Analisi strutturale e funzionale di proteine del glutine coinvolte in aspetti tecnologici e nutrizionali del frumento
Author(s)
Santagati, Vito Davide
Date Issued
June 29, 2016
Type
Doctoral Thesis
Abstract
Wheat is considered since long time the staple food for man, because it has unique nutritional and technological characteristics. Dough technological properties are mainly due to gluten proteins, which are composed by gliadins (monomeric) and glutenin (polymeric) that interact by hydrogen bonds and covalent bonds between cysteine residues. In particular, the size and the amount of glutenin polymers are the major determinants of dough technological properties. Since the size and the structure of glutenin polymers depend also on the number and position of the cysteine residues involved in disulfide bonds, the knowledge of their organization provides insight into the molecular basis of dough quality.
The nutritional properties of wheat-based products are due to the high amount of carbohydrates and proteins. Proteins, even if responsible of the technological quality, are linked to a large number and variety of allergies and intolerances in humans. Thus, obtaining plants with a reduced concentration or a total absence of protein involved in adverse reactions appears to be a good approach for obtaining more tolerable products.
The aims of this work were: i) the structural study of the High Molecular Weight gluten-subunit Bx20 and By20 (HMW-GS-Bx20 and HMW-GS-By20) in relationship with the technological aspects of the dough; ii) the proteomic characterization of wheat lines having deletions in α-gliadins genes located on the 6A and/or 6D chromosomes (as well as in the α–type low molecular weight glutenin subunits, that are part of the so-called LMW-GS type C). These genes are related to celiac disease, one of the major adverse reaction in human correlated to wheat.
Regarding the first aim, we studied the HMW-GS-Bx20 and HMW-GS-By20 coded by linked genes present at the Glu-B1 locus in the durum wheat cultivar Lira. The coding genes Bx20 and By20 were cloned and sequenced, and the corresponding subunits characterized by a mass spectrometry approach. The comparison between the deduced amino acid sequences and mass spectrometry results was performed. A total coverage of the protein sequence HMW-GS Bx20 and the 83.5% coverage for the HMW-GS By20 were obtained. The comparison between the structural features of Bx20 and By20, generally associated to negative technological properties, and other Glu-B1 coded pairs known to be associated to positive technological properties, allowed us to postulate a hypothesis regarding the role of number and position of the cysteine residues.
Afterwards, the Bx20/By20 heterodimers were isolated through partial reductions. Cysteines presumably involved in intra- and intermolecular disulfide bonds were differentially alkylated, trypsin digested and analyzed with mass spectrometry, obtaining indications regarding the organization of the disulfide bonds within and between these subunits. These data are not conclusive at the moment.
Regarding the second aim, a study in bread wheat lines obtained from cv. Pegaso in
which genes coding for α-gliadins and LMW-GS type C are deleted was conducted. In
particular, the wild type cv. Pegaso and its deletion lines Pegaso 6A- (Gli-A2 locus
deleted), Pegaso 6D- (Gli-D2 locus deleted) and Pegaso 6A-/6D- (Gli-A2 and Gli-D2 loci
deleted) were used.
A differentially proteomic analysis (APAGE X SDS-PAGE) of gliadins and
glutenins (LMW) type C was conducted, identifying the present/absent proteins linked
to a specific locus (either Gli-A2 or Gli-D2 ) and those proteins differentially expressed
likely for pleiotropic effects. Moreover, the gliadins extracts, the most likely triggering
factor in celiac disease, were chimotrypsin digested and analyzed through mass
spectrometry for the identification and quantification of peptides related to this
adverse reaction. Results showed that, as expected, the line with Gli-2A deleted loci
had a lower amount of immunogenic peptides. Finally, a parameter related to dough
technological properties (% UPP) was calculated in Pegaso and its derived deletion
lines, by showing no significantly difference. Thus, the deletion lines here analysed
represent a good starting plant material in order to develop new wheat genotypes with
a lower content of immunogenic peptides and with good technological properties.
Additional information
Dottorato di ricerca in Biotecnologie vegetali