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PubMed2005

Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of gamma-Gliadin.

The gene encoding the repetitive domain located in the N-terminal half of gamma-Gliadin from wheat endosperm has been subcloned into a thioredoxin expression system (pET102/D-Topo). It was over-expressed as fusion protein with thioredoxin in Escherichia coli. Thioredoxin was removed by enterokinase cleavage or by acid cleavage at the respective engineered recognition sites. The soluble N-terminal half of gamma-Gliadin was purified by affinity and reverse-phase chromatography. While, the enterokinase cleavage leaded to only one species detectable by mass spectroscopy, the acid cleavage resulted in a three different length polypeptides, due to the presence of the same number of acid cleavage sites. The secondary structure of the purified protein domain was analysed by circular dichroism, showing an spectral shape common to a Poly(Pro) II conformation. The spectrum is dominated by a large negative peak centred around 201 nm and a broad shoulder centred around 225 nm. Also, the temperature denaturation process was studied. The differences observed in the spectra show two main tendencies, the increment of the shoulder intensity, and the drop of the intensity of the peak around 201. When the sample was cooled down, the change on intensity of the shoulder around 225 was completely reversible and that around the 201 nm peak reached a reversibility of 90%. Such structure and thermal behaviour are characteristic of the repetitive domains of the wheat prolamins.

Chromatography, High Pressure Liquid↗

PubMed2005

Decreased accumulation of glutelin types in rice grains constitutively expressing a sunflower seed albumin gene.

Previous studies have shown differential accumulation of sulfur rich glutelins and sulfur poor prolamins in transgenic rice seeds expressing a sunflower seed albumin gene [Hagan, N.D., Upadhyaya, N., Tabe, L.M., Higgins, T.J., 2003. The redistribution of protein sulfur in transgenic rice expressing a gene for a foreign, sulfur-rich protein. Plant J 34, 1-11]. Here, we show, by two-dimensional electrophoresis, differential accumulation of three classes of glutelin proteins - type I, II and III - and a globulin, not previously resolved, in transgenic seeds grown under low and high sulfur nutrition. Several glutelin polypeptides were resolved and four identified as a type I glutelin, two type II glutelins and a type III glutelin. Although sulfur nutrition did not affect the accumulation of sunflower seed albumin, the levels of all four identified glutelins and the globulin were lower in mature seeds derived from transgenic plants grown under sulfur-optimum or sulfur limited conditions compared to non-transgenic rice seeds. The reduction of all four glutelin polypeptides and the globulin varied from 21% to 68%. The re-allocation of sulfur reserves from endogenous proteins to the sulfur sink in transgenic grain is suggestive of a transcriptional control of sulfur mobilization in plants.

Albumins↗

PubMed2001

Inhibitory serpins from rye grain with glutamine as P1 and P2 residues in the reactive center.

Six of seven serpins detected in grains of rye (Secale cereale) were purified and characterized. The amino acid sequence close to the blocked N-terminus, the reactive center loop sequence and the second order association rate constant (k(a)') for irreversible complex formation with chymotrypsin were determined for each serpin. Three of four serpins containing the unusual reactive center P2-P1' QQ/S and one with P2-P1' PQ/M were equally efficient inhibitors of chymotrypsin (k(a)' approximately 10(5) M(-1) s(-1)). One serpin with P2-P1' PY/M was a faster inhibitor (k(a)' approximately 10(6) M(-1) s(-1)). Similar but differently organized glutamine-rich reactive centers were recently found in grain serpins cloned from wheat [Ostergaard et al. (2000) J. Biol. Chem. 275, 33272] but not from barley. The prolamin storage proteins of cereal grains contain similar sequences in their glutamine-rich repeats. A possible adaption of hypervariable serpin reactive centers late in Triticeae cereal evolution as defence against insects feeding on cereal grains is discussed.

Amino Acid Sequence↗

PubMed2003

Wheat gliadin induces apoptosis of intestinal cells via an autocrine mechanism involving Fas-Fas ligand pathway.

Wheat gliadin and other cereal prolamins have been said to be involved in the pathogenic damage of the small intestine in celiac disease via the apoptosis of epithelial cells. In the present work we investigated the mechanisms underlying the pro-apoptotic activity exerted by gliadin-derived peptides in Caco-2 intestinal cells, a cell line which retains many morphological and enzymatic features typical of normal human enterocytes. We found that digested peptides from wheat gliadins (i) induce apoptosis by the CD95/Fas apoptotic pathway, (ii) induce increased Fas and FasL mRNA levels, (iii) determine increased FasL release in the medium, and (iv) that gliadin digest-induced apoptosis can be blocked by Fas cascade blocking agents, i.e. targeted neutralizing antibodies. This favors the hypothesis that gliadin could activate an autocrine/paracrine Fas-mediated cell death pathway. Finally, we found that (v) a small peptide (1157 Da) from durum wheat, previously proposed for clinical practice, exerted a powerful protective activity against gliadin digest cytotoxicity.

Apoptosis↗

PubMed1998

An innovative sandwich ELISA system based on an antibody cocktail for gluten analysis.

A cocktail sandwich ELISA based on the employ of two monoclonal antibodies (MAbs) as coating antibodies and a third MAb conjugated to horseradish peroxidase has been developed for the analysis of gluten in foods. Given that each MAb displays a wide specificity spectrum for wheat, barley, rye and oats prolamins, their combination for ELISA ensures a high crossreactivity with most of the potentially toxic gliadin, hordein, secalin and avenin protein family. One of the unprecedented features of the cocktail sandwich ELISA is that it permits for the first time analysis of barley hordeins in foods, which is unattainable using conventional or commercial ELISA kits. Besides, gliadins, hordeins and secalins are recognised to the same extent. The system provides a high detection sensitivity for gliadins, hordeins, secalins and avenins (1.5, 0.05, 0.15 and 12 ng/ml, respectively). The working linear range comprises 3-100 ng/ml with a gliadin detection limit of 1.5 ppm. This limit of detection is even better than that demanded in the latest Codex recommendation, 10 ppm. Cocktail ELISA data were contrasted with those of commercial ELISA kits and confirmed by mass spectrometry, a non-immunological technique which provides evidence for the occurrence of false positive results with the commercial kits.

Antibodies, Monoclonal↗

PubMed1996

Nitric oxide inhibition attenuates systemic hypotension produced by protamine.

BACKGROUND: Protamine reversal of heparin anticoagulation often causes systemic hypotension, and in vitro studies suggest that this may be mediated by release of nitric oxide from the endothelium. The present investigations were designed to evaluate the direct myocardial effects of protamine and to determine in vivo whether nitric oxide inhibition can prevent hypotension during protamine infusion. METHODS/RESULTS: Protamine sulfate (50 microg/ml) was added to perfusate of eight isolated rabbit heart preparations; in six other preparations, a similar concentration of prolamine was added to heparinized (5 U/ml) Krebs perfusate. Left ventricular developed pressure, maximum rate of pressure rise, and heart rate declined significantly (p < 0.01) in hearts exposed to protamine only (65.0% +/- 6.6%, 55.5% +/- 6.0%, and 87.6% +/- 2.5% of baseline, respectively), whereas protamine added to heparinized perfusate caused little change in developed pressure, maximum rate of pressure rise, and heart rate (85.3% +/- 5.4%, 84.9% +/- 5.5%, and 98.8% +/- 1.6%). To study systemic effects of protamine, we measured hemodynamic parameters in 12 heparinized dogs (150 U/kg). During protamine infusion (1.5 mg/kg intravenously over 30 seconds), mean blood pressure decreased by 46% +/- 7% from baseline (P < 0.05), cardiac output decreased by 38% +/- 4% (p < 0.05), and systemic vascular resistance decreased bv by 14& +/- 9%. After hemodynamic stabilization, Ng-monomethyl-L-arginine (2 mg/kg), a competitive inhibitor of nitric oxide synthesis, was administered to six dogs, and methylene blue (2 mg/kg), an inhibitor of cyclic guanosine monophosphate synthesis, was administered to the remaining six dogs. After treatment with Ng-monomethyl-L-arginine and methylene blue, the second infusion of protamine sulfate caused no significant change in blood pressure or cardiac output. In an additional six dogs, Ng-monomethyl-L-arginine pretreatment (5 mg/kg) blocked the effects of the first dose of protamine. The effect of Ng-monomethyl-L-arginine could be reversed by the addition of (6 mg/kg) L-arginine but not D-arginine. CONCLUSIONS: Protamine-heparin complex does not cause direct myocardial depression but does lead to severe hypotension in vivo. The finding that hypotension can be blocked by inhibitors of the nitric oxide pathway confirms previous in vitro studies indicating that the effects of protamine are mediated, in part, by the vascular endothelium. Further, these studies suggest a novel approach to prevention of hemodynamic complications caused by heparin reversal after cardiopulmonary bypass.

Animals↗

PubMed1994

Wheat peptide challenge in coeliac disease.

The exact nature of the cereal moiety that exacerbates coeliac disease is unknown. In-vitro studies have implicated both the N-terminal and far C-terminal domains of one of the wheat prolamins, A-gliadin. Peptides within these regions may act as epitopes that trigger immune events leading to enteropathy. We synthesized three peptides corresponding to amino-acids 3-21, 31-49, and 202-220 of A-gliadin. Four patients with coeliac disease were challenged by intraduodenal infusion of 1 g of gliadin or 200 mg of the synthetic peptides. Jejunal biopsies were taken before and at hourly intervals for 6 h after the infusion. Morphometric variables were measured and intraepithelial lymphocytes counted. Significant histological changes occurred in the small intestinal mucosa after challenge with a synthetic peptide corresponding to amino acids 31-49 of A-gliadin. The N-terminal peptide, residues 3-21 of A-gliadin, did not cause histological changes in any of the patients. In one of the four patients, minor histological changes following challenge with the peptide corresponding to residues 202-220 of A-gliadin were seen. Our results suggest that the oligopeptide corresponding to aminoacids 31-49 of A-gliadin is toxic in vivo, but there is no evidence of toxicity of the far N-terminal peptide, residues 3-21. The C-terminal peptide 202-220 may contain an epitope to which patients with coeliac disease display variable sensitivity. Since the oligopeptide corresponding to amino-acids 31-49 of A-gliadin is recognised by HLA DQ2-restricted T cells, the observed effects may be due to immune activation within the intestinal mucosa.

Adult↗

PubMed2002

Temperature-dependent binding of monoclonal antibodies to C hordein.

The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5 degrees C but not at 37 degrees C, as determined by enzyme-linked immunosorbent assay (ELISA). The K(d) of IFRN 0614 for the consensus peptide was found to be 1.2x10(12) mol(-1) at 12 degrees C, but no constant could be calculated at 37 degrees C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and beta-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12 degrees C.

Antibodies, Monoclonal↗

PubMed1998

Structural specificities and significance for coeliac disease of wheat gliadin peptides able to agglutinate or to prevent agglutination of K562(S) cells.

Two peptides corresponding to bread wheat A-gliadin fragments 31-43 and 44-55, well known for their ability to damage the coeliac disease intestinal mucosa both in vitro and in vivo, have been confirmed to be very active in inducing in vitro agglutination of K 562 (S) cells. Removal of six amino acid residues from the carboxy-terminal end of the 31-43 peptide, or of five amino acid residues from the amino terminal end of the 44-55 peptide, resulted in a lower, but still very significant, cell agglutination activity. The peptide consisting of ten amino acid residues with a molecular mass of 1157.5 Da, isolated from durum wheat gliadin, was able to prevent agglutination of K 562 (S) cells induced not only by prolamine peptic-tryptic digests from all the cereals toxic in coeliac disease (i.e. bread wheat, rye, barley and oats), but also by the 31-43 and 44-55 peptides. The ability to protect K 562 (S) cells from agglutination was exhibited to the fullest extent also by all the peptides derived from the 1157.5-Da peptide by five progressive deletions of the terminal carboxylic residue, whereas the sixth consecutive deletion yielded a completely inactive peptide. A similar total loss of activity was observed upon addition of a glycine residue to the amino terminal residue of the 1157.5-Da peptide and all the above-mentioned active peptides derived from it. The remarkable sequence homologies existing between peptides able to induce [Gln-Gln-Gln-Pro and -Pro-Ser-Gln-Gln-] or to prevent [H2N-Gln-Gln-Pro-Gln-Asp-COOH] induction of cell agglutination strongly suggest that all these peptides compete for identical or structurally related binding sites on the cell surface.

Agglutination Tests↗

PubMed2000

Induction of apoptosis in caco-2 cells by wheat gliadin peptides.

Recent experimental evidence suggests that enterocyte apoptosis is greater than hitherto assumed and may be responsible for villous atrophy in coeliac disease. We have previously demonstrated that a small peptide (M.W. 1157.5 Da), identified as the sequence H(2)N-gln-gln-pro-gln-asp-ala-val-gln-pro-phe-COOH from durum wheat gliadin, is able to prevent K 562 (S) cell agglutination induced by the peptic-tryptic digests (PT) of prolamin fractions from the cereals which are not tolerated in coeliac disease (i.e. bread wheat, rye, barley and possibly oats), and toxic A-gliadin peptides in coeliac disease. In the present study we have investigated the effects of the bread wheat gliadin digest (PT) on apoptosis of Caco-2 cells and whether the '1157.5' Da peptide may in any way interfere with them. We evaluated both earlier biochemical and later morphological nuclear apoptotic events in the human colon adenocarcinoma cell line Caco-2. After 48 h exposure to the PT gliadin digest and the '1157.5' Da peptide, apoptosis was detected both for the early-stage apoptotic cells (adherent cells) and the late-stage apoptotic ones (detached cells which were floating in the culture medium). Exposure to the PT gliadin digest resulted in a high percentage of adherent cells that underwent cell death by apoptosis (about 30%), independent of the concentration range used; while the presence in the culture medium of peptide '1157.5' Da determined complete inhibition of cell death. On the other hand, morphological nuclear modifications observed in the floating cells showed a difference in the rate of the apoptosis dependent on the PT concentration, with partial protection in the presence of the peptide. These findings show an action of bread wheat gliadin peptides leading to cell death by apoptosis in the Caco-2 cell line and that the '1157.5' Da peptide is capable of preventing such an effect.

Apoptosis↗

PubMed1986

Bilateral patent nasopalatine ducts. A new method of treatment.

A case of bilateral patent nasopalatine ducts, a rare congenital anomaly, has been described. A review of the literature does not permit dogmatic conclusions about the development and existence of patent nasopalatine ducts in both prenatal and postnatal life. A new, simple method of closing epithelium lined fistulae by instillation of alcoholic prolamin-acid solution is presented.

Adult↗

PubMed1998

Modulation of gene expression by DNA-protein and protein-protein interactions in the promoter region of the zein multigene family.

A common cis-acting element in the promoter region of many genes expressed during endosperm development of cereal seeds, the prolamine-box or P-box, is only 20bp upstream of the alpha-class 22-kDa zein gene-specific cis element, the O2-box, which is recognized by the b-ZIP transcription factor, Opaque-2 (O2). The proximity of these two boxes has prompted a study of how two DNA-binding proteins of a different hierarchy might be involved in the activation and modulation of the 22-kDa zein-encoding genes. This was accomplished by utilizing a highly purified P-box-binding-factor-1 (PBF-1) and a bacterially expressed truncated form of the O2 protein. After adding the recombinant O2 to the purified fraction of PBF-1, binding studies were performed with a series of DNA probes combining the P- and O2-boxes from zein promoters. These studies have revealed an interesting inhibitory effect of PBF-1 over O2 function dependent on their ratio, consistent with its in-vivo properties and the developmental expression profiles of zein genes. We also could show that the P-box is specifically recognized by topoisomerase II and single-strand DNA-binding proteins, indicating a possible additional linkage between P-box and the scaffold-attachment-region (SAR).

Base Sequence↗

PubMed2004

The bZIP region of the plant transcription factor opaque-2 forms stable homodimers in solution and retains its helical structure upon subunit dissociation.

Opaque-2 (O2) is a plant bZIP transcription factor that regulates the expression of alpha and beta prolamines, the main storage proteins in seeds of cereals such as maize and Coix. One of the main processes modulating O2 activity is the heterodimerization with other bZIP transcription factors, but the primary mechanism underlying the partner choice is still unknown. In this paper, we have characterized the bZIP domain of O2 by nuclear magnetic resonance (NMR), circular dichroism (CD), and size-exclusion chromatography. Results obtained from CD measurements suggested that the native O2bZIP has about 40 of its 49 leucine-zipper residues in helical structure, while the DNA-binding domain is completely unstructured. Diffusion-ordered NMR spectroscopy and size-exclusion chromatography showed that O2 forms homodimers in solution. Thermal denaturation experiments indicate that O2 reversibly undergoes dissociation and unfolding in a process that is fully dependent on the protein concentration. Subunit dissociation of O2bZIP dimers, upon dilution of the protein, led to partially folded monomers that retained approximately 80% of the native CD ellipticity at 222 nm. We believe that the existence of partially folded monomers could decrease the entropic penalty for helix formation involved in the DNA binding and in the subunit association of O2bZIP. Stabilization of partially folded monomers may also play a significant role in the dimerization of O2 with other bZIP transcription factors and, consequently, can be important for the regulation of the biological functions of O2 in plants.

Amino Acid Sequence↗

PubMedDate not supplied

Changes in protein secondary structure during gluten deformation studied by dynamic fourier transform infrared spectroscopy.

Fourier transform infrared (FT-IR) spectroscopy was used to monitor changes in the secondary structure of wheat prolamins, the main components of gluten, during mechanical deformation in a series of cycles of extension and relaxation. A sample derived from protein bodies isolated from developing grain showed a buildup of persistent beta-sheet structure. In gluten, the ratio of beta-sheet to random and beta-turn structures changed on extension. After the applied force was released, the sample recovered some of its original shape and structure, but the material became stiffer in consecutive extension cycles. The relationship between gluten structure and mechanical properties is discussed in terms of a model in which conversion of beta-turn to beta-sheet structure is a response to extension and a means by which elastic energy is stored in the system.

Glutens↗

PubMedDate not supplied

Zein adsorption to hydrophilic and hydrophobic surfaces investigated by surface plasmon resonance.

Zein, the prolamine of corn, has been investigated for its potential as an industrial biopolymer. In previous research, zein was plasticized with oleic acid and formed into sheets/films. Physical properties of films were affected by film structure and controlled in turn by zein-oleic acid interactions. The nature of such interactions is not well understood. Thus, protein-fatty acid interactions were investigated in this work by the use of surface plasmon resonance (SPR). Zein adsorption from 75% aqueous 2-propanol solutions, 0.05% to 0.5% w/v, onto hydrophilic and hydrophobic self-assembled monolayers (SAMs) formed by 11-mercaptoundecanoic acid and 1-octanethiol, respectively, was monitored by high time resolution SPR. Initial adsorption rate and ultimate surface coverage increased with bulk protein concentration for both surfaces. The initial slope of plotted adsorption isotherms was higher on 11-mercaptoundecanoic acid than on 1-octanethiol, indicating higher zein affinity for hydrophilic SAMs. Also, maximum adsorption values were higher for zein on hydrophilic than on hydrophobic SAMs. Flushing off loosely bound zein in the SPR cell allowed estimation of apparent monolayer values. Differences in monolayer values for hydrophobic and hydrophilic surfaces were explained in terms of zein adsorption footprint.

Adsorption↗

PubMed2001

Study of metallopeptidase isozymes from malted barley (Hordeum vulgare cv. Morex).

It has been reported that germinated barley contains peptidases that are sensitive to metal-chelating agents; however, none of these enzymes have been isolated, nor have their roles in germinated barley been investigated. Anion-exchange chromatography and chromatofocusing have been used to isolate a group of peptidases from barley (Hordeum vulgare cv. Morex) green malt that are sensitive to metal-chelating agents. Their activities were studied using one- and two-dimensional polyacrylamide gel electrophoresis. When analyzed on two-dimensional PAGE gels that contained gelatin as substrate, the enzymes separated into three major and approximately six minor activity spots with acidic pI values. The enzymes were optimally active against the gelatin substrate at pH 8.0 and were completely inhibited by 1,10-phenanthroline and EDTA, indicating that they belonged to the metallopeptidase class (EC 3.4.24.x). After the enzymes were inhibited with EDTA, the activities were recovered in the presence of low concentrations of metal ions. The hydrolysis of gelatin substrate was also impaired by the presence of reducing agents. The metallopeptidases readily digested, in vitro, the barley prolamine D hordein, indicating that they may be involved in degrading storage proteins during barley germination.

Anions↗

PubMed2003

Analysis of zein by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) was used to analyze the protein composition of corn prolamine (zein). Mass spectra were obtained from commercial zein and zein extracted with aqueous 2-propanol and aqueous ethanol from consumer corn meal. For the commercial zein, three major zein fractions with m/z 26.8k, 24.1k, and 23.4k were clearly seen with two minor fractions (m/z 14.5k and 20.4k) also present. As compared with the results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these three fractions were identified as alpha-zeins (24.1k and 23.4k combined as Z19; 26.8k as Z22). When extracted with 55% aqueous 2-propanol, three alpha-zein fractions with m/z 26.8k, 24.1k, and 23.4k were predominant. When extracted with ethanol, extraction temperature had an effect on the final products. When extracted with 75% aqueous ethanol at room temperature, alpha-zein and some 17-18k species were observed, whereas at 60 degrees C, a small amount of delta-zein was also present. Comparison of the MALDI/MS results with SDS-PAGE and gene sequence analysis shows that the MALDI/MS method is superior to SDS-PAGE in having higher resolution and mass accuracy.

2-Propanol↗

PubMed2004

Improving nutritional quality of maize proteins by expressing sense and antisense zein genes.

The predominant proteins in maize grain are a family of alcohol-soluble prolamin storage proteins called zeins. They account for >50% of total seed proteins but are deficient in several essential amino acids. As a result, the corn grain is considered to be nutritionally poor for monogastric animals with respect to key essential amino acids, most notably lysine, tryptophan, and methionine. Thus, corn mutants with reduced levels of zeins, such as opaque-2 (o2), have been demonstrated to possess grain with improved nutritional quality characteristics. The o2 mutant has a superior amino acid composition and has been used through conventional breeding to develop Quality Protein Maize (QPM) for human and animal consumption in developing countries. With the understanding of molecular genetics of zeins and progress in biotechnology, an alternative approach to zein reduction is explored here. Through the targeted reduction of the 19-kDa alpha-zeins, increased levels of lysine, tryptophan, and methionine have been engineered in grain of transgenic hybrids. Currently, the agronomic properties and nutritional values of these transgenic lines are being evaluated.

DNA, Antisense↗