is diluted by the increased role of birch pollen cross-reactivity with Cor a 1. Cor a 8 sensitization in the Mediterranean is probably driven by diet in combination with pollen exposure. Hazelnut oleosin sensitization is prevalent across Europe; however, the clinical relevance remains to be established.

Purification and structural stability of the peach allergens Pru p 1 and Pru p 3

Pru p 1 (a Bet v 1 homologue) and Pru p 3 (a nonspecific lipid transfer protein; nsLTP) are major allergenic proteins in peach fruit, but differ in their abundance and stability. Pru p 1 has low abundance and is highly labile and was purified after expression as a recombinant protein in Escherichia coli. Pru p 3 is highly abundant in peach peel and was purified by conventional methods. The identities of the proteins were confirmed by sequence analysis and their masses determined by MS analysis. The purified proteins reacted with antisera against related allergens from other species: Pru p 1 with antiserum to Bet v 1 and Pru p 3 with antiserum to Mal d 3 (from apple). The presence of secondary and tertiary structure was demonstrated by circular dichroism (CD) and high field NMR spectroscopy. CD spectroscopy also showed that the two proteins differed in their stability at pH 3 and in their ability to refold after heating to 95 degrees C. Thus, Pru p 1 was unfolded at pH 3 even at 25 degrees C but was able to refold after heating to 95 degrees C at pH 7.5. In contrast, Pru p 3 was unable to refold after heating under neutral conditions but readily refolded after heating at pH 3.

Promoter analysis and immunolocalisation show that puroindoline genes are exclusively expressed in starchy endosperm cells of wheat grain

The purolindolines are small cysteine-rich proteins which are present in the grain of wheat. They have a major impact on the utilisation of the grain as they are the major determinants of grain texture, which affects both milling and baking properties. Bread and durum wheats were transformed with constructs comprising the promoter regions of the Puroindoline a (Pina) and Puroindoline b (Pinb) genes fused to the uidA (GUS) reporter gene. Nine lines showing 3:1 segregation for the transgene and comprising all transgene/species combinations were selected for detailed analysis of transgene expression during grain development. This showed that transgene expression occurred only in the starchy endosperm cells and was not observed in any other seed or vegetative tissues. The location of the puroindoline proteins in these cells was confirmed by tissue printing of developing grain, using a highly specific monoclonal antibody for detection and an antibody to the aleurone-localised 8S globulin as a control. This provides clear evidence that puroindolines are only synthesised and accumulated in the starchy endosperm cells of the wheat grain.

Temporal and spatial changes in cell wall composition in developing grains of wheat cv. Hereward

A combination of enzyme mapping, FT-IR microscopy and NMR spectroscopy was used to study temporal and spatial aspects of endosperm cell wall synthesis and deposition in developing grain of bread wheat cv. Hereward. This confirmed previous reports that changes in the proportions of the two major groups of cell wall polysaccharides occur, with β-glucan accumulating earlier in development than arabinoxylan. Changes in the structure of the arabinoxylan occurred, with decreased proportions of disubstituted xylose residues and increased proportions of monosubstituted xylose residues. These are likely to result, at least in part, from arabinoxylan restructuring catalysed by enzymes such as arabinoxylan arabinofurano hydrolase and lead to changes in cell wall mechanical properties which may be required to withstand stresses during grain maturation and desiccation.

Isolation, cloning, and characterization of the 2S albumin: A new allergen from hazelnut

SCOPE 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. METHODS 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP-HPLC. After N-terminal sequencing, degenerated and poly-d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut-allergic patients. RESULTS N-terminal sequencing of a approximately 10 kDa peak from size exclusion chromatography/RP-HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N-terminal and a 10 aa internal peptide. The obtained clone (441 bp) encoded a 147 aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. CONCLUSION We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.

Role of polysaccharides in food, digestion, and health

ABSTRACT Polysaccharides derived from plant foods are major components of the human diet, with limited contributions of related components from fungal and algal sources. In particular, starch and other storage carbohydrates are the major sources of energy in all diets, while cell wall polysaccharides are the major components of dietary fiber. We review the role of these components in the human diet, including their structure and distribution, their modification during food processing and effects on functional properties, their behavior in the gastrointestinal tract, and their contribution to healthy diets.

Purification and characterisation of a panel of peanut allergens suitable for use in allergy diagnosis

Peanut is a major cause of type 1 hypersensitive reactions including anaphylaxis. This results from the presence of a number of protein allergens, six of which are being studied as part of the EU FP6 EuroPrevall programme. These are Ara h 1 (7S globulin), Ara h 2, Ara h 6 (2S albumins), Ara h 3/4 (11S globulins) and Ara h 8 (Bet v 1 homologue). Methods for the purification of Ara h 1, Ara h 3/4, Ara h 2 and Ara h 6 from peanut seeds and for the production of recombinant Ara h 8 in Escherichia coli are described with spectroscopic analyses being used to confirm that they are authentically folded. N-terminal sequencing of the proteins purified from peanut seeds also revealed details of the differences between isoforms and their generation by proteolytic processing within the seed. Preliminary IgE binding studies of the purified allergens confirmed that they retained their immunological properties indicating their suitability for use in allergy diagnosis.