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PubMed2001

Analysis and clinical effects of gluten in coeliac disease.

The prolamin working group coordinates research on laboratory gluten analysis in food and on clinical evaluation of patient sensitivity to prolamins. As an observer organization to the Codex Alimentarius Commission, the group summarizes current data on analysis and effects of gluten in coeliac disease. All types of gliadin, the ethanol-soluble fraction of gluten, contain the coeliac-active factor. However, coeliac toxicity and immunogenicity (humoral and cellular) of various prolamins are not identical in coeliac patients. There are no conclusive data on the threshold of gluten sensitivity of coeliac patients. Information as to the long-term risk to coeliac patients exposed to small doses of gliadin is lacking. Therefore, every effort should be made to keep the diet of coeliac patients as gluten-free as possible. The prolamin group is currently evaluating a new enzyme-linked immunosorbent assay (ELISA) protocol for gluten analysis that could serve as a basis for further Codex regulations. The group recommends adherence to a single Codex limit for gluten-free foods. The current limit of 200 ppm gluten is questionable and requires reconsideration based on new information that will be available soon.

Celiac Disease↗

PubMed1998

Measurement of gluten using a monoclonal antibody to a coeliac toxic peptide of A-gliadin.

BACKGROUND: Future European Community regulations will require a sensitive and specific assay for measurement of coeliac toxic gluten proteins in foods marketed as gluten-free. To avoid spurious cross reactions with non-toxic proteins, specific antibodies and target antigens are required. A synthetic 19 amino acid peptide of A gliadin has been shown to cause deterioration in the morphology of small intestinal biopsy specimens of coeliac patients in remission. AIMS: To develop an assay for detection of gluten in foods, based on measurement of a known toxic peptide. METHODS: A monoclonal antibody raised against the toxic A gliadin peptide, with a polyclonal anti-unfractionated gliadin capture antibody, was used to develop a double sandwich enzyme linked immunosorbent assay (ELISA) for the measurement of gluten in foods. RESULTS: Standard curves for gliadin and for rye, barley, and oat prolamins were produced. The sensitivity of the assay was 4 ng/ml of gliadin, 500 ng/ml for rye prolamins, and 1000 ng/ml for oat and barley prolamins. The assay could detect gluten in cooked foods, although at reduced sensitivity. Prolamins from coeliac non-toxic rice, maize, millet, and sorghum did not cross react in the assay. A variety of commercially available gluten-free foods were analysed; small quantities of gluten were detected in some products. CONCLUSION: The assay may form the basis of a sensitive method for measurement of gluten in foods for consumption by patients with coeliac disease.

Antibodies, Monoclonal↗

PubMed1980

[Comparative toxicity of different cereals for subjects intolerant of gluten].

Coeliac disease is caused by prolamines, the storage proteins of some cereals, located in the endosperm. Cereals do not all have the same toxicity. The four wheat prolamine groups (alpha, beta, gamma and omega gliadins), visible in electrophoresis at acid pH, have been isolated and their toxicities compared by observing the morphological changes in intestinal biopsies cultured in vitro when peptic-tryptic digests of the studied proteins were added to the culture medium. The toxicity was found to be mainly located in the alpha and beta-gliadins and in peptides of 5 to 10 000 molecular weight. Peptides, resulting from peptic-tryptic hydrolysis, varied in length as a direct function of their proline content. In fact, peptide bond splitting by pepsin and trypsin is known to be blocked by proline. Thus, proline content determines peptide length and toxicity. Wheat, rye and barley toxicities were compared on the basis of a correlation between toxicity and the alpha- and beta-gliadin-like prolamine contents of these cereals. Electrophoretic estimation of alpha- and beta-gliadin-like prolamine content gave the following prediction of relative toxicity (in decreasing order): wheat, triticale, rye, barley and oats.

Amino Acids↗

PubMed1992

[Comparative studies of the Osborne protein fraction of wheat varieties with different dough and baking properties].

The proteins of flours from the wheat varieties Clement (CLE) and Disponent (DIS), which are characterized by substitution of the wheat chromosome 1B by the rye chromosome 1R (CLE) and by 1BL/1RS-translocation of chromosome segments (DIS), respectively, and which yield sticky doughs, were extracted by the Osborne procedure and compared with the good variety Kolibri (KOL). CLE and DIS delivered significantly higher amounts of water-soluble proteins (31.2 and 23.2% of the total protein) than KOL (15.5%). The proportions of ethanol-soluble proteins were approximately constant for the three varieties (31.5-33.9%) while the amounts of residual proteins after extraction with ethanol were significantly different (CLE, 16.8%; DIS, 21.6%; KOL, 26.4%). Water extracts as well as residues exhibited also marked differences in their amino acid compositions. Investigation of the water extract from DIS by gel permeation chromatography and by RP-HPLC showed the presence of prolamins: omega 1,2-gliadins and/or omega-secalins could be detected on the basis of amino acid composition and N-terminal amino acid sequence. In the case of good varieties such as KOL, these prolamins seem to remain partially in the residue of the ethanol extract under the conditions of the classical Osborne procedure, whereas they are found in the water extract in the case of such varieties as CLE and DIS. On the other hand, a modified Osborne procedure (salt/ethanol/acetic acid) delivered the prolamins, which correspond to 39-40% of the total protein, completely within the ethanol fraction in the case of all three varieties.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1985

Molecular evolution of the seed storage proteins of barley, rye and wheat.

The major storage proteins (prolamins) of barley, rye and wheat are characterized by the presence of two or more unrelated structural domains, one of which contains repeated sequences. Because of this repetitive structure and their restricted distribution (only in grasses), it has been suggested that the prolamins are of recent origin. Contrary to this hypothesis, we show that parts of the non-repetitive domain of one group of prolamins are homologous with sequences present in a large group of seed proteins from monocotyledonous and dicotyledonous plants; including Bowman-Birk protease inhibitors, cereal inhibitors of alpha-amylase and trypsin, and 2 S globulin storage proteins of castor bean and oil seed rape. This implies an ancient origin for these non-repetitive domains. The origins of the repetitive domains are not known but may lie within the grasses.

Amino Acid Sequence↗

PubMed1993

Segregation of storage protein mRNAs on the rough endoplasmic reticulum membranes of rice endosperm cells.

Developing rice endosperm cells display two distinct rough endoplasmic reticula (ER), cisternal ER (C-ER) and protein body ER (PB-ER), the latter delimiting the prolamine protein bodies. These ER membranes are utilized for the simultaneous synthesis of glutelin and prolamine storage proteins, which are subsequently routed into separate protein bodies. We demonstrate by blot hybridization, and by visualization of the spatial distributions and densities of these transcripts in endosperm cells via high resolution in situ hybridization analysis, that prolamine transcripts are associated primarily with the PB-ER, while glutelin mRNAs are enriched on the C-ER. The results suggest that the initial targeting process of these storage proteins into distinct protein bodies is the segregation of their transcripts on the ER membranes.

Cell Compartmentation↗

PubMed2002

Changes induced by infestation on some chemical properties of cowpea seeds.

Three cowpea varieties were used in the present study, Insect susceptible popular variety, 'Ife-brown' (IFB), an improved variety for insect resistance IT 81D-975 (1975) and a local black cowpea variety with a white hilium adapted to diverse habitats, 'Akidi' (AKD). Cowpea seeds were infested with Callosobruchus maculatus. Crude protein content of infested and uninfested whole cowpea flour and protein concentrate was determined. Prolamin contents of flour and protein concentrates were evaluated. Fat content, free fatty acid and peroxide values of cowpea seeds were also determined. All determinations were carried out in infested and uninfested cowpea seeds in 3 replicates for each determination. Emulsion properties of cowpea seeds stored in 3 containers, plastic cans (PC) and polyethylene bags (white and black WPB/BPB) were monitored on a biweekly basis for 24 weeks. Varietal differences existed in the protein, fat and prolamin contents of cowpeas. Infestation reduced the contents of these nutrients in all cases. Free fatty acid contents and peroxide values were increased by > 100%. Infestation reduced the prolamin content by > 25% in all cases. Varietal differences also existed in emulsion capacities of cowpea flours; IFB and 975 formed better and more stable emulsions than AKD flour. Storage in plastic cans led to better retention of the emulsion properties of the cowpeas.

Animals↗

PubMed1998

The bifactorial endosperm box of gamma-zein gene: characterisation and function of the Pb3 and GZM cis-acting elements.

The proximal region of the gamma-zein promoter (gamma Z) has a functional bifactorial prolamin box element containing two cis-acting elements, a prolamin-box motif (Pb3) and a GCN4-like motif (GZM). By particle bombardment of maize endosperms with 5' deletions and internal deletions of gamma Z fused to the GUS gene, we have shown that a 135 bp region containing the bifactorial element is involved in the transcriptional activation of the gamma Z promoter. However, the 135 bp region was unable to activate the gamma Z promoter in the absence of a 84 bp downstream sequence. Using in vivo footprinting and gel mobility shift assays with 15 DAP endosperm nuclear extracts, we have demonstrated the presence of trans-acting factors that interact with Pb3 and GZM target sites. Base-substitution mutations within Pb3 and GZM decreased transcription activity of the gamma Z promoter suggesting a co-ordinated function between the two cis-acting elements. Two additional cis-motifs upstream of the bifactorial prolamin element have been identified: a motif with high homology to the AACA elements of rice glutelin genes and an AZM motif containing an ACGT core which binds nuclear proteins other than the Opaque 2 (O2).

Base Sequence↗

PubMed1994

Identification of sulphur-rich proteins which resist rumen degradation and are hydrolysed rapidly by intestinal proteases.

Several proteins with high proportions of S-containing essential amino acids were incubated in sheep rumen fluid in vitro and their rate of digestion was examined by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. The S-rich proteins rice prolamin (10 kDa), maize zein (10 kDa) and the 3.2 kDa pumpkin (Cucurbita maxima L.) trypsin inhibitor-1 (CMTI-1) were highly resistant to rumen fluid degradation, relative to control proteins of known degradation rate (casein, bovine serum albumin (BSA) and pea (Pisum sativum) albumin-1 (PA1)). Comparison of PA1 and a recombinant N-terminal epitope-tagged PA1 indicated that addition of the epitope caused a slight increase in resistance to rumen degradation. The proteins were also incubated with a mixture of trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). PA1, BSA and casein were hydrolysed less rapidly than rice prolamin, maize zein and CMTI-1. Digestion by these intestinal proteases appeared to be complete. Thus, the prolamin, zein and CMTI-1 proteins are suitable candidates for expression as foreign proteins in pasture plants to increase throughput and uptake of essential amino acids in sheep.

Amino Acids, Essential↗

PubMed2003

Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol.

OBJECTIVES: There is currently much call for a reliable enzyme-linked immunosorbent assay (ELISA) protocol for determining gluten in foods to serve as a basis for further Codex Alimentarius regulations. Given its ability to recognize the potential coeliac-toxic epitope QQPFP, which occurs repeatedly in alpha-, gamma- and omega-gliadins, hordeins and secalins, the monoclonal antibody R5 raised against a secalin extract may prove to be an essential tool for gluten analysis. This study was designed to develop a highly sensitive and specific sandwich ELISA to quantify low levels of wheat, barley and rye prolamins in foods for coeliacs. METHODS: Simple sandwich ELISA based on the use of a single monoclonal antibody (R5) as both the coating and detection was developed. A quantitative cocktail gluten-extraction procedure for heat-processed foods was also tested. RESULTS: R5-ELISA was able to identify gliadins, hordeins and secalins with assay sensitivities of 0.78, 0.39 and 0.39 ng/ml, respectively. The assay's detection limit was 1.5 ng gliadins/ml (1.56 ppm gliadins, 3.2 ppm gluten). The system proved insensitive to the non-coeliac-toxic cereals maize, rice and oats, and was non-cultivar-dependent. It was also able to detect gliadins and hordeins in unprocessed and heat-processed wheat- and barley-based products, and to estimate the gluten content of hydrolysed foods. CONCLUSION: We present a new generation of a robust sandwich R5-ELISA with good reproducibility (8.7%) and repeatability (7.7%). Its gluten-detection limit of 3.2 ppm is lower than the existing threshold of 20-200 ppm. The ELISA, which is equally sensitive to barley, wheat and rye prolamins, is compatible with the quantitative cocktail extraction procedure for heat-processed foods. Along with the cocktail procedure, the Working Group on Prolamin Analysis and Toxicity is currently evaluating an R5-ELISA system as proposed by the Codex Alimentarius Commission.

Antibodies, Monoclonal↗

PubMed2003

Dual regulated RNA transport pathways to the cortical region in developing rice endosperm.

Prolamine and glutelin RNAs are localized to two subdomains of the cortical endoplasmic reticulum (ER), the protein body ER and the cisternal ER, in developing rice seeds. The addition of nearly full-length prolamine sequences at the 3' untranslated region of a reporter RNA redirects its localization from the cisternal ER to the protein body ER. Deletion analysis of prolamine RNA sequences indicates the presence of two partially redundant cis elements required for protein body ER targeting. The addition of glutelin 3' untranslated region to protein body ER cis sequences, however, redirects RNA localization to the cisternal ER. These results indicate that there are at least two regulated RNA transport pathways as well as a constitutive pathway to the cortical ER.

3' Untranslated Regions↗

PubMed1984

An in vitro animal model for the study of cereal components toxic in celiac disease.

Peptic-tryptic-Cotazym (PTC) digests were obtained, simulating in vivo protein digestion, from rice, maize, rye, oats, barley, and sorghum prolamines and tested on small intestine cultures from rat fetus. The PTC digests of the prolamine fractions from rice and maize, even when tested at a concentration as high as 0.5 mg/ml, did not affect in vitro differentiation and maturation of fetal rat jejunum that took place in vitro in a way comparable to what happens in vivo. On the contrary, the PTC digests of prolamines from rye, oats, barley, and sorghum were very active in slowing down in vitro development of fetal rat intestine. These results further strengthen earlier findings and all together show that there is a strong correlation between toxicity results of cereal and/or cereal components assessed with clinical trials or in vitro systems based on bioptic specimens of intestinal mucosa from celiac patients and with the culture of rat fetal intestine. Therefore, the rat fetal intestine culture is considered to be an adequate model for screening and investigating cereal peptides which are toxic for the celiac small intestinal mucosa.

Animals↗

PubMed1994

Electrophoretic analysis of ruminal degradability of corn proteins.

Albumin, globulin, and prolamin fractions were extracted from corn meal in water, .5 M NaCl, and 50% (vol/vol) 1-propanol and examined by SDS-PAGE and densitometric scanning to investigate fractional degradation rates of corn proteins. Several protein fractions were identified for globulins, zein prolamins, and glutelins. Ruminal degradability of individual subfractions was evaluated by suspension of corn and corn gluten meal samples in the rumen of lactating dairy cows from 0 to 72 h. Electrophoretic and densitometric analysis of protein residues revealed that the prolamin fraction zein for corn and corn gluten meal was more resistant to ruminal degradation than albumins, globulins, and glutelins. Relative rates of degradation of zein and the fraction containing albumins, globulins, and glutelins were .060, .026, and .018, .015/h for corn and corn gluten meal, respectively. Total degradabilities of corn and corn gluten meal, measured by summation of degradability of subfractional components, were 52.2 and 18.6%. Quantitative measurement of ruminally degradable subfractions and estimation of their degradation rates by electrophoretic and densitometric scanning are useful in understanding ruminal degradability of corn proteins.

Albumins↗

PubMed1993

In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm.

The quality of the wheat grain is determined by the quantity and composition of storage proteins (prolamins) which are synthesized exclusively in endosperm tissue. We are investigating the mechanisms underlying the regulation of expression of a prolamin gene, the low molecular weight glutenin gene LMWG-1D1. The LMWG-1D1 promoter contains the endosperm box, a sequence motif highly conserved in the promoter region of a large number of storage protein genes, which is thought to confer endosperm-specific expression of prolamin genes. Here we show by in vivo DMS footprinting of wheat endosperm tissue that the endosperm box becomes occupied by putative trans-acting factors during grain ripening. During early stages of development the endosperm motif within the 5' half of the endosperm box becomes occupied first, followed by binding of a second activity to a GCN4/jun-like motif in the 3' half just prior to the stage of maximum gene expression. Occupancy of the endosperm box is highly tissue-specific: no protection was observed in husk and leaf tissues. Several binding activities were identified in vitro from nuclear protein extracts of wheat endosperm which bind specifically to the endosperm and GCN4/jun motifs identified by in vivo footprinting.

Base Sequence↗

PubMed2005

Cloning and molecular characterization of B-hordeins from Hordeum chilense (Roem. et Schult.).

One of the main limitations of cereal breeding is the lack of genetic variability within cultivated crops. Hordeum chilense is a wild relative of Hordeum vulgare, which has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. Among the agronomic traits of these new amphiploids, the allelic variation in the endosperm storage proteins and their influence on breadmaking and malting quality are of special interest. B-hordeins are sulfur rich prolamins, which account for 70-80% of the total hordein fraction in barley. In this work, rapid amplification of cDNA ends by PCR (RACE-PCR) has been used for the cloning of the full-length open reading frame (ORF) of six sequences of B3-hordeins from two lines of H. chilense. Two consensus sequences of 813 and 822 bp for the H1 and H7 lines, respectively, were determined by alignment of all the sequences generated. Between both lines, differences involving single base changes, which could correspond to single nucleotide polymorphisms (SNP), insertions and deletions were observed. Of these differences, only six out of the 13 within the ORF caused a change of amino acid. Two insertions/deletions of 9 and 12 bp were also observed between both lines. The derived amino acid sequences showed a similar structure to the B-hordeins from cultivated barley and other prolamins. The repetitive region is based on the repetition of the motif PQQPFPQQ. The copy number of the B3-hordeins was estimated as a minimum of nine and five copies for the H1 and H7 lines, respectively. The expression profile of the B-hordeins through the developing endosperm is also described in this work. This study of the storage proteins of H. chilense is a useful contribution to the knowledge of the genetic diversity available in wild relatives of cultivated barley. In addition, the origin of the different prolamins can be better understood with an in-depth knowledge of its wild equivalent.

Amino Acid Motifs↗

PubMed2006

Wheat Dof transcription factor WPBF interacts with TaQM and activates transcription of an alpha-gliadin gene during wheat seed development.

Wheat prolamin-box binding factor (WPBF), a DOF transcription factor previously was isolated from wheat endosperm and suggested to function as an activator of prolamin gene expression during seed development. In this study, we showed that WPBF is expressed in all wheat tissues analyzed, and a protein, TaQM, was identified from a wheat root cDNA library, to interact with the Dof domain of WPBF. The specific interaction between WPBF and TaQM was confirmed by pull-down assay and bimolecular fluorescence complementation (BiFC) experiment. The expression patterns of TaQM gene are similar with that of WPBF. The GST-WPBF expressed in bacteria binds the Prolamin box (PB) 5'-TGTAAAG-3', derived from the promoter region of a native alpha-gliadin gene encoding a storage protein. Transient expression experiments in co-transfected BY-2 protoplast cells demonstrated that WPBF trans-activated transcription from native alpha-gliadin promoter through binding to the intact PB. When WPBF and TaQM are co-transfected together the transcription activity of alpha-gliadin gene was six-fold higher than when WPBF was transfected alone. Furthermore, the promoter activities of WPBF gene were observed in the seeds and the vascular system of transgenic Arabidopsis, which was identical to the expression profiles of WPBF in wheat. Hence, we proposed that WPBF functions not only during wheat seed development but also during other growth and development processes.

Electrophoretic Mobility Shift Assay↗

PubMed1989

The production and characterisation of monoclonal antibodies to wheat gliadin peptides.

BALB/c mice maintained on a gluten-free diet were immunised with Frazer's Fraction III (FFIII, a peptic tryptic digest of wheat gluten that exacerbates coeliac disease) in order to overcome oral tolerance. A control group was maintained on normal diet. Serum antibody titres to FFIII were higher in the mice on a gluten-free diet (P less than 0.05). Three monoclonal antibodies to FFIII were produced from splenocytes obtained from mice maintained on a gluten-free diet. The antibodies were characterised by ELISA, immunodot assay and immunoblotting with prolamins from cereals toxic to coeliac patients (wheat, rye, barley, oats) and the non-toxic prolamins from maize and rice. The binding characteristics of the three antibodies to the cereal prolamins were different, implying that the antibodies recognise different cereal protein epitopes. Immunoblotting revealed FFIII to be comprised of antigenically dissimilar peptides.

Antibodies, Monoclonal↗

PubMed2005

Identification of dehydro-ferulic acid-tyrosine in rye and wheat: evidence for a covalent cross-link between arabinoxylans and proteins.

To monitor chemical reactions between ferulate and proteins during breadmaking, 8-(14)C-(E)-ferulic acid-(d-galactopyranose-6'-yl)ester was synthesized as a radiotracer and added to wheat and rye flour prior to breadmaking. Breads were lyophilized, extracted by means of a modified Osborne fractionation, and the radioactivity of the fractions was determined by scintillation analysis. The major portion of the radioactivity remained in the water-soluble fraction. However, a significant enrichment of the tracer was also detected in the prolamin and glutelin fractions in comparison to the control experiment. Separation of the prolamin fraction by RP-HPLC and scintillation measurement of the fractions gave evidence for a chemical modification of the tracer. To determine the structure of the reaction product, the prolamin fractions were completely hydrolyzed to free amino acids by means of an enzyme cocktail, and the digests were studied by LC-MS. In one fraction, a newly formed compound was detected. Comparison of its chromatographic and mass spectrometric behavior with a synthetic reference compound gave evidence that the newly identified compound was a dehydroferulic acid-tyrosine cross-link. It is likely that this cross-link represented a covalent linkage between arabinoxylans and cereal proteins. The newly identified cross-link was also identified in wheat and rye flour doughs, which had been prepared without addition of the ferulate tracer. The relative concentration of the dehydroferulic acid-tyrosine cross-link increased during wheat dough preparation.

Bread↗