Selective Neoglycosylation Increases the Structural Stability of Vicilin, the 7S Storage Globulin from Pea Seeds

Abstract

The effects of glycosylation on the stability and subunit interactions of vicilin, the major storage protein in pea seeds, were investigated. Glycosylated vicilin derivatives were prepared by alkylation of lysine ϵ-amino groups with various carbohydrates. Average modification levels of 13.4 ± 3.0, 11.1 ± 3.6, 7.5 ± 4.2, and 4.7 ± 0.3 moles of carbohydrate/mol of vicilin were obtained with glucose, galactose, galacturonic acid, and lactose, respectively. Nondenaturing polyacrylamide gel electrophoresis and size-exclusion chromatography indicated that the quaternary structure and hydrodynamic radius of vicilin were not affected by glycosylation at the levels used. We have previously shown that application of hydrostatic pressure causes dissociation of vicilin subunits [C. Pedrosa and S. T. Ferreira (1994) Biochemistry 33, 4046–4055]. Analysis of pressure dissociation data allowed determination of the Gibbs free energy change (ΔGdiss) and molar volume change (ΔVdiss) of dissociation of vicilin subunits. For unmodified vicilin, ΔGdiss = 18.2 kcal/mol and ΔVdiss = −102 ml/mol. Glycosylated vicilin derivatives were significantly stabilized against subunit dissociation, with ΔGdiss of 19.4, 19.2, 20.6, and 22.1 kcal/mol for glucose, galactose, lactose, and galacturonic acid derivatives, respectively. No changes in ΔVdiss were found for the glucose and galactose derivatives, whereas ΔVdiss of −128 and −135 ml/mol, respectively, were found for the lactose and galacturonic acid derivatives. The glycosylated derivatives also appeared more resistant to unfolding by guanidine hydrochloride than unmodified vicilin. Intrinsic fluorescence lifetime measurements showed that glycosylation caused a significant increase in heterogeneity of the fluorescence decay, possibly reflecting increased conformational heterogeneity of glycosylated derivatives relative to unmodified vicilin. These results indicate that the stability and subunit interactions of vicilin may be modulated by mild, selective glycosylation at low modification levels, an effect that may be of interest in the study of other oligomeric proteins.