Deciphering the immunogenic potential of wheat flour: a reference map of the salt-soluble proteome from the U.S. wheat Butte 86.

BACKGROUND: Within the complex wheat flour proteome, the gluten proteins have attracted most of the attention because of their importance in determining the functional properties of wheat flour doughs and their roles in human health conditions such as celiac disease and food allergies. However, certain non-gluten proteins also trigger immunological responses but may be present in flour in low amounts or obscured by the more abundant gluten proteins in two-dimensional gels of total protein preparations. METHODS: Non-gluten proteins were preferentially extracted from the flour with a dilute salt solution and separated by two-dimensional gel electrophoresis. Proteins in 173 gel spots were identified by tandem mass spectrometry after cleavage with trypsin or chymotrypsin. Transgenic wheat lines in which specific groups of gluten proteins were suppressed by RNA interference were used to estimate the amount of carry-over of gluten proteins in the salt-soluble protein fraction. RESULTS: Fifty-seven different types of non-gluten proteins were identified, including 14 types that are known or suspected immunogenic proteins. The predominant proteins in 18 gel spots were gluten proteins. Some of these also contained non-gluten proteins. Analysis of the salt-soluble proteins from a transgenic line in which omega-1,2 gliadins were eliminated by RNA interference indicated that certain omega-1,2 gliadins were present in large amounts in the salt-soluble fraction and obscured relatively small amounts of beta-amylase and protein disulfide isomerase. In comparison, analysis of a transgenic line in which alpha gliadins were absent revealed that glyceraldehyde-3 phosphate dehydrogenase was a moderately abundant protein that co-migrated with several alpha gliadins. CONCLUSIONS: In this study, we constructed a proteomic map of the non-gluten protein fraction of wheat flour from the US wheat Butte 86 that complements a proteomic map of the total flour proteins developed previously for the same cultivar. Knowing the identities of low abundance proteins in the flour as well as proteins that are hidden by some of the major gluten proteins on two-dimensional gels is critical for studies aimed at assessing the immunogenic potential of wheat flour and determining which wheat proteins that should be targeted in future gene editing experiments to reduce the immunogenic potential of wheat flour.

Reducing the Immunogenic Potential of Wheat Flour: Silencing of Alpha Gliadin Genes in a U.S. Wheat Cultivar.

The alpha gliadins are a group of more than 20 proteins with very similar sequences that comprise about 15%-20% of the total flour protein and contribute to the functional properties of wheat flour dough. Some alpha gliadins also contain immunodominant epitopes that trigger celiac disease, a chronic autoimmune disease that affects approximately 1% of the worldwide population. In an attempt to reduce the immunogenic potential of wheat flour from the U.S. spring wheat cultivar Butte 86, RNA interference was used to silence a subset of alpha gliadin genes encoding proteins containing celiac disease epitopes. Two of the resulting transgenic lines were analyzed in detail by quantitative two-dimensional gel electrophoresis combined with tandem mass spectrometry. Although the RNA interference construct was designed to target only some alpha gliadin genes, all alpha gliadins were effectively silenced in the transgenic plants. In addition, some off-target silencing of high molecular weight glutenin subunits was detected in both transgenic lines. Compensatory effects were not observed within other gluten protein classes. Reactivities of IgG and IgA antibodies from a cohort of patients with celiac disease toward proteins from the transgenic lines were reduced significantly relative to the nontransgenic line. Both mixing properties and SDS sedimentation volumes suggested a decrease in dough strength in the transgenic lines when compared to the control. The data suggest that it will be difficult to selectively silence specific genes within families as complex as the wheat alpha gliadins. Nonetheless, it may be possible to reduce the immunogenic potential of the flour and still retain many of the functional properties essential for the utilization of wheat.

Exploiting the reference genome sequence of hexaploid wheat: a proteomic study of flour proteins from the cultivar Chinese Spring.

Although the economic value of wheat flour is determined by the complement of gluten proteins, these proteins have been challenging to study because of the complexity of the major protein groups and the tremendous sequence diversity among wheat cultivars. The completion of a high-quality wheat genome sequence from the reference wheat Chinese Spring recently facilitated the assembly and annotation of a complete set of gluten protein genes from a single cultivar, making it possible to link individual proteins in the flour to specific gene sequences. In a proteomic analysis of total wheat flour protein from Chinese Spring using quantitative two-dimensional gel electrophoresis combined with tandem mass spectrometry, gliadins or low-molecular-weight glutenin subunits were identified as the predominant proteins in 72 protein spots. Individual spots were associated with 40 of 56 Chinese Spring gene sequences, including 16 of 26 alpha gliadins, 10 of 11 gamma gliadins, six of seven omega gliadins, one of two delta gliadins, and nine of ten LMW-GS. Most genes that were not associated with protein spots were either expressed at low levels in endosperm or encoded proteins with high similarity to other proteins. A wide range of protein accumulation levels were observed and discrepancies between transcript levels and protein levels were noted. This work together with similar studies using other commercial cultivars should provide new insight into the molecular basis of wheat flour quality and allergenic potential.

Elimination of Omega-1,2 Gliadins From Bread Wheat (Triticum aestivum) Flour: Effects on Immunogenic Potential and End-Use Quality.

The omega-1,2 gliadins are a group of wheat gluten proteins that contain immunodominant epitopes for celiac disease (CD) and also have been associated with food allergies. To reduce the levels of these proteins in the flour, bread wheat (Triticum aestivum cv. Butte 86) was genetically transformed with an RNA interference plasmid that targeted a 141 bp region at the 5' end of an omega-1,2 gliadin gene. Flour proteins from two transgenic lines were analyzed in detail by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry. In one line, the omega-1,2 gliadins were missing with few other changes in the proteome. In the other line, striking changes in the proteome were observed and nearly all gliadins and low molecular weight glutenin subunits (LMW-GS) were absent. High molecular weight glutenin subunits (HMW-GS) increased in this line and those that showed the largest increases had molecular weights slightly less than those in the non-transgenic, possibly due to post-translational processing. In addition, there were increases in non-gluten proteins such as triticins, purinins, globulins, serpins, and alpha-amylase/protease inhibitors. Reactivity of flour proteins with serum IgG and IgA antibodies from a cohort of CD patients was reduced significantly in both transgenic lines. Both mixing time and tolerance were improved in the line without omega-1,2 gliadins while mixing properties were diminished in the line missing most gluten proteins. The data suggest that biotechnology approaches may be used to create wheat lines with reduced immunogenic potential in the context of gluten sensitivity without compromising end-use quality.

Towards reducing the immunogenic potential of wheat flour: omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA.

BACKGROUND: Omega-5 gliadins are a group of highly repetitive gluten proteins in wheat flour encoded on the 1B chromosome of hexaploid wheat. These proteins are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). The elimination of omega-5 gliadins from wheat flour through biotechnology or breeding approaches could reduce the immunogenic potential and adverse health effects of the flour. RESULTS: A mutant line missing low-molecular weight glutenin subunits encoded at the Glu-B3 locus was selected previously from a doubled haploid population generated from two Korean wheat cultivars. Analysis of flour from the mutant line by 2-dimensional gel electrophoresis coupled with tandem mass spectrometry revealed that the omega-5 gliadins and several gamma gliadins encoded by the closely linked Gli-B1 locus were also missing as a result of a deletion of at least 5.8 Mb of chromosome 1B. Two-dimensional immunoblot analysis of flour proteins using sera from WDEIA patients showed reduced IgE reactivity in the mutant relative to the parental lines due to the absence of the major omega-5 gliadins. However, two minor proteins showed strong reactivity to patient sera in both the parental and the mutant lines and also reacted with a monoclonal antibody against omega-5 gliadin. Analysis of the two minor reactive proteins by mass spectrometry revealed that both proteins correspond to omega-5 gliadin genes encoded on chromosome 1D that were thought previously to be pseudogenes. CONCLUSIONS: While breeding approaches can be used to reduce the levels of the highly immunogenic omega-5 gliadins in wheat flour, these approaches are complicated by the genetic linkage of different classes of gluten protein genes and the finding that omega-5 gliadins may be encoded on more than one chromosome. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.

The flowering time regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via a unique cis-element.

CONSTANS is an evolutionarily-conserved central component of the genetic pathway that controls the onset of flowering in response to daylength. However, the specific biochemical mechanism by which the CONSTANS protein regulates the expression of its target genes remains largely unknown. *By using a combination of cell-based expression analysis and in vitro DNA binding studies, we have demonstrated that CONSTANS possesses transcriptional activation potential and is capable of directly binding to DNA. *CONSTANS was found to bind DNA via a unique sequence element containing a consensus TGTG(N2-3)ATG motif. This element is present in tandem within the FLOWERING LOCUS T promoter and is sufficient for CO binding and activity. The conserved CCT (CONSTANS, CONSTANS-like and TOC1) domain of CONSTANS was shown to be required for its recruitment to the DNA motif and other CCT-containing proteins were also found to have the ability to regulate gene expression via this element. *The CCAAT box, which has been previously hypothesized as a recruitment site for complexes containing the CONSTANS protein, potentiated CONSTANS-mediated activation but was not essential for CONSTANS recruitment to a target promoter or for its activity as a transcriptional factor.

Cell wall reactive proteins in the coat and wall of maize pollen: potential role in pollen tube growth on the stigma and through the style.

The surface of a pollen grain consists of an outermost coat and an underlying wall. In maize (Zea mays L.), the pollen coat contains two major proteins derived from the adjacent tapetum cells in the anthers. One of the proteins is a 35-kDa endoxylanase (Wu, S. S. H., Suen, D. F., Chang, H. C., and Huang, A. H. C. (2002) J. Biol. Chem. 277, 49055-49064). The other protein of 70 kDa was purified to homogeneity and shown to be a beta-glucanase. Its gene sequence and the developmental pattern of its mRNA differ from those of the known beta-glucanases that hydrolyze the callose wall of the microspore tetrad. Mature pollen placed in a liquid medium released about nine major proteins. These proteins were partially sequenced and identified via GenBank trade mark data bases, and some had not been previously reported to be in pollen. They appear to have wall-loosening, structural, and enzymatic functions. A novel pollen wall-bound protein of 17 kDa has a unique pattern of cysteine distribution in its sequence (six tandem repeats of CX3CX10-15) that could chelate cations and form signal-receiving finger motifs. These pollen-released proteins were synthesized in the pollen interior, and their mRNA increased during pollen maturation and germination. They were localized mainly in the pollen tube wall. The pollen shell was isolated and found to contain no detectable proteins. We suggest that the pollen-coat beta-glucanase and xylanase hydrolyze the stigma wall for pollen tube entry and that the pollen secrete proteins to loosen or become new wall constituents of the tube and to break the wall of the transmitting track for tube advance.

Maize tapetum xylanase is synthesized as a precursor, processed and activated by a serine protease, and deposited on the pollen.

Pollen coat contains ingredients that interact with the stigma surface during sexual reproduction. In maize (Zea mays L.) pollen coat, the predominant protein is a 35-kDa endoxylanase, whose mRNA is located in the tapetum cells enclosing the maturing pollen in the anthers. This 2.0-kb mRNA was found to have an open reading frame of 1,635 nucleotides encoding a 60-kDa pre-xylanase. In developing anthers, the pre-xylanase protein appeared prior to the 35-kDa xylanase protein and enzyme activity and then peaked and declined, whereas the 35-kDa xylanase protein and activity continued to increase until anther maturation. An acid protease in the anther extract converted the inactive pre-xylanase to the active 35-kDa xylanase in vitro. The protease activity was inhibited by inhibitors of serine proteases but unaffected by inhibitors of cysteine, aspartic, or metallic proteases. Sequence analysis revealed that the 60-kDa pre-xylanase was converted to the 35-kDa xylanase with the removal of 198 and 48 residues from the N and C termini, respectively. During in vitro and in vivo conversions, no intermediates of 60-35 kDa were observed, and the 35-kDa xylanase was highly stable. The pre-xylanase was localized in the tapetum-containing anther wall, whereas the 35-kDa xylanase was found in the pollen coat. The significance of having a large non-active pre-xylanase and the mode of transfer of the xylanase to the pollen coat are discussed. A gene encoding the barley (Hordeum vulgare L.) tapetum xylanase was cloned; this gene and the gene encoding the seed aleurone-layer xylanase had strict tissue-specific expressions.