Maize Prolamin Research Articles

Zeolin is a recombinant storage protein with different solubility and stability properties according to its localization in the endoplasmic reticulum or in the chloroplast

Several strategies have been exploited to maximize heterologous protein accumulation in the plant cell. Recently, it has been shown that a portion of a maize prolamin storage protein, gamma-zein, can be used for the high accumulation of a recombinant protein in novel endoplasmic reticulum (ER)-derived protein bodies of vegetative tissues. In this study, we investigate whether this protein can be expressed in the chloroplast. Our long-term purpose is to use zeolin to produce value-added proteins by fusing these polypeptides with its gamma-zein portion and targeting the recombinant proteins to the ER or to the chloroplast. We show here that zeolin accumulates in the chloroplast to lower levels than in the ER and its stability is compromised by chloroplast proteolytic activity. Co-localization of zeolin and the ER chaperone BiP in the chloroplast does not have a beneficial effect on zeolin accumulation. In this organelle, zeolin is not stored in protein bodies, nor do zeolin polypeptides seem to be linked by inter-chain disulfide bonds, which are usually formed by the six cysteine of the gamma-zein portion, indicating abnormal folding of the recombinant protein. Therefore, it is concluded that to accumulate zeolin in the chloroplast it is necessary to facilitate inter-chain disulfide bond formation.

New strategy for the determination of gliadins in maize- or rice-based foods matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: fractionation of gliadins from maize or rice prolamins by acidic treatment.

A procedure for determining small quantities of gliadins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) in gluten-free foods containing relatively large amounts of prolamin proteins from maize or rice is described. We report for the first time that gliadins, the ethanol-soluble wheat prolamin fraction, can be quantitatively solubilized in 1.0 M acetic acid, while the corresponding ethanol-soluble maize or rice prolamin fraction remains insoluble in acetic acid. We describe a methodology for the detection of gliadins in maize and rice foods based on a two-step procedure of extraction (60% aqueous ethanol followed by 1 M acetic acid). Subsequent MALDI-TOFMS analysis of the resulting acidic extract from these gluten-free foods clearly confirms the presence of a typical mass pattern corresponding to gliadin components, ranging from 30 to 45 kDa. Depending on the percentages of maize or rice flours employed in the elaboration of these foods, the combined procedure enables levels of gliadins from 100 to 400 ppm to be detected. The efficiency of this combined procedure corroborates enzyme-linked immunosorbent assay data for a large number of maize/rice gluten-free foods by means of direct visualization of the characteristic gliadin mass pattern in maize or rice foods.

Improvement of Sorghum‐Wheat Composite Dough Rheological Properties and Breadmaking Quality Through Zein Addition

ABSTRACTAddition of sorghum flour to wheat flour produces marked negative effects on rheological properties of dough and loaf volume. Although there are notable differences in the chemical composition of sorghum proteins (kafirins) compared with wheat gluten that might imply poor functionality in breadmaking systems, a larger constraint may be the unavailability of kafirins due to encapsulation in protein bodies. In this study, zein, the analogous maize prolamin to kafirin, was used to determine the potential effects of protein‐body‐free prolamins on dough rheology and baking quality of wheat‐sorghum composite flour. Mixograms run at 35°C (above the glass transition temperature of zein) were significantly (P < 0.01) improved with addition of zein. Mixogram peak heights increased while mixing time decreased uniformly with addition of zein. Dough extensibility studies showed an increase in maximum tensile stress, while baking studies showed an increase in loaf volume with increasing amounts of added zein. These data are supported by a previous study showing that, in a model system, zein mixed with starch can form viscoelastic networks, and suggest that kafirin, if made available, could contribute to dough formation.

N-terminal amino acid sequences of prolamins encoded by the alleles at the Pro1 and Pro2 loci in foxtail millet, Setaria italica (L.) P. Beauv.

N-terminal amino acid sequences of six prolamins encoded by seven alleles at two loci, Pro1 and Pro2, of foxtail millet (Setaria italica (L.) P. Beauv.) were analyzed and compared with other prolamins of subfamily Panicoideae. Based on the N-terminal amino acid sequences, band 3 (the prolamin purified from band 3) which is controlled by an allele at the Pro1 locus and bands 1, 2, 4, 5 and 6 which are controlled by alleles at the Pro2 locus could be classified into three groups. Band 3 was found to be homologous to the prolamin of pearl millet (Pennisetum americanum) and is designated as the "pennisetin-like prolamin". Bands 2 and 4, and bands 1, 5 and 6 were subdivided into "x-type prolamin" and "y-type prolamin". Both of the x-type and y-type prolamins showed homology with prolamin of Echinochloa crus-galli and alpha-zein-like prolamins of maize, sorghum and Job's tears. Therefore, these prolamins were designated as "alpha-zein-like prolamin". These results suggest that alleles at the Pro1 locus and those at the Pro2 locus have not arisen from an identical ancestral gene, and that the Pro2 locus comprise two tightly linked genes, which encode similar prolamins. Hypotheses on the diversification of alleles at the Pro2 locus are discussed based on the N-terminal amino acid sequences of the respective bands, combinations of bands controlled by the alleles, and frequencies of the alleles.

Characterization of the maize prolamin box-binding factor-1 (PBF-1) and its role in the developmental regulation of the zein multigene family.

A maize prolamin box (P-box)-binding factor (PBF-1) has been purified and characterized from immature endosperm tissue. PBF-1 has a molecular weight of 38kDa. It is detected only in endosperm, but not in root or leaf tissues, consistent with its tissue-specific function. Site-directed mutagenesis experiments reveal that both the P-box and its flanking sequences are important for PBF-1 DNA binding. Developmental studies show that PBF-1 accumulates in the endosperm from 8 to at least 30days after pollination (DAP). From 16 to 24DAP, however, multiple shifted bands of protein(s)-DNA complexes can be observed, which correlate with an increase in zein gene expression. PBF-1 can also bind to the P-box from '22-kDa' and '19-kDa' zein promoters, but at a lower affinity than to the '27-kDa' zein promoter. The effects of protein dephosphorylation and zinc ion chelators on PBF-1 DNA binding activity are also shown. A model is proposed where PBF-1 serves as a 'recruiter' of class-specific transcription factors like Opaque2 (O2).

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2.

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5'-TGTAAAG-3') found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

Coeliac disease: characterisation of monoclonal antibodies raised against a synthetic peptide corresponding to amino acid residues 206-217 of A-gliadin.

A dodecapeptide of A-gliadin, which shares amino acid homologies with the E1b protein of adenovirus 12, was used to produce murine monoclonal antibodies. Five monoclonal antibodies were produced and were screened by enzyme linked immunosorbant assay, immunodot assay, and immunoblotting. The antibodies were tested against whole wheat gliadin and its alpha, beta, gamma, and omega subfractions, and the prolamins of rye, barley, oats, maize, millet, rice, and sorghum. Four of the five antibodies cross reacted with one or more of the coeliac non-toxic cereals--maize, millet, sorghum, and rice. The monoclonal antibody that did not cross react with these non-toxic cereals, did not recognize Frazer's fraction III, a peptic-tryptic digest of wheat gluten which is known to be toxic. The results suggest that the A-gliadin dodecapeptide shares a region of homology with cereals that do not exacerbate coeliac disease. This study does not support the hypothesis that prior infection with adenovirus 12 is a precipitating factor in coeliac disease.

Gliadin induced changes in the expression of MHC-class II antigens by human small intestinal epithelium. Organ culture studies with coeliac disease mucosa.

Jejunal biopsies from 16 treated coeliac disease patients and from nine controls were cultured with and without a peptic-tryptic digest of gliadin. Cultures with a peptic-tryptic digest of maize prolamins were also undertaken. Frozen sections of baseline and cultured mucosa were stained by immunofluorescence with an anti-HLA-DR monoclonal antibody. Before culture the villous epithelium from both controls and treated coeliac disease expressed DR molecules while the crypt epithelium did not. When biopsies from treated coeliac disease were cultured with gliadin the expression of DR was enhanced in the crypt epithelium in eight of 14 cultures and in 11 of 14 was reduced or absent on the villous epithelium. No change was observed in control cultures. We conclude that gliadin is capable of inducing HLA-DR on the crypt epithelium of in vitro cultured coeliac disease mucosa, providing indirect evidence that gliadin may activate cell mediated immune mechanisms within the small bowel mucosa. This model could prove useful in identifying the immunogenic sequence(s) of gliadins and related prolamins.

IMMUNOCYTOCHEMICAL LOCALIZATION OF δ‐ZEIN IN THE PROTEIN BODIES OF MAIZE ENDOSPERM CELLS

The δ‐zein, a minor component of the maize prolamin, shows extensive immunological cross‐reactivity with α‐ and β‐zeins. The adsorption of an anti‐δ‐zein serum sequentially with cross‐reacting antigens revealed that only about 18% of the reactivity of the antiserum was directed to epitopes unique to δ‐zein. The localization of the various zein classes within the protein bodies of endosperm cells is important to understanding the synthesis, sequestering, and utilization of these storage proteins. Sections of 28 days after pollination (DAP) isolated protein bodies and 18 and 40 DAP whole endosperms were reacted sequentially with whole anti‐δ‐zein serum and gold‐conjugated protein A. The results showed intense gold labeling in the core (inside the peripheral zone) and weak labeling in the periphery of the sections. This localization was not definitive in view of the above‐mentioned cross‐reactivities. To obtain an unequivocal localization, the whole antiserum was adsorbed with α‐, β‐, and γ‐zeins and rendered monospecific for δ‐zein. Immunostaining of protein body sections with monospecific antiserum showed that gold label was exclusively in the core region of the protein body and appeared to be in discrete lines and zones especially in 18 DAP protein bodies. The data from localizations using the monospecific antiserum indicated that δ‐zein occurs throughout the core region of the protein body, probably interspersed with α‐ and β‐zeins. The location of δ‐zein is consistent with that predicted from its order of degradation during seed germination.

Reaction of anti-α-zein sera with synthetic peptides derived from selected regions of α-zein polypeptides

Twenty-three 17-mer oligopeptides were synthesized that correspond to the predicted antigenic sites and to other selected regions of the α-zein fraction of maize (Zea mays L.) prolamin. The peptides were reacted, after conjugation to BSA, with seven different antisera to intact α-zein polypeptides of the Mr 23·8k, 25k and 26·7k subclasses using an enzyme-linked immunosorbent assay (ELISA) to identify continuous antigenic sites and the regions of zein primary structure that partially or fully reside on the surface of the folded protein. Two of the seven antisera were raised against the native proteins while five were against the denatured proteins. Sixteen of the 23 peptides yielded varying intensities of positive reactions with one or more of the seven different antisera. The results suggested that the reactive peptides contain continuous epitopes that reside partly or entirely on the surface of the folded protein. They also suggest that most of the immune response to α-zeins is directed to shared epitopes. Seven unreactive peptides were judged not to be antigenic, either because the regions of the protein corresponding to them are not exposed and therefore not immunogenic, or because the peptides have a conformation different from that in the intact protein. Only a few peptides reacted with the antisera to native α-zein polypeptides; also, only a few of the peptides corresponding to variants of the 20-mer α-zein repeat unit reacted with antisera to the native protein.

An immunodominant site of γ-zein1 is in the region of tandem hexapeptide repeats

The immunochemical data from studies with polyclonal antisera to gamma-zein1, the 27 kD component of the maize prolamin, indicated that the region containing 8 tandem repeats of the sequence PPPVHL is an immunodominant site. In one case, the entire antibody repertoire of an antiserum recognized epitope(s) within this region. Three 17-mer oligopeptides corresponding to the predicted antigenic epitopes of gamma-zein1 were synthesized and reacted with three different anti-gamma-zein1 sera in order to map antigenic sites in the intact protein. These antisera yielded positive reactions with a 17-mer peptide (peptide 37), which was not in a hydrophilic maximum but derived from the repeat region. The same antisera gave little or no reaction with other peptides (peptides 38 and 39), both of which were in a hydrophilic maximum. In addition, an antiserum to peptide 37 reacted strongly with both the homologous antigen and the intact gamma-zein1. Peptide 37 also blocked the binding of antisera to gamma-zein1 in competition assays. Subsequently, the shorter 6-mer (peptide 82) and 12-mer (peptide 80) versions of peptide 37 were synthesized, and both reacted with anti-peptide 37 serum and also with each of the three anti-gamma-zein1 sera. In these reactions and in competition assays, the reactivity and the blocking ability increased in proportion to the length of the peptide. Based on these data, it was concluded that the repeat region of gamma-zein1 is the site of one or more continuous immunodominant epitopes. The data also suggest that the repeat region is exposed on the surface of the folded protein and probably occur as a mobile, random coil.

The prolamin storage proteins of cereal seeds: structure and evolution

With the exception of rice, the major cereals falls into two groups. The temperate cereals comprise barley, wheat, rye and oats, and the tropical cereals maize, sorghum and millets. Only the prolamins of wheat, barley and maize have been studied in detail at the molecular and physicochemical levels, and we will therefore focus on these. We will, however, also draw comparisons with the prolamins of other species where appropriate.

Homologies between grain storage proteins of different cereal species. 1. Monoclonal antibody reaction with total protein extracts

The interactions of a library of monoclonal antibodies, prepared to wheat ( Triticum aestivum L.) gluten proteins, with extracts of total seed storage protein from a number of related cereals was studied qualitatively using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and immunoblotting techniques and studied quantitatively using indirect enzyme-immunoassay. Antibodies were of four cross-reaction types: (1) some antibodies, such as many of those binding α- and β-gliadins, bound selectively to wheat grain proteins, (2) others with similar gliadin specificities, bound to prolamins from other Festucoid species (rye, barley and oats), (3) other antibodies specific for certain γ- and ω-gliadins and high-molecular-weight glutenins, bound very well to proteins from wheat, rye and barley grain, while (4) some antibodies showed anomalous cross-reactivities, binding well to wheat and maize prolamins but very weakly to rye or barley grain proteins. These homologies were generally in keeping with structural homologies of proteins from various cereal grains obtained from DNA sequencing and cross-hybridization studies. In some cases, new homologies were identified.

Unusual features of cereal seed protein structure and evolution.

The alcohol-soluble (prolamin) storage proteins of barley, wheat and rye vary in their structures, but all have two features in common: the presence of distinct structural domains differing in amino acid compositions, and of repeats within one of these domains. Detailed comparisons of amino acid sequences show that all appear to have evolved from a single ancestral gene consisting of three short related regions (called A, B and C). Regions related to A, B and C are also present in the minor prolamins of maize and in three other groups of seed proteins: inhibitors of alpha-amylase and/or trypsin from cereals. 25 storage globulins from several dicotyledonous species and a 2S albumin from sunflower. It is suggested that these proteins together constitute a protein superfamily with limited sequence homology.

Enzyme-linked immunosorbent assay for quantitation of cereal proteins toxic in coeliac disease

Coeliac disease is revealed by polypeptides in the prolamin fraction of wheat, barley, rye and oats. Recovery depends on adherence to a strict cereal-free diet. A few methods for quantitation of the wheat prolamin, gliadin, have been described. In order to assess the suitability of food products for inclusion in the coeliac diet an assay should measure the total amount of potentially toxic cereal proteins. An inhibition ELISA was developed, by use of a purified, polyclonal prolamin-antibody, reacting with gliadin and gliadin-like polypeptides. The antibody did not react with maize, millet, rice or soya prolamins. The assay had a detection limit of 1 ng antigen with a very high degree of accuracy. The interassay coefficient of variation including the day-to-day variation, was close to 30%, which is acceptable for the clinical applications of the assay. The flour of buckwheat was analyzed for antigen content. An amount of 39.5 μg gliadin-like polypeptides/g flour was measured, which corresponds to 0.06% of the gliadin content in wheat flour.

A proposed nomenclature for the alcohol-soluble proteins (zeins) of maize ( Zea mays L.)

A nomenclature system for maize (Zea mays L.) prolamins, zeins, is proposed. According to this system, the protein that is rich in proline, glutamine, leucine and/or alanine, and is extracted with aqueous alcohol solutions containing a reducing agent, is considered to be zein. Zein is separated into three distinct classes (α, β and γ) based on differences in solubility, amino acid composition, electrophoretic, chromatographic, and immunological properties, N-terminal amino sequences and complete amino acid sequences deduced from nucleic acid sequences. α-Zein constitutes 75–85% of the total zein and is made up of Mr-21,000–25,000 polypeptides plus a Mr-10,000 minor polypeptide. β-Zein includes Mrr-17,000–18,000 methionine-rich polypeptides and constitutes 10–15% of the total zein. γ-Zein is made up of one size class, Mr-27,000 proline-rich polypeptide, that constitutes 5–10% of the total zein.

In vitro activation of adenylate cyclase of atrophic celiac intestinal mucosa by wheat gliadin-derived peptides.

In order to demonstrate that gliadin peptides may interact with cell membranes of celiac small intestinal mucosa, the capacity of these peptides to activate the cell membrane enzyme adenylate cyclase was tested. The addition of peptides from bread wheat purified A-gliadin and whole gliadin (proteins that are toxic for celiac patients) enhanced the adenylate cyclase activity of crude cell membrane preparations obtained from atrophic small intestinal mucosa of celiac patients. No activation of adenylate cyclase of this tissue was observed with peptides from proteins nontoxic for celiac patients (bread wheat albumin and maize prolamin). Gliadin peptides did not activate adenylate cyclase of morphologically normal small intestinal mucosa from normal subjects or from celiac patients in remission. These results, therefore, suggest that peptides from bread wheat gliadin may interact with cell membrane of atrophic small intestinal mucosa of celiac patients.

Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize

The B-hordeins are the major group of prolamin storage proteins in barley (Hordeum vulgare L.) and they are encoded by a small multigene family that is expressed specifically in the developing endosperm. We report the complete nucleotide sequence of a clone of one B-hordein gene (pBHR184). The cloned gene contains no introns and belongs to the B1 sub-family of B-hordein genes. Comparison of the 5'-flanking sequences of pBHR184 with those of related S-rich prolamin genes from wheat shows that several short sequences within 600 bp upstream of the translation initiation codon are strongly conserved. A sequence that is conserved at around -300 bp in the S-rich prolamins is also conserved at similar locations in genes encoding the two major classes of maize prolamin (the Z19 and Z21 zeins) and appears to be unique to prolamin genes. We discuss the possible role of this '-300 element' in the control of gene expression in the developing cereal endosperm.

Immunochemical studies on barley seed storage proteins

The antigenic relationships between the prolamins of barley, rye and wheat have been studied by examining the specificity of an antibody to 'C' hordein in a quantitative study using a laser nephelometer. The antibody reacts weakly with 'B' hordein and strongly with 75-kdalton and 40-kdalton γ-secalins from rye and γ3 some ω-gliadins from wheat. Absorption experiments and immunodiffusion tests indicate that there are shared antigenic determinants for most of the prolamins. All the species with reacting prolamins belong to the sub-family Festucoideae of the Gramineae. The prolamins of maize, pearl millet and sorghum, species of the sub-family Panicoideae, do not react. The results confirm the known lack of homology between the prolamins of the two sub-families and also indicate the presence of relationships not yet established between 'C' hordein, the 75-kdalton and 40-kdalton γ-secalins and also γ3 gliadin.

LECTIN-LIKE ACTIVITY OF CEREAL PROLAMIN-DERIVED PEPTIDES

It has been suggested that cereal peptides may damage the celiac intestinal mucosa by a cytotoxic action on immature or im mature-like enterocytes (Auricchio et al.Ped. Res.In press).Peptic-tryptic peptides (PTP) of prolamins from bread wheats, rye, barley and oats and of bread wheat A-gliadin (gift of Dr. D. Kasarda) were very active in agglutinating undifferentiated in vitro cultured K 562 S cells(Lozzio et al.Blood 45,321,1975). The minimal concentrations required to agglutinate 30% of total cells (MAC) were: 3.2mg/l of PTP of wheat gliadins, 0.3mg/l of PTP of A-gliadin, 4mg/l of concanavalin A and 20mg/l of wheat germ agglutinin. The MAC of PTP of wheat gliadins was much higher (412mg/l)when tested on erythroid differentiated K 562 S cells. The agglutinating activity was inhibited by N-acetylglucosamine, mannose and mannan and was reversed by mannan.PTP of rice and maize prolamins did not agglutinate undifferentiated K 562 S cells. There is therefore a good correlation between toxicity for celiacs of cereal prolamins and their lectin-like activity on undifferentiated cells.