Selective Neoglycosylation Increases the Structural Stability of Vicilin, the 7S Storage Globulin from Pea Seeds Cristiana Pedrosa, 1 Fernanda G. De Felice, 1 Cristina Trisciuzzi, and Se ´rgio T. Ferreira 2 Departamento de Bioquı ´mica Me ´dica, Instituto de Cie ˆncias Biome ´dicas, Universidade Federal do Rio de Janeiro, RJ 21941-590, Brazil Received March 6, 2000, and in revised form July 14, 2000 The effects of glycosylation on the stability and sub- unit interactions of vicilin, the major storage protein in pea seeds, were investigated. Glycosylated vicilin derivatives were prepared by alkylation of lysine -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 polyacryl- amide gel electrophoresis and size-exclusion chroma- tography 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 (G diss ) and molar vol- ume change (V diss ) of dissociation of vicilin subunits. For unmodified vicilin, G diss  18.2 kcal/mol and V diss 102 ml/mol. Glycosylated vicilin derivatives were significantly stabilized against subunit dissocia- tion, with G diss of 19.4, 19.2, 20.6, and 22.1 kcal/mol for glucose, galactose, lactose, and galacturonic acid de- rivatives, respectively. No changes in V diss were found for the glucose and galactose derivatives, whereas V diss of 128 and 135 ml/mol, respectively, were found for the lactose and galacturonic acid de- rivatives. The glycosylated derivatives also appeared more resistant to unfolding by guanidine hydrochlo- ride than unmodified vicilin. Intrinsic fluorescence lifetime measurements showed that glycosylation caused a significant increase in heterogeneity of the fluorescence decay, possibly reflecting increased con- formational 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 inter- est in the study of other oligomeric proteins. © 2000 Academic Press Key Words: glycosylation; stability; subunit dissocia- tion; hydrostatic pressure; guanidine hydrochloride; vicilin. Protein glycosylation may affect the hydrophilic/hy- drophobic balance and/or net charge at the protein surface, leading to changes in protein–solvent and pro- tein–protein interactions. These, in turn, may lead to changes in folding, stability, protease resistance, or biological activity of glycosylated proteins (1–3). In sev- eral cases, the structural effects of glycosylation have been well characterized. For example, glycosylation of ribonuclease A decreases the overall conformational dynamics of the enzyme and increases its stability towards proteinases (4, 5). Similarly, fucosylation of a proteinase inhibitor causes an overall decrease in dy- namic structural fluctuations, which correlates with an increase in stability monitored by thermal denatur- ation (6). Covalently bound carbohydrate has also been shown to play a stabilizing role in the folding of human T lymphocyte CD2 cell surface glycoprotein (7). How- ever, despite the relative abundance of data on the effects of glycosylation on protein folding and stability, relatively little is known on the effects of carbohydrate moieties on subunit interactions in oligomeric proteins. Selective neoglycosylation has been used as a strat- egy to modify the physicochemical properties of pro- teins in a controlled manner. For example, enzyme– carbohydrate conjugates have been prepared to in- crease thermal stability and resistance to proteolysis (8, 9), and a few studies have dealt with neoglycosyla- 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed. Fax: (+5521)270- 8647. E-mail: ferreira@bioqmed.ufrj.br. 0003-9861/00 $35.00 203 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 382, No. 2, October 15, pp. 203–210, 2000 doi:10.1006/abbi.2000.2024, available online at http://www.idealibrary.com on