Characterization of a low-molecular-weight glutenin subunit gene from bread wheat and the corresponding protein that represents a major subunit of the glutenin polymer

Abstract

Both high- and low-molecular-weight glutenin subunits (LMW-GS) play the major role in determining the viscoelastic properties of wheat (Triticum aestivum L.) flour. To date there has been no clear correspondence between the amino acid sequences of LMW-GS derived from DNA sequencing and those of actual LMW-GS present in the endosperm. We have characterized a particular LMW-GS from hexaploid bread wheat, a major component of the glutenin polymer, which we call the 42K LMW-GS, and have isolated and sequenced the putative corresponding gene. Extensive amino acid sequences obtained directly for this 42K LMW-GS indicate correspondence between this protein and the putative corresponding gene. This subunit did not show a cysteine (Cys) at position 5, in contrast to what has frequently been reported for nucleotide-based sequences of LMW-GS. This Cys has been replaced by one occurring in the repeated-sequence domain, leaving the total number of Cys residues in the molecule the same as in various other LMW-GS. On the basis of the deduced amino acid sequence and literature-based assignment of disulfide linkages, a computer-generated molecular model of the 42K subunit was constructed.

Figure 1

Comparison between SDS-PAGE (A) and the PCR-amplification pattern (B) of durum wheat cv Lira biotype 45 (lane 1) and bread wheat cvs Red River (lane 2), Cheyenne (lane 3), Yecora Rojo (lane 4), and Solar (lane 5). Both the 42K LMW-GS protein band and the 1.15-kb PCR product (both indicated) were present in all bread wheat cultivars except cv Cheyenne.

Figure 2

Deduced amino acid sequence of the lmw-gs gene corresponding to the 1.15-kb PCR product. Underlines show those peptides that confirmed correspondence with the 42K LMW-GS. The peptide numbers are keyed to Figure 5. The double-underlined sequence represents the final part of the signal peptide typical of LMW-GS. Question marks (?) indicate unidentified amino acids. The complete sequence is reported under accession no. Y17845.

Figure 3

Alignment of the amino acid repeats found in the repetitive domain of the 42K LMW-GS encoded by the 1.15-kb PCR product. The figure shows the regular distribution of the repeats from amino acids 13 to 163 of the deduced mature protein. Cons, Consensus sequence.

Figure 4

RP-HPLC separation of the Lys-C digests of the 42K LMW-GS (A) and the SDS-PAGE pattern of the peaks collected (B).

Figure 5

Amino acid sequences of Cys-containing chymotryptic peptides obtained after covalent chromatography (A) and peptides obtained after chymotryptic digestion of the Lys-C N-terminal fragment of the 42K LMW-GS (B) (peak 2 of Fig. 4A). The alternative amino acids at positions where heterogeneity is present are reported.

Figure 6

Computer-generated molecular model of the 42K subunit. A, Entire 42K LMW-GS shown in space-filling form (van der Waals's radii for atoms) with all atoms shown in blue, except the sulfur atoms of Cys or cystine side chains, which are shown in yellow. B, Simplified model of the region containing the intramolecular disulfide linkages and Cys-295, which presumably forms one of two intermolecular disulfide cross-linkages. Residues 200 to 369 of the 42K subunit are shown in protein cartoon format, which displays the α-helical structure as a helical ribbon. There is also a specific display of selected side chains in a licorice (stick) bond format. The sulfur atoms of the Cys and cystine residues are shown in yellow. The main polypeptide chain is shown in red for residues 200 to 248 and in blue from there on to the C-terminal end at residue 369. The numbers of connected (intramolecular) Cys residues are shown.

Figure 7

Flexibility of the polypeptide chain of the 42K LMW-GS, as predicted by the method of Karplus and Schulz (1985). A coefficient of 1.0 represents an average flexibility, and lower values indicate less-than-average flexibility. Positive values of 1.15 seen for some regions of the 42K LMW-GS are equivalent to highly flexible regions of the polypeptide chain, based on the model proteins used in the development of the predictive method. Arrows indicate the positions of the two Cys residues in the 42K subunit that are supposed to form intermolecular disulfide bonds.