Three exoglucanases (Exgs), ExgIa, ExgIb and Exg325, are secreted by Saccharomyces cerevisiae cells. They share a common protein portion with two potential glycosylation sites (sequons) but differ in the amount of N-linked carbohydrate [Basco, R.D., Muñoz, M.D., Hernández, L.M., Váquez de Aldana, C. and Larriba, G. (1993) Yeast 9, 221-234]. ExgIb contains two short oligosaccharides attached to asparagines (Asn) 165 and 325 of the primary translation product [Hernández, L.M., Olivero, I., Alvarado, E. and Larriba, G. (1992) Biochemistry 31, 9823-9831]. Exg325 carries a single, short oligosaccharide bound to Asn325 whereas ExgIa has at least one large oligosaccharide, since it has not been produced by mutant mnn9. To address the question of the origin of ExgIa, both sequons were individually mutated by substituting Gln for Asn. An ExgIa-like isoenzyme was still secreted by mutant Exg165 but not by mutant Exg325. Additional studies on sequential deglycosylation of ExgIa with endo-beta-N-acetylglucosaminidase H (endo H), the susceptibility of both oligosaccharides to the endoglycosidase, and analysis of the presence of GlcNAc at both asparagine residues after total deglycosylation with endo H, indicated that ExgIa contained two oligosaccharides, a short one bound to Asn165 and a large one bound to Asn325, and, accordingly, originated from ExgIb. The elongation of the second oligosaccharide did not result in a higher stability towards thermal inactivation or unfolding, or in an increased resistance to proteases as compared with ExgIb; however, the affinity of the enzyme towards laminarin decreased by 50%. This site-specific elongation occurred in the oligosaccharide that was less susceptible to endo H, indicating that these properties are determined by different conformational constraints.
Selective elongation of the oligosaccharide attached to the second potential glycosylation site of yeast exoglucanase: effects on the activity and properties of the enzyme
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R D Basco, L M Hernández, M D Muñox, I Olivero, E Andaluz, F Del Rey, G Larriba; Selective elongation of the oligosaccharide attached to the second potential glycosylation site of yeast exoglucanase: effects on the activity and properties of the enzyme. Biochem J 15 December 1994; 304 (3): 917–922. doi: https://doi.org/10.1042/bj3040917
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