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PubMed1984

An enzyme-linked immunosorbent assay for zein and other proteins using unconventional solvents for antigen adsorption.

An enzyme-linked immunosorbent assay (ELISA) performed in polystyrene microtiter plates that can detect and quantitate the maize prolamin zein is described. The assay yields positive reactions with as little as 1 ng of antigen and uses solvents not ordinarily employed in ELISA methods. A systematic investigation of zein adsorption to polystyrene in various solvents supports the hypothesis that antigen binding occurs through nonpolar interactions. The method was also used to determine structural relationships among three zein polypeptides differing in size and charge. Additional experiments indicate that a number of soluble proteins are absorbed to polystyrene in the denaturing agent urea and retain immunological reactivity. The retention of antigen reactivity after solubilization in 6-8 M urea suggests that ELISA methods may be applicable to other proteins which are insoluble, or rendered insoluble, in aqueous buffers.

Cross Reactions↗

PubMed1987

Detection of wheat gliadin contamination of gluten-free foods by a monoclonal antibody dot immunobinding assay.

Unfractionated wheat gliadin was used to produce murine monoclonal antibodies to gliadin. A dot immunobinding assay, using these antibodies, was developed to detect possible gliadin contamination of nominally gluten-free flour, using dilute ethanol extracts spotted onto nitrocellulose membranes. The sensitivity of the assay was less than 10 micrograms/ml of unfractionated gliadin which permitted the detection of trace amounts of gliadin present in certain wheat starch based 'gluten-free' products. The assay detected not only wheat gliadin, but also prolamine extracts of rye, barley and oats; maize, soya and potato extracts as well as the control proteins casein and ovalbumin, gave negative results. The assay is of value as a simple and rapid method of screening foods for their suitability for consumption by patients with coeliac disease.

Animals↗

PubMed1991

Cellular and humoral responses in coeliac disease. 2. Protein extracts from different cereals.

The humoral and cellular immune responses to grain protein extracts from coeliac-toxic and non-toxic cereals were compared by use of a number of ELISA and immunoblotting methods and the indirect leucocyte migration inhibition factor (LMIF) assay. Both adult and child coeliacs had elevated levels of serum antibody to proteins from the coeliac-toxic cereals, namely bread wheat, durum wheat, rye and barley and low levels of proteins from other cereals. Using protein blotting techniques, antibody binding was greatest to gliadins/low mol mass glutenin subunits and homologous prolamins from rye and barley, consistent with the ELISA findings. Competition ELISA and preabsorption tests indicated that antibody reaction to maize storage proteins did not simply result from cross-reaction of antigliadin antibodies. In LMIF assays, only the wheat extracts had activity in coeliac patients. This is most likely partly due to loss of some of T-cell epitopes from the extraction technique required for these proteins, as well as the relatively small effects seen for even very active fractions in the LMIF assay.

Adult↗

PubMed1994

Gamma-type gliadins cause secretion of prostaglandin E2 in patients with coeliac disease.

Coeliac disease is induced by polypeptides in the prolamin fraction of wheat, termed gliadin. It has previously been demonstrated that the alpha-, the beta- and the gamma-gliadin fractions contain toxic components and it has furthermore been strongly indicated that alpha-type gliadins are toxic. Due to insufficient protein separation methods there has been no information as to whether also the gamma-type gliadins are injurious in coeliac disease. We have therefore purified one alpha-type (alpha-39) and two gamma-type gliadins (gamma-36 and gamma-47) in a preparative scale by a combination of different ion exchange chromatographies. The purity was analyzed by high performance liquid chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, while the typing was based on determination of N-terminal amino acid sequence. Six patients with coeliac disease in remission were included in the study. Each of the purified gliadins was given by an intestinal perfusion technique to two patients. The perfusion fluid was collected and analyzed for the concentration of prostaglandin E2 (PGE2) as a marker for a toxic effect. All patients reacted with increased PGE2 secretion. For the first time it is clearly demonstrated that gamma-type gliadins are active in coeliac disease.

Amino Acid Sequence↗

PubMed1992

Conformation of wheat gluten proteins. Comparison between functional and solution states as determined by infrared spectroscopy.

The conformation of wheat gluten proteins in their functional hydrated solid state (doughy state) has been studied for the first time using attenuated total reflection infrared spectroscopy. The amide I band of functional gluten proteins reveals that, in addition to beta-turns and alpha-helices, these proteins contain a significant amount of intra- and intermolecular extended beta-sheet structures. It appears that the solubilization of gluten proteins results in a major decrease of the amount of beta-sheet structures accompanied by an increase of the content of the beta-turn and alpha-helical conformations. In addition, the alpha-helices appears to be more distorted in solution than in the functional state. Furthermore, spectra of omega- and gamma-gliadins, which are two types of prolamins of differing amino acid sequence and conformation, confirm the results obtained on the functional protein system. These results suggest that viscoelastic gluten proteins may interact through aligned beta-sheets corresponding to their repetitive domains.

Fourier Analysis↗

PubMed1986

A sandwich enzyme immunoassay for wheat gliadin.

A sandwich double antibody solid-phase enzyme immunoassay (EIA) that can detect and quantitate gliadin is described. Quantitative analysis could be carried out in the range of 5-400 ng/ml and the assay was more sensitive than inhibition EIA. Gliadin possessed weak cross-reactivity with oats, maize and rice prolamines. In the presence of anti-gliadin antibody (serum from a patient with active coeliac disease) gliadin could not be detected, but the sensitivity of the assay was restored by heat treatment of the serum. This EIA can therefore be employed to assess the gliadin content of biological fluids where specific antibody may coexist.

Antibody Specificity↗

PubMedDate not supplied

Long-term pancreatic duct occlusion impairs the entero-insular axis in the dog--failure of plasma VIP to respond as "incretin".

The response of VIP to either an oral glucose load (OGT) or intravenous glucose (IV glucose), aimed at reproducing the plasma glucose level after OGT, was studied in trained, conscious, sham-operated (Sham; n = 6) dogs, and dogs having initially (12 months before the glucose experiments) undergone occlusion of the pancreatic duct by the prolamine glue technique (Occ; n = 5). As a result, prior to glucose studies, the exocrine pancreas function was found subtotally reduced, as indirectly evaluated by the para-aminobenzoic acid (PABA) test, but no signs of diabetes were detected. The two studies with glucose administration designed to demonstrate the release of insulin, VIP, somatostatin into plasma as modified by enteric signals (represented by the difference of plasma peptide concentration during OGT minus peptide concentration during IV glucose) revealed the following: (1) basal plasma glucose, insulin, VIP, somatostatin did not differ between Sham and Occ dogs; (2) after OGT in Occ dogs the plasma glucose was elevated, whereas plasma insulin was markedly reduced, and VIP, somatostatin were largely unchanged; (3) the integrated output of insulin only was impaired when considering the so-called entero-insulin axis, while integrated VIP, somatostatin were unaltered. It was concluded (a) the Occ procedure in the dog has the capacity to subtotal destruction of the pancreatic acinar tissue, and of the entero-insular axis of insulin, the latter through yet unknown pathways, (b) the Occ technique may be a useful tool for investigation of the nature of "incretin," (c) VIP and somatostatin do not respond to elevated blood glucose and may have no role in the "incretin" concept of enteric modulation of the B-cell.

Animals↗

PubMed1988

Nucleotide sequence and endosperm-specific expression of the structural gene for the toxin alpha-hordothionin in barley (Hordeum vulgare L.).

A barley genomic library, obtained by cloning in the vector lambda EMBL-4, was screened with a cDNA probe encoding the alpha-hordothionin toxin. A positive clone, designated lambda TH1, was selected for further characterization. The coding and flanking regions of the alpha-hordothionin gene (Hth-1) were sequenced. Hth-1 has two introns of 420 and 91 nucleotides (nt), respectively. The promoter region has the following main features: one TATA box; three CATC boxes; an enhancer-like sequence, starting at nt position -282 from the first ATG codon, which is homologous to sequences appearing at similar positions in other endosperm genes; two versions of an 18-nt sequence that is more highly repeated in structural domains of several prolamin genes; two extensive regions close to the first ATG codon that are homologous to a sequence located much further upstream in the B-hordein promoter. The transcription start point was determined at nt positions -46 to -47, both by the S1 nuclease-protection and by the primer-extension assays. A maximum of 2-4 copies of the Hth-1 gene per haploid genome was determined by Southern-blot hybridization. Expression of the Hth-1 gene was detected during the cell proliferation stage of endosperm development (maximum at 13-16 days after pollinization) and was not detected in either etiolated or green coleoptiles.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of wheat alpha-gliadin synthesized in the yeast, Saccharomyces cerevisiae.

The development of efficient methods for production and purification of plant seed storage proteins in heterologous microbial hosts would facilitate structure-function studies of these proteins. This report describes such methods applied to the production and isolation of wheat alpha-gliadin, a prolamine-type seed storage protein, from Saccharomyces cerevisiae. Beginning with the vector, growth conditions, and extraction methods of Neill et al. [Gene 55 (1987) 303-317], we implemented several improvements to increase the yields of alpha-gliadin per volume of yeast cell culture. The CYCl::Gli-A2-Y transcriptional fusion vector, pAY31 (Neill et al., 1987), was modified by replacing the ARS1 region of replication with that of the 2 mu plasmid of yeast. We formulated a new medium, a derivative of synthetic defined (SD) medium supplemented with several nitrogen sources, that allows both selection for maintenance of plasmids and growth to high cell densities. Stationary phase cultures of cells bearing the modified expression vector, and grown in this medium with glycerol and lactate as carbon sources, contain significantly higher levels of alpha-gliadin than log-phase cultures grown in SD glucose. Sonication in 80% ethanol selectively and efficiently extracts the alpha-gliadin from cell pellets of small- or large-scale cultures, allowing the purification of several hundred micrograms of the wheat protein per liter in just a few high-yield steps. The alpha-gliadin isolated from yeast elutes at the same position in HPLC as the A-gliadin fraction purified from wheat flour. N-terminal amino acid (aa) sequencing reveals that the signal peptide is removed from the gliadin precursor in yeast cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Coeliac disease in adults.

Coeliac disease is a chronic disease characterized by small bowel villous atrophy which impairs nutrient absorption and improves on withdrawal of wheat gliadins and barley, rye and oat prolamins from the diet. Knowledge of the adult form of coeliac disease has greatly improved in recent years. Although this knowledge is not yet sufficiently widespread among referring clinicians, it has, over the past few years, allowed an increasing number of patients to be diagnosed with subclinical forms characterized by minor, transient or apparently unrelated symptoms. As a consequence, our views on the clinical and epidemiological aspects of this condition, the prevalence of which in the general population is believed to be close to 1 in 300, have changed and are still changing. Since it has been demonstrated that a strict gluten-free diet is protective against the complications of adult coeliac disease, it is important that even subclinical and silent forms are diagnosed and treated as early as possible. Non-invasive screening tests, such as anti-gliadin and anti-endomysium antibody estimation, should therefore be used systematically in groups considered to be at risk of coeliac disease. These include first-degree relatives of coeliac patients and patients with insulin-dependent diabetes mellitus, iron-deficiency anaemia, epilepsy with cerebral calcification, recurrent aphthous stomatitis and dental enamel hypoplasia. Other conditions will probably be identified in the near future.

Adult↗

PubMed1993

Assembly and transport of seed storage proteins.

Plant seeds store nitrogen by accumulating storage proteins in protein bodies within various compartments of the endomembrane system. The prolamin storage proteins of some cereal species are normally retained and assembled into protein bodies within the ER. Yet, these proteins lack a C-terminal KDEL/HDEL signal, suggesting that their retention is regulated by novel mechanisms. Furthermore, in other cereal species, such protein bodies formed within the ER may be subsequently internalized into vacuoles by a special route that does not utilize the Golgi complex. Thus, studies of the routing of seed storage proteins are revealing novel mechanisms of protein assembly and transport in the endomembrane system.

Journal Article↗

PubMed2003

High microbial production and characterization of strictly periodic polymers modelled on the repetitive domain of wheat gliadins.

Primary structures of wheat prolamins contain repetitive domains involved in the mechanical properties of gluten. In order to experience the ability of recombinant strictly periodic polypeptides, modelled on a consensus sequence of wheat gliadins (PQQPY)(8) and (PQQPY)(17) (SPR8 and SPR17 polypeptides, respectively), to be formulated in film solutions, their heterologous expression conditions, in batch culture and low cell densities, were optimized to match the high requirements of this process. A convenient and general purification procedure was also devised. Moreover, FTIR-ATR characterizations indicated that these periodic polypeptides prepared as hydrated doughy state and dried have the tendency to form a protein network through intermolecular beta-sheets, strongly maintained by hydrogen bonds. Accordingly, these recombinant polypeptides are assumed to be a suitable candidate for potential application.

Dimerization↗

PubMed2006

High prevalence of coeliac disease: need for increasing awareness among physicians.

BACKGROUND: Coeliac disease is a wheat gluten and related prolamines-induced disease with a prevalence that may be underestimated in many geographical regions and populations. AIM: To investigate the prevalence of coeliac disease in a population of schoolchildren of Estonia using tissue transglutaminase antibodies for screening. SUBJECTS AND METHODS: The study was designed as cross-sectional. Serum samples from 1160 randomly selected schoolchildren (636 female and 564 male, aged 9 or 15 years) were studied using a novel tissue transglutaminase antibody immunoassay (EliA Celikey IgA assay). Antibody-positive subjects were investigated for coeliac disease. RESULTS: A total of five subjects had antibodies. Four of them agreed for further investigations. By small-bowel biopsy they all were confirmed to have active coeliac disease, including three subjects with symptoms that were not considered by their family doctors. The prevalence of coeliac disease is at least 1 case per 290 (0.34% with CI 0.09-0.88%) in Estonia. It is much higher than that in our previous screening studies but is comparable with data from other European countries. CONCLUSION: The prevalence of coeliac disease might have increased during the last decade in Estonia. This study clearly shows that the awareness of coeliac disease among physicians is low. Thus, there is a need for more epidemiological studies and education related to coeliac disease.

Adolescent↗

PubMed2005

Rapid disruption of intestinal barrier function by gliadin involves altered expression of apical junctional proteins.

Coeliac disease is a chronic enteropathy caused by the ingestion of wheat gliadin and other cereal prolamines derived from rye and barley. In the present work, we investigated the mechanisms underlying altered barrier function properties exerted by gliadin-derived peptides in human Caco-2 intestinal epithelial cells. We demonstrate that gliadin alters barrier function almost immediately by decreasing transepithelial resistance and increasing permeability to small molecules (4 kDa). Gliadin caused a reorganisation of actin filaments and altered expression of the tight junction proteins occludin, claudin-3 and claudin-4, the TJ-associated protein ZO-1 and the adherens junction protein E-cadherin.

Actins↗

PubMed2006

Cys155 of 27 kDa maize gamma-zein is a key amino acid to improve its in vitro digestibility.

Twenty-seven kilodalton gamma-zein is a subclass of the maize zein storage proteins and, due to its localization at the protein body periphery, is critical to digestibility characteristics of all zeins. This protein had low in vitro digestibility, presumably due to its high Cys content (7.35 mol%) that is similar to the hard-to-digest analogous sorghum protein, gamma-kafirin. Therefore, each of the conserved disulfide-bonded Cys' was mutated to create C144A, C148A, C155A, and C156A maize gamma-zein mutants. The C155A showed a remarkable increase in digestibility to proteases - pepsin, chymotrypsin, and trypsin. A high conservation of this Cys among cereal gamma-prolamins indicates the utility of this finding.

Amino Acid Sequence↗

PubMed2005

Molecular properties of food allergens.

Plant food allergens belong to a rather limited number of protein families and are also characterized by a number of biochemical and physicochemical properties, many of which are also shared by food allergens of animal origin. These include thermal stability and resistance to proteolysis, which are enhanced by an ability to bind ligands, such as metal ions, lipids, or steroids. Other types of lipid interaction, including membranes or other lipid structures, represent another feature that might promote the allergenic properties of certain food proteins. A structural feature clearly related to stability is intramolecular disulfide bonds alongside posttranslational modifications, such as N-glycosylation. Some plant food allergens, such as the cereal seed storage prolamins, are rheomorphic proteins with polypeptide chains that adopt an ensemble of secondary structures resembling unfolded or partially folded proteins. Other plant food allergens are characterized by the presence of repetitive structures, the ability to form oligomers, and the tendency to aggregate. A summary of our current knowledge regarding the molecular properties of food allergens is presented. Although we cannot as yet predict the allergenicity of a given food protein, understanding of the molecular properties that might predispose them to becoming allergens is an important first step and will undoubtedly contribute to the integrative allergenic risk assessment process being adopted by regulators.

Allergens↗

PubMed2005

Pollen allergens are restricted to few protein families and show distinct patterns of species distribution.

BACKGROUND: Inhalative allergies are elicited predominantly by pollen of various plant species. However, a classification of the large number of identified pollen allergens is still missing. OBJECTIVE: To analyze pollen allergen sequences with respect to protein family membership, taxonomic distribution of protein families, and interspecies variability. METHODS: Protein family memberships of all plant allergen sequences from the Allergome database were determined by using the Protein Families Database of Alignments and Hidden Markov Models. The taxonomic distribution of pollen allergens was established from the Integrated Taxonomic Information System. Members of abundant pollen allergen families were compared with allergenic and nonallergenic homologues by database similarity searches and multiple sequence alignments. RESULTS: Pollen allergens were classified into 29 of 7868 protein families. Expansins, profilins, and calcium-binding proteins constitute the major pollen allergen families, whereas most plant food allergens belong to the prolamin, cupin, or profilin families. Pollen allergens were revealed to be ubiquitous (eg, profilins), present in certain plant families (eg, pectate lyases), or limited to a single taxon (eg, thaumatin-like proteins). Allergenic plant profilins constitute a highly conserved family with sequence identities of 70% to 85% among each other but low identities of 30% to 40% with nonallergenic profilins from other eukaryotes, including human beings. Similarly, allergenic polcalcins possess sequence identities of 64% to 92% but show low identities of 39% to 42% to related nonallergenic calmodulins and calmodulin-like proteins from vegetative plant tissues and man. CONCLUSION: This classification of pollen allergens into protein families will aid in predicting cross-reactivity, designing comprehensive diagnostic devices, and assessing the allergenic potential of novel proteins.

Allergens↗

PubMed2004

cDNA microarray analysis of gene expression in coeliac disease jejunal biopsy samples.

In coeliac disease in the jejunum of a genetically susceptible person wheat gliadin and related prolamins from rye and barley trigger an immunological reaction, which induces small-bowel mucosal transformations, villous atrophy and crypt hyperplasia. Though CD4+ specific T cells, intraepithelial lymphocytes, infiltrating plasma cells and autoantibodies are known to have an effect on the coeliac disease, the pathogenic mechanisms leading to the tissue injury remain to be elucidated. Our aim was to find novel gene transcripts, which might have a role in coeliac disease pathogenesis. The gene expression in duodenal biopsy samples from untreated coeliac patients (n=4), patients on gluten-free diet (n=4) and healthy controls (n=4) was studied by cDNA microarray analysis. The method allows monitoring of the expression of thousands of genes simultaneously. Compared to healthy controls, the expression of 156 and 60 genes was changed in untreated and treated coeliac disease, respectively. Between treated and untreated coeliac disease, 98 genes had altered expression. Of the 5184 genes or expressed sequence tags, altogether 263 were affected. Many of these genes are directly or indirectly connected to T-cell activation, B-cell maturation or epithelial cell differentiation. By the microarray method, numerous genes were found to evince altered mRNA expression in coeliac disease. The method holds promise in exploring the pathogenetic mechanisms in the small bowel and may reveal new target genes for the therapy of coeliac disease.

Adult↗