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PubMed1997

Molecular cloning, expression and subcellular localization of a BiP homolog from rice endosperm tissue.

The ER luminal binding protein, BiP, has been linked to prolamine protein body formation in rice. To obtain further information on the possible role of this chaperone in protein body formation we have cloned and sequenced a BiP cDNA homolog from rice endosperm. The rice sequence is very similar to the maize BiP exhibiting 92% nucleotide identity and 96% deduced amino acid sequence identity in the coding region. Substantial amino acid sequence homology exists between rice BiP and BiP homologs from several other plant and animal species including long stretches of conservation through the amino-terminal ATPase domain. Considerable variation, however, is observed within the putative carboxy-terminal peptide-binding domain between the plant and nonplant BiP sequences. A single hand of approximately 2.4 kb was visible when RNA gel blots of total RNA purified from seed tissue were probed with radiolabeled rice BiP cDNA. This band increased in intensity during seed development up to 10 days after flowering, and then decreased gradually until seed maturity. Protein gel blots indicated that BiP polypeptide accumulation parallels that of the prolamine polypeptides throughout seed development. Immunocytochemical analysis demonstrated that BiP is localized in a non-stochastic fashion in the endoplasmic reticulum membrane complex of developing endosperm cells. It is abundant on the periphery of the protein inclusion body but not in the central portion of the protein body or in the cisternal ER membranes connecting the protein bodies. These data support a model which proposes that BiP associates with the newly synthesized prolamine polypeptide to facilitate its folding and assembly into a protein inclusion body, and is then recycled.

Amino Acid Sequence↗

PubMed1986

Structural Relationship among the Rice Glutelin Polypeptides.

When the glutelin protein fraction of rice (Oryza sativa L.) seeds was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, three size classes of proteins, 51 kilodaltons (kD), 34 to 37 kD, and 21 to 22 kD, as well as a contaminating prolamine polypeptide of 14 kD were detected. Antibodies were raised against these proteins and employed in studies to determine whether a precursor-product relationship existed among the glutelin components. Antibodies of the 34 to 37 kD and 21 to 22 kD polypeptides strongly reacted with the 51 kD protein, and conversely, anti-51 kD protein cross reacted with both of the putative subunits. Immunoprecipitation of in vitro translated products resulted in the synthesis of only the precursor form, indicating that the alpha and beta subunits are proteolytic products of the 51 kD precursor protein. The poly(A)(+) RNA directed in vitro translated product was about 2000 daltons larger than both the authentic glutelin precursor and the in vitro translated product from polysome run-off synthesis. Western blot analysis of the 34 to 37 kD and 21 to 22 kD polypeptides partially digested with Staphylococcus aureus V8 protease revealed distinct patterns indicating that these proteins are structurally unrelated. As observed for the glutelins, the rice prolamines are also synthesized as a precursor of 16 kD, 2000 daltons larger than the mature polypeptide. Addition of dog pancreatic microsomal membranes to a wheat germ protein translation system resulted in the processing of the prolamine preprotein but not the preproglutelin to the mature form.

Journal Article↗

PubMed2004

Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice.

Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa prolamin gene was the only one tested that required both the 5' and 3' flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.

Journal Article↗

PubMed2006

Cloning and structural analysis of an Indian little millet (Panicum sumatrense) zein-like storage protein: implications for molecular assembly.

Zeins are prolamin storage proteins that accumulate in kernel endosperm of several cereals. For cloning of genes coding for zein-like proteins that accumulate in enhanced quantities in the filling stages of little millet (Panicum sumatrense Roth.) developing grains, RT-PCR was performed using specific primers. A 750-bp cDNA was directly sequenced and in silico analysis showed high identity degree to alpha-prolamins. This family is composed of zeins from Zea mays, coixins from Coix lachryma-jobi, and alpha-kafirins from Sorghum bicolor. The putative conserved domain of zein-like proteins was identified by primary structure comparisons. Furthermore, threading analyses indicated that the millet zein-like protein forms an anti-parallel alpha-helical hairpin with two opposite surfaces: one hydrophobic and the other hydrophilic that probably could be involved in protein storage assembly. Knowledge about zein-like alpha-prolamins in little millet will lead to cloning and transfer of this gene to other major food crops, such as cereals and legumes, with inferior nutritional quality for monogastric animals.

Amino Acid Sequence↗

PubMed2004

Colocation between a gene encoding the bZip factor SPA and an eQTL for a high-molecular-weight glutenin subunit in wheat (Triticum aestivum).

The quality of wheat grain is largely determined by the quantity and composition of storage proteins (prolamins) and depends on mechanisms underlying the regulation of expression of prolamin genes. The endosperm-specific wheat basic region leucine zipper (bZIP) factor storage protein activator (SPA) is a positive regulator that binds to the promoter of a prolamin gene. The aim of this study was to map SPA (the gene encoding bZIP factor SPA) and genomic regions associated with quantitative variations of storage protein fractions using F7 recombinant inbred lines (RILs) derived from a cross between Triticum aestivum "Renan" and T. aestivum "Récital". SPA was mapped through RFLP using a cDNA probe and a specific single nucleotide polymorphism (SNP) marker. Storage protein fractions in the parents and RILs were quantified using capillary electrophoresis. Quantitative trait loci (QTLs) for protein were detected and mapped on six chromosome regions. One QTL, located on the long arm of chromosome 1B, explained 70% of the variation in quantity of the x subunit of Glu-B1. Genetic mapping suggested that SPA is located on chromosome arm 1L and is also present in the confidence interval of the corresponding QTL for Glu-B1x on 1BL, suggesting that SPA might be a candidate gene for this QTL.

Base Sequence↗

PubMed1983

Effects on the exocrine and endocrine pancreas of duct occlusion with two different tissue glues in the rat.

Exocrine and endocrine pancreatic function was studied in the rat after occlusion of the pancreatic ducts with acrylate or prolamine, respectively. After 5 months the effect of these procedures was a clear-cut exocrine insufficiency as evidenced by reduced enzyme activities in pancreatic tissue and in intestinal content, while the endocrine function was not impaired. The insulin secretory response to an intravenous glucose load was rather increased in duct-occluded rats, and the glucose tolerance was improved. The liver glycogen levels in duct occluded rats were decreased. As judged from the present investigation there was no striking difference between the effects of acrylate and prolamine on either exocrine or endocrine pancreas. The results suggest that long-term duct occlusion in the rat by acrylate or prolamine is harmless to the endocrine pancreas. The improvement of the glucose-induced insulin response and the glucose tolerance is probably due to an enteropancreatic feedback mechanism elicited by low levels of intestinal trypsin.

Animals↗

PubMed1994

Demonstration of the presence of coeliac-activating gliadin-like epitopes in malted barley.

A peptide B3144, derived after peptic tryptic digestion of alpha-gliadin and corresponding to residues 3-56 from the coeliac-activating domain I, was previously used to produce monoclonal antibodies. A dot immunobinding assay was developed using these antibodies to detect gluten in wheat, rye, barley and oats. The limit of sensitivity of the assay was 1 microgram/ml for unfractionated wheat gliadin and rye prolamins, and 5 micrograms/ml for barley and oat prolamins. Extracts of flours from coeliac non-toxic rice, maize, millet and sorghum gave negative results. Malt, which represents a partial hydrolysate of barley prolamins, was shown to contain the equivalent of 100-200 mg of barley prolamins/100 g of malt. The assay demonstrates the presence of intact epitopes from the coeliac-activating domain I of alpha-gliadins in malted barley, suggesting toxicity.

Celiac Disease↗

PubMed1989

Pancreatic duct occlusion in the management of acute necrotizing pancreatitis in a canine model.

Based on results reported by others in using prolamine (Ethibloc; Ethicon Gmb H) to treat patients with chronic pancreatitis, without any observed pancreatic complications, we decided to use pancreatic duct occlusion experimentally in dogs in which we induced acute pancreatitis by the injection of calcium chloride into the pancreatic duct. This injection proved to be 100% lethal in the 5 animals constituting the control group, none of which were treated after the injection. Acute pancreatitis was then induced in 57 animals, also by injection of calcium chloride. These dogs were then treated with one of three substances that share the same physical properties: prolamine; Tissucol (Immuno AG, Vienna), a biologic tissue adhesive; silicone (Xantopren, Bayer Dental D-5090 Leverkusen). Thirty-seven dogs were treated with prolamine, 10 dogs with Tissucol, and 10 with silicone. The mortality rate in the 57 treated animals was 12.2%, compared to the 100% rate in the untreated control group. The mechanical action achieved by blocking the pancreatic duct shows how the evolution of acute pancreatitis at different stages could be modified. This specific treatment limits the pathophysiologic process of acute pancreatitis in dogs. These findings provide us with a promising outlook for the treatment of this severe illness.

Acute Disease↗

PubMed1989

Anti-gliadin antibody specificity for gluten-derived peptides toxic to coeliac patients.

The specificities of serum and intestinal antibodies from coeliac and normal individuals towards gluten-derived peptides, known to be toxic in coeliac disease, has been investigated. Though untreated coeliacs had high serum antibody levels towards gliadin and some gluten-derived peptides, antibody specificities to various toxic gluten-derived peptides were similar to normal patients. Further, no significant binding in any patient group was found to the alpha-gliadin-derived peptides B1342 (Wieser, Belitz & Ashkenazi, 1984) or the 12 amino-acid A-gliadin peptide (Kagnoff, 1985). There appears to be no direct relationship between the toxicities and the antigenic reactivity of gluten-derived peptides. Thus, the intestinal damage in coeliac disease is probably not primarily caused by antibody-dependent mechanisms. The specificities of several monoclonal antibodies which bound to wheat prolamins as well as prolamins from other coeliac-toxic cereals have also been investigated with these toxic gluten-derived peptides, in order to identify possible common epitopes. No monoclonal antibody tested bound the B1342 and 12-amino-acid A-gliadin peptide. However the monoclonal antibodies which were specific for the coeliac-toxic cereal prolamins did show the strongest binding to other coeliac-toxic gluten-derived peptides.

Adult↗

PubMed1994

[Electrophoretic analysis of buckwheat flour compared with regular wheat flour].

The composition of the alcohol soluble proteins (prolamins) obtained from buckwheat meal and common wheat flour by two procedures were analysed by electrophoresis at pH 3.1 and, after dissociation, in the presence of sodium dodecyl sulphate at pH 8. The profiles obtained from the prolamin fraction of buckwheat were very different, qualitatively and quantitatively, from those of the prolamin of common wheat. It is probable therefore that the adverse effects associated with the presence of wheat gliadin in diets of patients with celiac disease would be reduced and possibly avoided if wheat flour were replaced by flour from buckwheat.

Celiac Disease↗

PubMed1997

Identification of the major water/salt insoluble wheat proteins involved in cereal hypersensitivity.

BACKGROUND: Several studies have investigated water/salt soluble proteins which comprise 50% of the proteins in wheat. The remaining 50% of wheat proteins, are water/salt insoluble proteins of which there is limited information on their role in cereal hypersensitivity. OBJECTIVES: To investigate the allergenicity of the water/salt insoluble gliadin and glutenin proteins (prolamins). METHODS: RAST, electrophoresis and Western blotting were used to identify water/salt insoluble wheat allergens. Competitive RAST inhibition was conducted to investigate cross-reactivity between prolamins and water/salt soluble wheat proteins. RESULTS: Specific IgE to alpha-gliadin and to total glutenins were detected in all sera. IgE to beta-, gamma-, fast omega-, and slow omega-gliadin were present in lower numbers of sera. Prolamin allergens of 90-11 kDa were identified by immunoblotting. Water/salt soluble proteins crossreacted with alpha-gliadin and total glutenins. CONCLUSIONS: Individuals who are hypersensitive to water/salt soluble wheat proteins produce specific IgE to water/salt insoluble wheat proteins. Western blotting has shown that gliadins, glutenins and proteins with similar molecular weights as the endogenous water/salt soluble wheat enzyme inhibitors are important allergens. Alpha and fast omega- are the most allergenic gliadins. The water/salt insoluble proteins share cross-reacting epitopes with water/salt soluble proteins. These data show that the numbers of proteins involved in the development of cereal hypersensitivity is greater than previously believed and that the development of specific IgE to alpha-gliadin may in part depend on the presence of cross-reacting antibodies to water/salt soluble flour allergens.

Antibodies, Blocking↗

PubMed1989

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.

Amino Acid Sequence↗

PubMed1995

Protein, calcium, and iron content of wild and cultivated species of Echinochloa.

Two species of Echinochloa millets and their direct wild ancestor species were analyzed for proximate composition, and amino acid, calcium, and iron content. Additionally, lactate polyacrylamide gel electrophoresis (PAGE) was performed to separate and resolve prolamin polypeptide present in the wild and domesticated species. The protein, calcium, and iron content of the four species were comparable to or greater than in other major cereals. Calcium was higher in each of the wild species than their domesticated counterpart. Essential amino acid values for the three species analyzed were generally higher than the FAO/WHO standards, except for lysine. Densitometric analysis of lactate PAGE gels revealed that the domesticated species contained prolamin, polypeptides that were either absent or present in smaller amounts in the wild species. The results indicate a wide variation in the content of examined nutrients and suggest that there is opportunity for improvement in the nutritional value of the Echinochloa millets via selective crossbreeding of wild and domesticated species.

Amino Acids↗

PubMed2004

A classification of plant food allergens.

Plant food allergens can be classified into families and superfamilies on the basis of their structural and functional properties. The most widespread groups of plant proteins that contain allergens are the cupin and prolamin superfamilies and the protein families of the plant defense system. The cupin superfamily includes allergenic seed storage proteins of the vicilin and legumin type present in soybeans, peanuts, and tree nuts. The prolamin superfamily includes several important types of allergens of legumes, tree nuts, cereals, fruits, and vegetables, such as the 2S albumin seed storage proteins, the nonspecific lipid transfer proteins, and the cereal alpha-amylase and protease inhibitors. Plant food allergens are also found among the various groups of defense proteins that enable plants to resist biotic and abiotic stress, such as the pathogenesis-related proteins, certain proteases, and protease inhibitors. This review focuses on a classification system of plant food allergens that is emerging from the synopsis of allergology and protein evolution.

Allergens↗

PubMed1993

The nitrogen response of a barley C-hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box.

The 431 bp C-hordein promoter of lambda-1-17 exhibits a specific response to amino acids and NH4NO3 in developing barley (Hordeum vulgare L.) endosperms. With the aid of particle bombardment it is shown that the GCN4 motif ATGA(C/G)TCAT is the dominating cis-acting element in this response. But synergistic interaction with the neighbouring endosperm motif TGTAAAGT within the bifactorial prolamin element and cooperation with upstream sequences including a second prolamin-like element is an absolute requirement for a strong, positive regulation by an optimal nitrogen regime. Low nitrogen levels convert the GCN4 box into a negative motif. In contrast the endosperm box on its own exerted a silencing activity, independent of nitrogen nutrition. Sequence comparisons revealed that GCN4- and endosperm-like motifs are widely distributed among plant promoters. Their putative role in nitrogen regulation is discussed.

Base Sequence↗

PubMed2004

The maize O2 and PBF proteins act additively to promote transcription from storage protein gene promoters in rice endosperm cells.

A transient expression assay system was employed to investigate the possible use of the maize Opaque 2 (O2) and prolamin box binding factor (PBF) proteins as transcriptional activators of rice and wheat storage protein gene promoters. When assayed in developing rice endosperm cells, either O2 or PBF alone could increase transcription from the promoter of the rice glutelin gene, Gt1. However, mutant forms of O2 and PBF that are defective in DNA binding could not. Co-transfection with both transcriptional activators resulted in an additive increase in transactivation of the Gt1 promoter. Co-bombardment of a Gt1::GUS construct with plasmids expressing the DNA binding domains of O2 and PBF in antisense orientation resulted in a decrease of GUS expression below background levels. Similar stimulatory and additive effects of O2 and PBF could be observed on the promoters from other storage protein genes including rice globulin (Glb), prolamins (RP6 and PG5a) and a wheat glutenin (Bx7). However, responsiveness of the promoters from non-storage protein genes like rice actin and CaMV 35S to O2 and PBF was insignificant. Our results indicate that the maize O2 and PBF proteins can act singly or additively as effective stimulators of heterologous storage protein promoters in developing rice endosperm cells. These data support the use of well-characterized transcription factors from maize as an effective means of increasing the expression level of recombinant proteins in developing rice seeds.

Antisense Elements (Genetics)↗

PubMed2004

Unexpected deposition patterns of recombinant proteins in post-endoplasmic reticulum compartments of wheat endosperm.

Protein transport within cereal endosperm cells is complicated by the abundance of endoplasmic reticulum (ER)-derived and vacuolar protein bodies. For wheat storage proteins, two major transport routes run from the ER to the vacuole, one bypassing and one passing through the Golgi. Proteins traveling along each route converge at the vacuole and form aggregates. To determine the impact of this trafficking system on the fate of recombinant proteins expressed in wheat endosperm, we used confocal and electron microscopy to investigate the fate of three recombinant proteins containing different targeting information. KDEL-tagged recombinant human serum albumin, which is retrieved to the ER lumen in leaf cells, was deposited in prolamin aggregates within the vacuole of endosperm cells, most likely following the bulk of endogenous glutenins. Recombinant fungal phytase, a glycoprotein designed for secretion, was delivered to the same compartment, with no trace of the molecule in the apoplast. Glycan analysis revealed that this protein had passed through the Golgi. The localization of human serum albumin and phytase was compared to that of recombinant legumin, which contains structural targeting information directing it to the vacuole. Uniquely, legumin accumulated in the globulin inclusion bodies at the periphery of the prolamin bodies, suggesting a different mode of transport and/or aggregation. Our results demonstrate that recombinant proteins are deposited in an unexpected pattern within wheat endosperm cells, probably because of the unique storage properties of this tissue. Our data also confirm that recombinant proteins are invaluable tools for the analysis of protein trafficking in cereals.

6-Phytase↗

PubMed2005

The critical role of disulfide bond formation in protein sorting in the endosperm of rice.

Many seed storage proteins, including monomeric 2S albumin and polymeric prolamin, contain conserved sequences in three separate regions, termed A, B, and C, which contain the consensus motifs LxxC, CCxQL, and PxxC, respectively. Protein-sorting mechanisms in rice (Oryza sativa) endosperm were studied with a green fluorescent protein (GFP) fused to different segments of rice alpha-globulin, a monomeric, ABC-containing storage protein. The whole ABC region together with GFP was efficiently transported to protein storage vacuoles (type II protein bodies [PB-II]) in the endosperm cells and sequestered in the matrix that surrounds the crystalloids. Peptide Gln-23 to Ser-43 in the A region was sufficient to guide GFP to PB-II. However, GFP fused with the AB or B region accumulated in prolamin protein bodies. Substitution mutations in the CCxQL motif in the B region significantly altered protein localization in the endosperm cells. Furthermore, protein extracts containing these substituted proteins had increased amounts of the endoplasmic reticulum (ER) chaperons BiP (for binding protein), protein disulfide isomerase, and calnexin as a part of protein complexes that were insoluble in a detergent buffer. These results suggest that the ER chaperons and disulfide bonds formed at the dicysteine residues in CCxQL play critical roles in sorting fused proteins in the endosperm cells.

Alpha-Globulins↗