Xylosyl Residues Research Articles

Structural features of arabinoxylans from Sonalika variety of wheat: comparison between whole wheat flour and wheat bran

AbstractArabinoxylans (AX) were extracted from Sonalika variety of wheat (whole wheat flour and wheat bran) with barium hydroxide and sodium hydroxide and purified by a combination of alcohol precipitation and glucoamylase digestion. Structural features of purified AX were elucidated by methylation analysis,13C NMR, FT‐IR, periodate oxidation and optical rotation measurements. The AX showed a backbone of xylose residues with β(1–4) linkages and were branched mainly through O‐3 of xylose residues. Completely branched xylosyl residues were also present. Copyright © 2003 Society of Chemical Industry

Cross-reactive N-glycans of Api g 5, a high molecular weight glycoprotein allergen from celery, are required for immunoglobulin E binding and activation of effector cells from allergic patients.

Allergy diagnosis relying on the determination of specific IgE is frequently complicated by the presence of cross-reacting IgE of unclear clinical relevance. Particularly, the anaphylactogenic activity of IgE directed to cross-reactive carbohydrate moieties of glycoproteins from plants and invertebrates has been a matter of debate. In this study, we present the biochemical and immunological characterization of Api g 5, a glycoprotein allergen from celery with homology to FAD containing oxidases. Carbohydrate analysis of the allergen revealed the presence of glycans carrying fucosyl and xylosyl residues, structures previously shown to bind IgE. Chemical deglycosylation of the protein completely abolished binding of serum IgE from all 14 patients tested. Likewise, basophils from a patient allergic to mugwort pollen and celery were stimulated only by native Api g 5, whereas the deglycosylated allergen did not trigger release of histamine. IgE inhibition immunoblots showed that native Api g 5 other than the deglycosylated protein completely inhibited IgE binding to high molecular weight allergens in protein extracts from birch pollen, mugwort pollen, and celery. A similar inhibition was accomplished using the IgE binding oligosaccharide, MUXF, coupled to bovine serum albumin. All these observations taken together confer convincing evidence that IgE directed to cross-reactive carbohydrates is capable of eliciting allergic reactions in vivo.

Positive and negative electrospray ionisation tandem mass spectrometry as a tool for structural characterisation of acid released oligosaccharides from olive pulp glucuronoxylans

Xylo-oligosaccharides with degrees of polymerisation 5–13, formed by partial acid hydrolysis from an extract representative of olive pulp glucuronoxylans (GX), were analysed by electrospray ionisation mass spectrometry (ESI-MS), both in positive and negative modes. The positive spectrum showed the presence of xylo-oligosaccharides in the mass range between m/z 500 and 1500 corresponding to singly [M+Na] + charged ions of neutral (Xyl 7–9) and acidic xylo-oligosaccharides (Xyl 5–9MeGlcA), and doubly [M+2Na] 2+ charged ions of Xyl 9–13 and Xyl 7–11MeGlcA. Ammonium adducts [M+NH 4] + were also observed for Xyl 5–9MeGlcA. The negative spectra showed the contribution of ions in the mass range between m/z 600 and 1400, ascribed to the deprotonated molecules [M−H] − of Xyl 3–9MeGlcA. Tandem mass spectrometry (MS/MS) of the major ions observed in the MS spectra was performed. The MS/MS spectra of the [M+Na] + adducts showed the loss of MeGlcA residues as the major fragmentation pathway and glycosidic fragment ions of Xyl n and Xyl n MeGlcA structures. The MS/MS spectra of the [M+NH 4] + adducts suggests the occurrence of isomers of Xyl 5–9MeGlcA oligosaccharides with the MeGlcA residue at the reducing end and at the non-reducing end of the molecules, although other structural isomers can also occur. Both glycosidic bond and cross-ring cleavages in the MS/MS spectra of the [M−H] − ion suggest the occurrence of Xyl 3–9MeGlcA with the substituting group at the reducing end position of the xylose backbone, as the main fragmentation ions. The results obtained by ESI-MS/MS, both in positive and negative modes, of Xyl 7–13- and Xyl 5–11MeGlcA, allow to identify fragmentation patterns of the structural isomers with MeGlcA linked to the terminal xylosyl residues of the oligosaccharides. The occurrence of these higher molecular weight oligosaccharides with a low substitution pattern allows to infer a scatter and random distribution of MeGlcA along the xylan backbone of olive pulp.

Solubilization of an arabinan arabinosyltransferase activity from mung bean hypocotyls.

The biosynthesis of polysaccharides destined for the plant cell wall and the subsequent assembly of the cell wall are poorly understood processes that are currently the focus of much research. Arabinan, a component of the pectic polysaccharide rhamnogalacturonan I, is composed of arabinosyl residues connected via various glycosidic linkages, and therefore, the biosynthesis of arabinan is likely to involve more than one arabinosyltransferase. We have studied the transfer of [(14)C]arabinose (Ara) from UDP-L-arabinopyranose onto polysaccharides using microsomal membranes isolated from mung bean (Vigna radiata) hypocotyls. [(14)C]arabinosyl and [(14)C]xylosyl residues were incorporated into endogenous products due to the presence of UDP-Xyl-4-epimerase activity. Enzymatic digestion of endogenous products with endo-arabinanase released very little radiolabeled sugars, whereas digestion with arabinofuranosidase released some [(14)C]Ara. Microsomal membranes solubilized with the detergent octyl glucoside were able to add a single [(14)C]Ara residue onto (1-->5)-linked alpha-L-arabino-oligosaccharide acceptors. The reaction had a pH optimum of 6.5 and a requirement for manganese ions. However, enzymatic digestion of the radiolabeled oligosaccharides with endo-arabinanase and arabinofuranosidases could not fully release the radiolabeled Ara residue, indicating that the [(14)C]Ara residue was not a (1-->2)-, (1-->3)-, or (1-->5)-linked alpha-L-arabinofuranosyl residue. Rather, mild acid treatment of the product suggested that the radiolabeled Ara residue was in a pyranose conformation, and this result was confirmed by thin-layer chromatography of radiolabeled partially methylated sugars. Using microsomal membranes separated on a discontinuous sucrose gradient, the arabinosyltransferase activity appears to be mainly localized to Golgi membranes.

Location of O-acetyl substituents in xylo-oligosaccharides obtained from hydrothermally treated Eucalyptus wood

A combination of techniques was used to localise the O-acetyl substituents in xylo-oligosaccharides, which are present in hydrolysates of hydrothermally treated Eucalyptus wood. Reversed-phase (RP)-high performance liquid chromatography (HPLC) coupled on-line to both a mass spectrometer and an evaporating light scattering (ELS) detector provided data about the order of elution of the various O-acetylated oligomers. The retention of the oligomers on the column depended on the number and position of the O-acetyl substituents within the xylo-oligosaccharides. One dimensional (1D)- and two dimensional (2D)- 1H NMR spectroscopy was used to study the structural features of several xylotetramers separated by RP-HPLC, each having one O-acetyl substituent. O-Acetyl migration was proven to have occurred in these xylo-oligosaccharides. Mainly O-acetyl migration within the same xylosyl residue was observed. RP-HPLC–NMR was performed in order to study the structural features of the acetylated oligomers ‘on-line’ avoiding O-acetyl migration. Finally, the precise location of the 2- O- or 3- O-acetyl substituent in 6 xylotetramers and 4 xylotrimers separated by RP-HPLC was determined.

An Arabidopsis gene encoding an alpha-xylosyltransferase involved in xyloglucan biosynthesis.

Microsomal membranes catalyze the formation of xyloglucan from UDP-Glc and UDP-Xyl by cooperative action of alpha-xylosyltransferase and beta-glucan synthase activities. Here we report that etiolated pea microsomes contain an alpha-xylosyltransferase that catalyzes the transfer of xylose from UDP-[(14)C]xylose onto beta(1,4)-linked glucan chains. The solubilized enzyme had the capacity to transfer xylosyl residues onto cello-oligosaccharides having 5 or more glucose residues. Analysis of the data from these biochemical assays led to the identification of a group of Arabidopsis genes and the hypothesis that one or more members of this group may encode alpha-xylosyltransferases involved in xyloglucan biosynthesis. To evaluate this hypothesis, the candidate genes were expressed in Pichia pastoris and their activities measured with the biochemical assay described above. One of the candidate genes showed cello-oligosaccharide-dependent xylosyltransferase activity. Characterization of the radiolabeled products obtained with cellopentaose as acceptor indicated that the pea and the Arabidopsis enzymes transfer the (14)C-labeled xylose mainly to the second glucose residue from the nonreducing end. Enzymatic digestion of these radiolabeled products produced results that would be expected if the xylose was attached in an alpha(1,6)-linkage to the glucan chain. We conclude that this Arabidopsis gene encodes an alpha-xylosyltransferase activity involved in xyloglucan biosynthesis.

A xylosyltransferase that synthesizes beta-(1-->4)-xylans in wheat (Triticum aestivum L.) seedlings.

A particulate preparation from 6-day-old seedlings of wheat (Triticum aestivum L.) was found to contain a xylosyltransferase (XylTase) which incorporated xylose (Xyl) from UDP-xylose into exogenous beta-(1-->4)-xylooligosaccharides with 2-aminopyridine-derivatized reducing end groups. High-performance liquid chromatographic analysis showed that the chain elongation of pyridylaminated beta-(1-->4)-xylotriose (Xyl3-PA) occurred by attachment of a series of one, two, or three xylosyl residues, depending on substrate concentrations and reaction times. Methylation analysis and beta-xylosidase digestion of the newly synthesized Xyl4-PA confirmed that the xylosyl residues were incorporated through beta-(1-->4)-linkages. The enzyme was maximally active at pH 6.8 and 20 degrees C, and required Triton X-100, which enhanced activity 5-fold at a concentration of 0.05-2%. Divalent ions, including Mn2+ and Mg2+, did not affect activity. Enzyme activity increased with increasing polymerization of xylosyl residues of the acceptor substrates: for instance, Xyl5-PA was almost 7 times as efficient as Xyl2-PA. The apparent Michaelis constants of the enzyme for Xyl3-PA and UDP-xylose were 13.5 and 7.9 mM, respectively. The enzyme also catalyzed incorporation of radioactive sugars (Xyl together with a small portion of L-arabinose) from UDP-[14C]xylose into higher beta-(1-->4)-xylooligosaccharides (degree of polymerization > 7) with or without (4-O-methyl-)glucuronosyl side chains at activities comparable to those observed for pyridylaminated xylooligosaccharides, and into several heteroxylans but with much lower efficiency. Enzymatic hydrolysis of the product with a beta-xylanase degraded it into mainly xylobiose, providing further evidence that the xylosyl residues are incorporated through beta-(1-->4)-linkages.

Barley arabinoxylan arabinofuranohydrolases: purification, characterization and determination of primary structures from cDNA clones.

A family 51 arabinoxylan arabinofuranohydrolase, designated AXAH-I, has been purified from extracts of 7-day-old barley (Hordeum vulgare L.) seedlings by fractional precipitation with (NH(4))(2)SO(4) and ion-exchange chromatography. The enzyme has an apparent molecular mass of 65 kDa and releases L-arabinose from cereal cell wall arabinoxylans with a pH optimum of 4.3, a catalytic rate constant (k(cat)) of 6.9 s(-1) and a catalytic efficiency factor (k(cat)/K(m)) of 0.76 (ml x s(-1) x mg(-1)). Whereas the hydrolysis of alpha-L-arabinofuranosyl residues linked to C(O)3 of backbone (1-->4)-beta-xylosyl residues proceeds at the fastest rate, alpha-L-arabinofuranosyl residues on doubly substituted xylosyl residues are also hydrolysed, at lower rates. A near full-length cDNA encoding barley AXAH-I indicates that the mature enzyme consists of 626 amino acid residues and has a calculated pI of 4.8. A second cDNA, which is 81% identical with that encoding AXAH-I, encodes another barley AXAH, which has been designated AXAH-II. The barley AXAHs are likely to have key roles in wall metabolism in cereals and other members of the Poaceae. Thus the enzymes could participate in the modification of the fine structure of arabinoxylan during wall deposition, maturation or expansion, or in wall turnover and the hydrolysis of arabinoxylans in germinated grain.

Barley arabinoxylan arabinofuranohydrolases: purification, characterization and determination of primary structures from cDNA clones

A family 51 arabinoxylan arabinofuranohydrolase, designated AXAH-I, has been purified from extracts of 7-day-old barley (Hordeum vulgare L.) seedlings by fractional precipitation with (NH4)2SO4 and ion-exchange chromatography. The enzyme has an apparent molecular mass of 65kDa and releases l-arabinose from cereal cell wall arabinoxylans with a pH optimum of 4.3, a catalytic rate constant (kcat) of 6.9s−1 and a catalytic efficiency factor (kcat/Km) of 0.76 (ml·s−1·mg−1). Whereas the hydrolysis of α-l-arabinofuranosyl residues linked to C(O)3 of backbone (1 → 4)-β-xylosyl residues proceeds at the fastest rate, α-l-arabinofuranosyl residues on doubly substituted xylosyl residues are also hydrolysed, at lower rates. A near full-length cDNA encoding barley AXAH-I indicates that the mature enzyme consists of 626 amino acid residues and has a calculated pI of 4.8. A second cDNA, which is 81% identical with that encoding AXAH-I, encodes another barley AXAH, which has been designated AXAH-II. The barley AXAHs are likely to have key roles in wall metabolism in cereals and other members of the Poaceae. Thus the enzymes could participate in the modification of the fine structure of arabinoxylan during wall deposition, maturation or expansion, or in wall turnover and the hydrolysis of arabinoxylans in germinated grain.

Extraction, purification and chemical characterisation of xylogalacturonans from pea hulls

Pea hulls contained 925 mg/g sugar including 659 mg/g cellulosic glucose and 90 mg/g uronic acid. They were de-esterified by NaOH (pH>13 at 4°C, 2 h) and treated with HCl (0.1 mol/l, 80°C, 24 h). The HCl-soluble fraction represented 95 mg/g initial pea hulls. It was rich in galacturonic acid (259 mg/g), xylose (93 mg/g) and rhamnose (91 mg/g), which co-eluted in anion-exchange chromatography. The HCl-soluble fraction was degraded by a rhamnogalacturonan-hydrolase and the reaction products were fractionated by size-exclusion chromatography. Two fractions, representing together 18 mg/g initial pea hulls, were composed almost exclusively of galacturonic acid and xylose and could be defined as xylogalacturonans. The first fraction exhibited a high molar mass, a molar ratio Xyl/GalA of 1 and contained almost 5% of rhamnose. The molar mass of the second fraction was much lower and the molar ratio Xyl/GalA was 0.6. Methylation analysis showed the presence in both fractions of a α(1→4) galacturonan backbone highly substituted on O-3 either by terminal xylosyl residues or by short side-chains of (1→2) linked xylosyl residues.

Water-soluble p-carboxybenzylated beechwood 4- O-methylglucuronoxylan: structural features and properties

The structural features of the water-soluble p-carboxybenzyl 4- O-methylglucuronoxylan derivative (CBGX), prepared by reaction of xylan with p-carboxybenzyl bromide in aqueous alkali, were characterised by various one-dimensional (1D)- and 2D-NMR techniques applied to the intact derivative as well as to both the oligosaccharide (XO) and resistant (XR) fractions obtained by digestion of CBGX with endo-β- d-xylanase. The real substitution of the xylan backbone with carboxybenzyl (CB) groups was unambiguously determined by the HMBC-NMR technique. The CB groups were located predominantly at the O-2 position of the xylosyl residues. The degrees of substitution of CBGX with 4- O-methylglucuronic acid units (DS U) and CB groups (DS CB) were 0.10 and 0.11, respectively. Thus, every fifth xylose unit of the main chain, on an average, bears either an acidic side chain or a CB substituent. The different proportions of both the substituents in XO and XR fractions suggested the distribution along the xylan backbone to be blockwise rather than uniform. The physicochemical properties of CBGX were characterised by means of viscometry, sedimentation velocity and HPGPC giving a relatively low intrinsic viscosity (53 ml g −1) and similar values of the mean molecular mass ( M w) 27,000 g mol −1 and ( M app) 34,000 g mol −1, respectively. CBGX exhibited remarkable emulsifying and protein foam-stabilising activities.

X-ray crystallographic study of xylopentaose binding to Pseudomonas fluorescens xylanase A.

The structure of the complex between a catalytically compromised family 10 xylanase and a xylopentaose substrate has been determined by X-ray crystallography and refined to 3.2 A resolution. The substrate binds at the C-terminal end of the eightfold betaalpha-barrel of Pseudomonas fluorescens subsp. cellulosa xylanase A and occupies substrate binding subsites -1 to +4. Crystal contacts are shown to prevent the expected mode of binding from subsite -2 to +3, because of steric hindrance to subsite -2. The loss of accessible surface at individual subsites on binding of xylopentaose parallels well previously reported experimental measurements of individual subsites binding energies, decreasing going from subsite +2 to +4. Nine conserved residues contribute to subsite -1, including three tryptophan residues forming an aromatic cage around the xylosyl residue at this subsite. One of these, Trp 313, is the single residue contributing most lost accessible surface to subsite -1, and goes from a highly mobile to a well-defined conformation on binding of the substrate. A comparison of xylanase A with C. fimi CEX around the +1 subsite suggests that a flatter and less polar surface is responsible for the better catalytic properties of CEX on aryl substrates. The view of catalysis that emerges from combining this with previously published work is the following: (1) xylan is recognized and bound by the xylanase as a left-handed threefold helix; (2) the xylosyl residue at subsite -1 is distorted and pulled down toward the catalytic residues, and the glycosidic bond is strained and broken to form the enzyme-substrate covalent intermediate; (3) the intermediate is attacked by an activated water molecule, following the classic retaining glycosyl hydrolase mechanism.

Xylanase and β-xylosidase separation by fractional precipitation

β-Xylosidase (EC 3.2. 1.37) belongs to an enzyme complex, the xylanases, which cleave xylans and release xylosyl residues by endwise attack of xylo-oligosaccharides. Precipitation was performed with ethanol at concentrations of 20, 40, 60 and 80% at different pH values (4.6, 5.9, 6.3 and 7.0), in order to compare the behaviour of β-xylosidase with the behaviour of total xylanase present in the medium. In addition, a fractional precipitation of these enzymes was performed in three steps under the most suitable of all the conditions tested (pH 4.6, 4°C and 20, 60 and 80% ethanol concentrations). In the first step (20% ethanol), around 10% of β-xylosidase and 80% of total protein precipitated, and in the second step (60% ethanol), 75% of the same enzyme precipitated. The remaining xylanolitic complex (82%) precipitated in the third step (80% ethanol). The electrophoresis analysis showed that the enzymes can be selectively separated by ethanol precipitation according to their molecular weights. The kinetic parameters ( K M and V m) of β-xylosidase before and after the precipitations remained constant. The separation process did not affect the kinetic characteristics of the enzyme.

Crystal Structure of Cardosin A, a Glycosylated and Arg-Gly-Asp-containing Aspartic Proteinase from the Flowers ofCynara cardunculus L.

Aspartic proteinases (AP) have been widely studied within the living world, but so far no plant AP have been structurally characterized. The refined cardosin A crystallographic structure includes two molecules, built up by two glycosylated peptide chains (31 and 15 kDa each). The fold of cardosin A is typical within the AP family. The glycosyl content is described by 19 sugar rings attached to Asn-67 and Asn-257. They are localized on the molecular surface away from the conserved active site and show a new glycan of the plant complex type. A hydrogen bond between Gln-126 and Manbeta4 renders the monosaccharide oxygen O-2 sterically inaccessible to accept a xylosyl residue, therefore explaining the new type of the identified plant glycan. The Arg-Gly-Asp sequence, which has been shown to be involved in recognition of a putative cardosin A receptor, was found in a loop between two beta-strands on the molecular surface opposite the active site cleft. Based on the crystal structure, a possible mechanism whereby cardosin A might be orientated at the cell surface of the style to interact with its putative receptor from pollen is proposed. The biological implications of these findings are also discussed.

(2- O-α- d-Galactopyranosyl-4- O-methyl-α- d-glucurono)- d-xylan from Eucalyptus globulus Labill

An unusual heteroxylan composed of galactosyl, 4- O-methyl-glucuronosyl and xylosyl residues with molar ratio 1:3:30 was isolated from the wood of Eucalyptus globulus Labill. The results of linkage analysis, supported by data of 1H, 2D 1H– 1H COSY and 13C NMR spectroscopy, revealed that the polysaccharide is a (2- O-α- d-galactopyranosyl-4- O-methyl-α- d-glucurono)- d-xylan with a (1→4)-linked β- d-xylopyranosyl backbone branched at O-2 by short side chains composed of terminal 4- O-methyl-α- d-glucuronic acid and of 4- O-methyl-α- d-glucuronic acid substituted at O-2 with α- d-galactose.

Cell-wall biosynthesis in differentiating cells of pine root tips

The patterns of incorporation of radioactivity from d-[U- 14C]glucose into pectins, hemicelluloses and cellulose synthesised in differentiating cells of pine root-tips were analysed after sequential solvent extractions. The percentage composition of the radioactive glycosyl residues of hot-H 2O- and Na 2EDTA- + NH 4-oxalate- + NaBH 4-extractable cell-wall polymers was very similar. All sections of pine roots investigated synthesise a remarkably large amount of polymers rich in galactosyl and arabinosyl residues. These polymers may create in the wall a highly hydrated gel compartment structurally and physiologically important for the roots. The KOH-extractable polymers are rich in glucosyl and xylosyl residues.

Xylose-rich polysaccharides from the primary walls of embryogenic cell line of Pinus caribaea

Embryogenic cell lines of Pinus caribaea were isolated from somatic embryogenesis from zygotic embryos. Previous studies showed that the proteins and glycoproteins were characteristic of the embryogenic state. In the present work we were seeking typical feature in the polysaccharide from the cell walls of embryogenic calli at nine days of culture. Sequential extraction with water, ammonium oxalate, dimethyl sulfoxide, sodium borohydride and 4.3 M potassium hydroxide revealed that the extracted polysaccharides contained high proportions of arabinose and significant amounts of xylose. Fractionation of the hydrosoluble polymers on DEAE cellulose afforded a xylose-rich fraction (80% xylose, 24% glucose and lower properties of fucose and mannose). Methylation analysis and 13C-NMR spectra showed that the glycan backbone consisted of β 1→4 linked xylosyl residues Similar study of the fractions extracted respectively with DMSO and 4.3 M KOH showed the presence of polydisperse glycoxylans but excluded the presence of xyloglucan in significant amount. This could be a characteristic feature of embryogenic cells walls of Pinus caribaea or could be typical of cells grown as calluses. In the various fractions obtained from DEAE cellulose chromatography of the alkaline extract the infrequent occurrence of fucoxylans beside an arabinogalactan showed again the unusual nature of the cell wall polymers of this embryogenic lines, which seems to differ greatly from those found in the primary wall of cells from suspension cultures.

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (I. A Model of Adhesives Based on Chemical Characterization and Localization of Polysaccharides from the Marine Diatom Achnanthes longipes and Other Diatoms).

Extracellular adhesives from the diatoms Achnanthes longipes, Amphora coffeaeformis, Cymbella cistula, and Cymbella mexicana were characterized by monosaccharide and methylation analysis, lectin-fluorescein isothiocyanate localization, and cytochemical staining. Polysaccharide was the major component of adhesives formed during cell motility, synthesis of a basal pad, and/or production of a highly organized shaft. Hot water-insoluble/hot 0.5 M NaHCO3-soluble anionic polysaccharides from A. longipes and A. coffeaeformis adhesives were primarily composed of galactosyl (64-70%) and fucosyl (32-42%) residues. In A. longipes polymers, 2,3-, t-, 3-, and 4-linked/substituted galactosyl, t-, 3-, 4-, and 2-linked fucosyl, and t- and 2-linked glucuronic acid residues predominated. Adhesive polysaccharides from C. cistula were EDTA-soluble, sulfated, consisted of 83% galactosyl (4-, 4,6-, and 3,4-linked/substituted) and 13% xylosyl (t-, 4f/5p-, and 3p-linked/substituted) residues, and contained no uronosyl residues. Ulex europaeus agglutinin uniformly localized [alpha](1,2)-L-fucose units in C. cistula and Achnanthes adhesives formed during motility and in the pads of A. longipes. D-Galactose residues were localized throughout the shafts of C. cistula and capsules of A. coffeaeformis. D-Mannose and/or D-glucose, D-galactose, and [alpha](t)-L-fucose residues were uniformly localized in the outer layers of A. longipes shafts by Cancavalia ensiformis, Abrus precatorius, and Lotus tetragonolobus agglutinin, respectively. A model for diatom cell adhesive structure was developed from chemical characterization, localization, and microscopic observation of extracellular adhesive components formed during the diatom cell-attachment process.

Role of complex asparagine-linked glycans in the allergenicity of plant glycoproteins.

Many plant proteins, particularly those found in foods and pollen, are known to act as sensitizing agents in humans upon repeated exposure. Among the cereal flour proteins involved in asthmatic reactions, those members of the alpha-amylase inhibitor family which are glycosylated, polypeptides, BMAI-1, BTAI-CMb*, and WTAI-CM16* are particularly reactive both in vivo and in vitro. We show here that these major glycoprotein allergens carry a single asparagine-linked complex glycan that contains both beta 1-->2 xylose and alpha 1-->3 fucose. Evidence is presented that the xylosyl residue and, to a lesser extent, the fucosyl residue are key IgE-binding epitopes and largely responsible for the allergenicity of these and unrelated proteins from plants and insects. Our results suggest that the involvement of xylose- and fucose-containing complex glycans in allergenic responses may have been underestimated previously; these glycans provide a structural basis to help explain the cross-reactivities often observed between pollen, vegetable food, and insect allergens.

Extraction of phenolic-carbohydrate complexes from graminaceous cell walls

Barley straw and a preparation of perennial ryegrass were sequentially extracted with oxalic acid, dimethyl sulphoxide (DMSO) and a “cellulolytic” enzyme preparation Driselase and the fractions studied by methylation analysis, 13C NMR and other methods. Oxalic acid, as expected, solubilised the bulk of the arabinose and ferulic acid in both samples, although appreciable amounts of xylose were also solubilised. DMSO yielded polymeric lignin-carbohydrate complexes (LCC), both of which consisted predominantly of a β(1 → 4) xylan. From the results of methylation analysis, sensitivity to oxalic acid hydrolysis and size-exclusion chromatography after alkaline hydrolysis, it was evident that lignin polymers were attached to arabinosyl and xylosyl residues by both ester and aryl-ether linkages. After cellulolytic hydrolysis of the residues, thioacidolysis analysis of the lignin component in the DMSO-soluble and Driselase-insoluble fractions from ryegrass revealed differences in every measurable aspect. The DMSO-soluble lignin was found to be more highly condensed, have a higher S/G ratio and have a higher terminal G/internal G ratio.