Evidence for the Non-Glycoprotein Nature of High Molecular Weight Glutenin Subunits of Wheat |
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Authors: | S.P. Roels J.A. Delcour |
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Affiliation: | Research Unit Food Chemistry, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, Heverlee, B-3001, Belgium |
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Abstract: | An isolation and purification procedure for wheat high molecular weight glutenin subunits (HMW-GS) involving no steps expected to cause deglycosylation of glycoproteins was developed to allow removal of the maximum amount of carbohydrates not covalently linked to HMW-GS. Flour was defatted and then extracted with 50%n-propanol (50%n-PrOH) to remove arabinogalactans and glucose-containing material. HMW-GS were extracted with 50%n-PrOH containing 5% β-mercaptoethanol, precipitated in 60%n-PrOH, alkylated and recovered by precipitation. The partially purified HMW-GS were separated by cation exchange chromatography and resulting fractions purified by RP–HPLC. The purified HMW-GS contained only 0·1% of glucose (corresponding to 51–59 monosaccharide residues per 100 HMW-GS molecules) and no other amino or neutral sugars. The HMW-GS gave a positive reaction in a periodate–digoxigenin glycan detection assay when labelled in solution (with or without periodate oxidation) but not when labelled directly on-the-blot. HMW-GS expressed inEscherichia coli, a micro-organism without glycosylation capabilities, reacted in an identical way. Possible reasons are given for the difference in results obtained after labelling in solution or on-the-blot. No positive reaction between the purified HMW-GS and mannose-specificGalanthus nivalisagglutinin was observed. The presence of N-acetylglucosamine in HMW-GS has been previously reported20. Since neither mannose nor N-acetylgalactosamine were found in the HMW-GS, and these proteins lack the sequon necessary for N-glycosylation, only O-linked GlcNAc moieties are possible. This modification could not be detected using a galactosyltransferase labelling assay. Taken together these results suggest that HMW-GS are not glycosylated. |
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Keywords: | glycosylation high molecular weight glutenin subunits periodate oxidation glycan detection |
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