Migliorare il grano per rimuovere gli epitopi dannosi per i celiaci mantenendone la funzionalità

Un interessante studio che esplora le possibilità di ottenere grani a bassa tossicità investigando qualità, quantità e distribuzione delle frazioni tossiche (per i celiaci) del grano

“…..omissis. Wheat gluten proteins are traditionally classified into two groups based on their solubility. The gliadins are readily extracted from flour with alcohol:water mixtures, such as 60% (v/v) ethanol or 50% (v/v) propan-1- ol, while the glutenins were traditionally extracted with dilute acid or alkali. However, these fractions contain related proteins and the differences in solubility are determined by their presence as monomers or polymers. Thus, the gliadin fraction comprises mainly proteins which are present as monomers, with small amounts of polymeric components, while the glutenins comprise “subunits” assembled into high molecular mass polymers stabilized principally by inter-chain disulphide bonds. When these disulphide bonds are reduced the monomeric glutenin subunits resemble the gliadins in being soluble in alcohol: water mixtures. Hence, the protein subunits present in both fractions correspond to alcohol-soluble prolamin proteins as defined in the classic studies of Osborne (1924). more in full text”. Improving wheat to remove coeliac epitopes but retain functionality. Peter R. Shewry and Arthur S. Tatham. Journal of Cereal Science 2016 Jan

Estratto dallo studio:
1.2. Gliadine e glutenine
1.3. La distribuzione delle subunità nei geni
2.2. L’individuazione delle frazioni tossiche per i celiaci
2.3. La distrubuzione delle frazioni tossiche per i celiaci
3.1. La diversità genetica del grano
3.2. La possibile individuazione di varietà (genetic diversity) a basso livello di tossicità
3.3. Il possibile sviluppo di varietà idonee per celiaci

1.2. Gliadine e glutenine
The use of wheat for most food products in underpinned by the gluten proteins. These correspond to the major group of grain storage proteins which are deposited in the starchy endosperm cells to support germination and seedling growth. They account for up to 80% of the total grain proteins, which in turn account for between 10 and 15% of the dry weight of grain grown commercially. These proteins form a continuous matrix surrounding the starch granules in the mature starchy endosperm cells, and are brought together to form a continuous network when flour is mixed with water to give dough. This network confers a unique combination of elasticity and viscosity which enable the dough to be processed into the range of products discussed above.
…….omissis. Wheat gluten proteins are traditionally classified into two groups based on their solubility. The gliadins are readily extracted from flour with alcohol:water mixtures, such as 60% (v/v) ethanol or 50% (v/v) propan-1ol, while the glutenins were traditionally extracted with dilute acid or alkali. However, these fractions contain related proteins and the differences in solubility are determined by their presence as monomers or polymers. Thus, the gliadin fraction comprises mainly proteins which are present as monomers, with small amounts of polymeric components, while the glutenins comprise “subunits” assembled into high molecular mass polymers stabilized principally by inter-chain disulphide bonds.
……….omissis. The gliadins are traditionally divided based on their mobility in electrophoresis at low pH into three groups: the S-rich α-type gliadins and γ-type gliadins (which contain three and four inter-chain disulphide bonds, respectively) and the S-poor ω-gliadins (which lack cysteine residues and hence do not form disulphide bonds) (Fig. 1). Similarly, the glutenin subunits are separated by sodium dodecylsulphate polyacrylamide electrophoresis (SDS-PAGE) into low molecular weight subunits (LMW subunits) and high molecular weight subunits (HMW prolamins) of glutenin (Fig. 1). The LMW and HMW subunits form inter-chain disulphide bonds which stabilise the glutenin polymers, while intra-chain disulphide bonds are also formed by LMW subunits and at least some HMW subunits.

1.3. La distribuzione delle subunità nei geni
Genetic and molecular analyses indicate that the individual gluten proteins are encoded by multiple genes at complex loci. The classical genetics has been reviewed in detail by Shewry et al. (2003a). The HMW subunits of glutenin are encoded by three loci on the long arms of the group 1 chromosomes (Glu-A1, GluB1, Glu-D1), each comprising two genes encoding one x-type and one y-type HMW subunit. Similarly, the α-type gliadins are encoded by three loci on the sort arms of the group 6 chromosomes (Gli-A2, Gli-B2, Gli-D2), 6B, 6D), but these loci are more complex and may together comprise over 50 genes (most of which do not appear to be expressed) (Anderson and Greene, 1997, Van Herpen et al., 2006).

2.2. L’individuazione delle frazioni tossiche per i celiaci
Currently thirty-one, nine amino acid peptide sequences in the prolamins of wheat and related species have been defined as being coeliac toxic: these are often referred to as coeliac “epitopes”. However, mapping is incomplete and the number of distinct epitopes a matter of on-going discussion (Sollid et al., 2012). These epitopes are located in the repetitive domains of the prolamins, which are proline and glutamine-rich, and the high levels of proline in their sequences may reduce their susceptibility to protease activity in the GI tract.
………..omissis. Tye-Din et al. (2010) reported a hierarchy of coeliac-stimulating peptides after challenge with wheat, barley and rye. This showed that the immunodominant sequence after wheat challenge corresponds to a well-characterised 33 residue peptide from α-gliadin that contains the overlapping T-cell epitopes DQ2.5-gliaα1a, b and DQ2.5-glia-α2. This “33-mer” is resistant to gastrointestinal digestion (with pepsin and trypsin) and was initially identified as the major coeliac toxic peptide in the gliadins.

2.3. La distrubuzione delle frazioni tossiche per i celiaci
Information on the frequency, distribution and immunodominance of coeliac toxic epitopes is required to underpin the use of conventional breeding or genetic engineering to develop lines with reduced CD toxicity. Table 2 shows the current list of T-cell epitopes for wheat, barley and rye (Sollid et al., 2012) and Fig. 3 their distribution in the amino acid sequences of representative prolamins.

3.1. La diversità genetica del grano
Wheat occurs in a range of diploid, tetraploid and hexaploid forms (summarised in Table 1). The earliest cultivated forms were the A genome diploid einkorn (T. monococcum var monococcum) and tetraploid emmer (T. turgidum var. dicoccum) with the A and B genomes. These are closely related to wild forms: diploid T. monococcum var. monococcum and T. ururtu and tetraploid T. turgidum var. dicoccoides, respectively. Modern tetraploid durum (pasta) wheat (T. turgidum var. durum) probably arose from mutations in cultivated emmer. Hexaploid bread wheat (Triticum aestivum) (genomes ABD) has never existed as a wild species and no wild hexaploid wheats are known. It probably arose by hybridization of cultivated emmer with the related wild grass T. tauschii (goat grass, also called Aegilops tauschii and Ae. squarossa). This hybridization probably occurred in south-eastern Turkey about 9000 years ago (Feldman, 1995, Dubcovsky and Dvorak, 2007) and contributed the D genome. All cultivated hexaploid wheats, including spelt, are forms of T. aestivum.

A major difference between “ancient” cultivated wheats (einkorn, emmer, spelt) and their wild relatives and modern durum and bread wheats is whether the grain are hulled or free threshing. In hulled wheats the glumes and palea adhere to the grain and the threshed material consists of intact spikelets. By contrast, these structures are removed in “free threshing” durum and bread wheats and the harvested material consists of caryopses. The hulled einkorn, emmer and spelt are together called “faro” in Italy. The reader is referred to Feldman, 1995, Dubcovsky and Dvorak, 2007 and chapters in Elsayed and Wood (2005) for detailed discussions of the evolutionary relationships of wheat species and the characteristics of the “ancient” cultivated forms.

3.2. La possibile individuazione di varietà (genetic diversity) a basso livello di tossicità
The improvement of wheat and other crops by plant breeding is based on the identification of genetic variation in traits of interest and incorporating this variation into lines which are commercially competitive in terms of their yield, agronomic performance and quality. In the case of coeliac toxicity, the trait of interest is the number and distribution of coeliac toxic sequences (epitopes). It is therefore logical to determine the relative distribution of coeliac epitopes within different gluten protein types (which is discussed above), and how this distribution varies between proteins coded by the three genomes of hexaploid bread wheat and between the genotypes, including modern commercial cultivars, exotic lines from other parts of the world and “land races” which were cultivated before the use of modern intensive breeding.
……omissis. Carroccio et al. (2011) have since described the production of a wheat line (C1173) which lacks the α-type gliadins encoded by Gli-A2 locus and also γ-gliadins and ω-gliadins encode by the complex Gli-D1/Glu-D3 locus. Prolamins from this line showed significantly lower toxicity in vitro than a similar fraction from the control line San Pastore. van den Broeck et al. (2009) have screened gluten protein fractions from lines of Chinese Spring wheat with partial deletions of the long and short arms of the group 6 chromosomes using monoclonal antibodies that recognise T-cell epitopes. Loss of the α-gliadin locus from the short arm of chromosome 6D resulted in a significant decrease in the presence of T-cell stimulatory epitopes but also a significant loss of dough functionality. This is consistent with the studies of van Herpen et al. (2006) who showed that T-cell stimulatory epitopes were more abundant in α-gliadins encoded by the D genome, and Molberg et al. (2005) who demonstrated that the immunodominant 33mer fragment of α-gliadin was encoded by chromosome 6D (and hence absent from diploid einkorn and tetraploid wheats). A subsequent study also showed that the detrimental effect of the loss of chromosome 1DS on functionality could be compensated for by adding coeliac-safe avenin proteins from oats (van den Broeck et al., 2011).
……….omissis. As discussed above, the absence of the D genome from durum wheat could result in lower coeliac activity due to the absence of the T-cell stimulatory epitopes at the Gli-D2 locus. van den Broeck et al. (2010a) therefore screened 103 accessions of tetraploid wheat by immunoblotting of gluten protein extracts with monoclonal antibodies against the Glia-α9 and Glia-α20 epitopes. This identified three accessions with significantly reduced levels of both epitopes. Further analysis of 61 durum wheat accessions by high throughput transcript sequencing similarly identified some accessions with lower abundances of transcripts containing coeliac disease epitopes (Salentjin et al., 2013). Finally, van den Broeck et al. (2010b) compared the abundance of the major Glia-A9 coeliac disease epitope in 36 modern wheat cultivars and 50 land races by immunoblotting of gluten protein extracts, using the minor Glia-A20 epitope as a technical reference. The modern cultivars tended to show higher reactivity with the Glia-A9 antibody and lower reaction with the Glia-A20 antibody, although lines showing high and low reactions with both antibodies were present in both sets of germplasm.

3.3. Il possibile sviluppo di varietà idonee per celiaci
The detailed studies discussed above clearly demonstrate that there is extensive variation in the occurrence of coeliac-toxic epitopes within and between the sequences of gliadins encoded by the three genomes of bread wheat (and the corresponding genomes of diploid and tetraploid species), and in the levels of expression of these proteins. Although in some cases these include the existence of forms that lack the currently defined coeliac epitopes, it cannot be ruled out that they contain sequences which will stimulate a response in some individuals. Nevertheless it should be possible to exploit this variation to develop wheat cultivars with low levels of these epitopes, if not their absence. However, there are several factors to consider.

Approfondimento
Improving wheat to remove coeliac epitopes but retain functionality