Identification of glycosylated forms of wheat storage proteins using two-dimensional electrophoresis and blotting.
Two-dimensional electrophoresis with acid-polyacrylamide gel electrophoresis (PAGE), followed by sodium dodecyl sulfate (SDS)-PAGE and SDS-PAGE of unreduced polypeptides followed by SDS-PAGE under reducing conditions, were used to separate and identify the different subgroups of gliadins and glutenins and to distinguish between covalent and noncovalent polymers of glutenins. Gels were blotted under semidry conditions according to Laurière (Anal. Biochem. 1993, 212, 206-211) to allow large polymers of glutenins to be transferred efficiently. Glycosylated polypeptides were detected on blots using either the method of Haselbeck and Hösel (Glycoconjugate J. 1990, 7, 63-74), or using anti-(xylose-containing N-glycan) antibodies (Laurière et al., Plant Physiol 1989, 90, 1182-1188). High and low molecular weight glutenin subunits were shown to aggregate through both disulfide bridges and noncovalent protein-to-protein interactions. Aggregated gamma-gliadins were also demonstrated. Glycans were detected on both gliadin and glutenin polypeptides. Covalently aggregated low molecular weight glutenins were shown to contain N-glycans with xylose, which demonstrated their sorting in the Golgi apparatus.
Laurière
M
M
Laboratoire de Chimie Biologique, Centre INRA de Grignon, Thiverval-Grignon, France. lauriere@cardere.grignon.inra.fr
Bouchez
I
I
Doyen
C
C
Eynard
L
L
D000906:Antibodies / Q000276:immunology; D002241:Carbohydrates / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005903:Gliadin / Q000032:analysis; D005983:Glutens / Q000031:analogs & derivatives / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis*; D015151:Immunoblotting; D010940:Plant Proteins / Q000032:analysis*; D011134:Polysaccharides / Q000276:immunology; D014908:Triticum / Q000737:chemistry*; D014994:Xylose / Q000276:immunology
D000906:Antibodies; D002241:Carbohydrates; D006023:Glycoproteins; D010940:Plant Proteins; D011134:Polysaccharides; C038234:storage protein, Triticum aestivum; D005983:Glutens; D005903:Gliadin; D014994:Xylose; C037302:glutenin