EVIDENCE FOR THE NON-GLYCOPROTEIN NATURE OF HIGH-MOLECULAR-WEIGHT GLUTENIN SUBUNITS OF WHEAT

An isolation and purification procedure for wheat high molecular weigh t glutenin subunits (HMW-GS) involving no steps expected to cause degl ycosylation of glycoproteins was developed to allow removal of the max imum amount of carbohydrates not covalently linked to HMW-GS. Flour wa s defatted and then extracted with 50% n-propanol (50% n-PrOH) to remo ve arabinogalactans and glucose-containing material. HMW-GS were extra cted with 50% n-PrOH containing 5% beta-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 HM W-GS gave a positive reaction in a periodate-digoxigenin glycan detect ion assay when labelled in solution (with or without periodate oxidati on) but not when labelled directly on-the-blot. HMW-GS expressed in Es cherichia coli a micro-organism without glycosylation capabilities, re acted in an identical way. Possible reasons are given for the differen ce in results obtained after labelling in solution or on-the-blot. No positive reaction between the purified HMW-GS and mannose-specific Gal anthus nivalis agglutinin was observed. The presence of N-acetylglucos amine in HMW-GS has been previously reported(20). Since neither mannos e nor N-acetylgalactosamine were found in the HMW-GS, and these protei ns 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. (C) 1996 Academic Press Limi ted