D. D. Kasarda

Characterization of the 1B-type ω-gliadins from Triticum aestivum cultivar Butte.

ABSTRACT ω-Gliadins were purified from wheat (Triticum aestivum L. ‘Butte’) flour and characterized. Although reversed-phase HPLC (RP-HPLC) separated the 1B-encoded ω-gliadins into two fractions, 1B1 and 1B2, these fractions had nearly identical amino acid compositions, three similar protein bands in SDS-PAGE, 10 similar spots in two-dimensional PAGE, and two similar N-terminal amino acid sequences. The main components had a range in mass of 48,900–51,500 when estimated by mass spectrometry, significantly less than the mass estimated by SDS-PAGE. The 1B fractions were digested with thermolysin, the peptides were separated by RP-HPLC, the peptide mass was determined, and nine peptides from each fraction were sequenced with nearly identical results for the 1B1 and 1B2 digests. A possible consensus sequence of the 1B-encoded ω-gliadin internal repeat was QQQXP, where X was F, I, or L in descending order of occurrence. The 1D-encoded ω-gliadins were purified by RP-HPLC as a single fraction that had one band i...

Similarities of omega gliadins from Triticum urartu to those encoded on chromosome 1A of hexaploid wheat and evidence for their post-translational processing

The ω-gliadins encoded on chromosome 1 of the A genome were purified from Triticum aestivum L. (2n=6x=42, AABBDD) cv. Butte86, nullisomic 1D-tetrasomic 1A of cv. Chinese Spring (CS N1DT1A), and the diploid T. urartu (2n=2x=14, AA). Reverse-phase high-performance liquid chromatography combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis of gliadin extracts from CS nullisomic-tetrasomic (NT) lines confirmed the assignment to chromosome 1A. The purified ω-gliadins were characterized by mass spectrometry and N-terminal sequencing. The 1A-encoded ω-gliadins were smaller than 1B- or 1D-encoded ω-gliadins. The N-terminal amino acid sequences for 1A ω-gliadin mature peptides were nearly identical to those for the T. urartu ω-gliadins and were more similar to 1D ω-gliadin sequences than to sequences for T. monococum ω-gliadins, barley C-hordeins, or rye ω-secalins. They diverged greatly from the N-terminal sequences for the 1B ω-gliadins. The data suggest that T. urartu is the A-genome donor, and that post-translational cleavage by an asparaginyl endoprotease produces those ω-gliadins with N-terminal sequences beginning with KEL.

Quality-related endosperm proteins in sulfur-deficient and normal wheat grain

Specific groups of proteins associated with the baking properties of flour were identified by extracting flour from normal and sulfur-deficient wheat with a sodium dodecylsulfate (SDS)-Tris buffer either with or without reducing agent. Total proteins (with reducing agent), extract proteins (without reducing agent), and residue proteins (re-extraction of sediment with reducing agent) were fractionated by SDS-polyacrylamide gel electrophoresis (with reducing agent present for all fractions). Significant correlations were obtained between the proportions of certain groups of polypeptides and flour-quality characteristics. The proportion of high molecular weight (HMW) components (Mr 80,000; corresponding to HMW-glutenin subunits) in the residue was strongly positively correlated with resistance to extension and negatively correlated with dough extensibility and breakdown. Of the proteins extracted mainly by SDS without reduction, ω-gliadins in the Mr range 51,000 to 80,000 showed the same correlations as the HMW-glutenin subunits, whereas proteins inthe Mr range 38,000 to 50,000 and 28,000 to 39,000 were positively correlated with dough extensibility. The proteins of these latter ranges correspond mainly to α-, β-, and γ-gliadins and LMW-glutenin subunits. The proteins of the lowest Mr range (Mr

Molecular cloning of the barley seed protein CMd: a variant member of the α-amylase/trypsin inhibitor family of cereals

The nucleotide and deduced amino-acid sequences of a cDNA clone encoding the barley seed protein CMd are described. The sequence is homologous with those of a family of inhibitors of alpha-amylase and trypsin, except for two short insertions. The longest of these (14 residues) is at the junction between the three proposed ancestral regions that comprise this family of proteins, and has limited identity with alpha-amylases of bacterial origin.

Surface and optical properties of wheat glutenin monolayers

Glutenin complexes isolated from wheat cultivars of different baking quality were spread as thin films at an air-water interface. The surface properties of the spread films were compared. Films of glutenin from both good and poor quality wheat varieties exhibited surface behavior similar to one another but different from that of other wheat proteins such as gliadin. The films showed no evidence of collapse at high compression. Glutenin films were also transferred from the surface to quartz plates for spectroscopic analysis. Circular dichroism studies showed that the glutenin complexes contain a significant amount of secondary structure that is likely to be in the form of β-turns. Furthermore, qualitative analysis of the circular dichroic difference spectrum suggested that the glutenin originating from a good quality wheat contained a greater proportion of β-turns than an equivalent sample obtained from a poor quality wheat.

An asparagine residue at the N-terminus affects the maturation process of low molecular weight glutenin subunits of wheat endosperm

BackgroundWheat glutenin polymers are made up of two main subunit types, the high- (HMW-GS) and low- (LMW-GS) molecular weight subunits. These latter are represented by heterogeneous proteins. The most common, based on the first amino acid of the mature sequence, are known as LMW-m and LMW-s types. The mature sequences differ as a consequence of three extra amino acids (MET-) at the N-terminus of LMW-m types. The nucleotide sequences of their encoding genes are, however, nearly identical, so that the relationship between gene and protein sequences is difficult to ascertain.It has been hypothesized that the presence of an asparagine residue in position 23 of the complete coding sequence for the LMW-s type might account for the observed three-residue shortened sequence, as a consequence of cleavage at the asparagine by an asparaginyl endopeptidase.ResultsWe performed site-directed mutagenesis of a LMW-s gene to replace asparagine at position 23 with threonine and thus convert it to a candidate LMW-m type gene. Similarly, a candidate LMW-m type gene was mutated at position 23 to replace threonine with asparagine. Next, we produced transgenic durum wheat (cultivar Svevo) lines by introducing the mutated versions of the LMW-m and LMW-s genes, along with the wild type counterpart of the LMW-m gene.Proteomic comparisons between the transgenic and null segregant plants enabled identification of transgenic proteins by mass spectrometry analyses and Edman N-terminal sequencing.ConclusionsOur results show that the formation of LMW-s type relies on the presence of an asparagine residue close to the N-terminus generated by signal peptide cleavage, and that LMW-GS can be quantitatively processed most likely by vacuolar asparaginyl endoproteases, suggesting that those accumulated in the vacuole are not sequestered into stable aggregates that would hinder the action of proteolytic enzymes. Rather, whatever is the mechanism of glutenin polymer transport to the vacuole, the proteins remain available for proteolytic processing, and can be converted to the mature form by the removal of a short N-terminal sequence.