Characterization of a Monoclonal Antibody Specific for HMW Subunits of Glutenin and Its Use To Investigate Glutenin Polymers

Abstract

A monoclonal antibody, IFRN 1602, has been developed to a synthetic peptide based on the sequence (94)GSVTCPQQV(101) of HMW subunit 1Dx5. The antibody bound strongly to the synthetic peptide based on the cognate sequence of HMW subunit 1Dx2 which contains a serine instead of a cysteine residue. However, it recognized the immunizing peptide by enzyme-linked immunosorbent assay (ELISA) only poorly, probably because the peptide exists as a disulfide-bonded dimer under the assay conditions. From immunoblotting studies against a wide range of wheat varieties, IFRN 1602 was shown to primarily recognize x-type HMW subunits of glutenin encoded on chromosomes 1A and 1D, cross-reacting weakly with the 1A and 1D y-type subunits. It did not bind to any of the 1B-encoded subunits. The Mab also recognized a small number of polypeptides of greater mobility than HMW subunits which were not visible on the stained gels and occurred only in the presence of specific 1A and 1D x-type HMW subunits. Such polypeptides were not present in a preparation of recombinant subunit 2, suggesting that they are modified forms of the subunits which arise in the seed perhaps by processing of the associated subunits. When used to probe partially reduced glutenin, IFRN 1602 bound to 1Dx5-1Dy10 dimers. As the Mab reacted primarily with Cys(97) of 1Dx5 in a reduced form, these data suggest that this residue is not involved in either intra- or intermolecular disulfide bond in the HMW subunit dimers. Thus, Cys(97) of 1Dx5 may be present in gluten in a reduced form, involved in intramolecular disulfide bonds, or linking of the HMW subunit dimers into larger polymers.

Characterization of monoclonal antibodies specific to wheat glutenin subunits and their correlation with quality parameters

“…The results showed that all mAbs could not discriminate 1Dx5 and 1Dx2 subunits of hexaploid wheat. This was consistent with the results of Mills et al (2000), in which a monoclonal antibody developed to a synthetic peptide of HMW subunit 1Dx5 was found bound strongly to the synthetic peptide based on the cognate sequence of HMW subunit 1Dx2, while recognizing the immunizing peptide by ELISA only poorly. The reason may be that the gene sequences of 1Dx5 and Dx2 were highly identical (Anderson et al 1989), so it was difficult to differentiate them by these mAbs.…”

Unexpected Deposition Patterns of Recombinant Proteins in Post-Endoplasmic Reticulum Compartments of Wheat Endosperm

“…1C). The signal obtained after labeling corresponds to the x-type HMW subunits of glutenin (Mills et al, 2000), which generally appeared to be distributed uniformly within the protein body, with the exception of a few dark, well-defined round areas that corresponded to inclusion bodies. No clusters or special distribution patterns were observed (Fig.…”

Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize

“…2A and B, lane 1). The larger of the two bands in wheat can be explained by crossreaction of the monoclonal antibody with the 1A HMW glutenin subunits (Mills et al 2000). In maize, the two bands on the immuno-blot could result from partial degradation of the protein.…”