Glycosylation of the overlapping sequons in yeast external invertase: effect of amino acid variation on site selectivity in vivo and in vitro.
Yeast invertase contains 14 sequons, all of which are glycosylated to varying degrees except for sequon 5 which is marginally glycosylated, if at all. This sequon overlaps with sequon 4 in a sequence consisting of Asn92-Asn93-Thr94-Ser95(Reddy et al., 1988, J. Biol. Chem., 263, 6978-6985). To determine whether glycosylation at Asn93is sterically hindered by the oligosaccharide on Asn92, the latter amino acid was converted to a glutamine residue by site-directed mutagenesis of the SUC2 gene in a plasmid vector which was expressed in Saccharomyces cerevisiae. A glycopeptide encompassing sequons 3 through 6 was purified from a tryptic digest of the mutagenized invertase and sequenced by Edman degradation, which revealed that Asn93 of sequon 5 contained very little, if any, carbohydrate, despite the elimination of sequon 4. When Ser and Thr were inverted to yield Asn-Asn-Ser-Thr carbohydrate was associated primarily with the second sequon, in agreement with numerous studies indicating that Asn-X-Thr is preferred to Asn-X-Ser as an oligosaccharide acceptor. However, when the invertase overlapping sequons were converted to Asn-Asn-Ser-Ser, both sequons were clearly glycosylated, with the latter sequon predominating. These findings rule out steric hindrance as a factor involved in preventing the glycosylation of sequon 5 in invertase. Comparable results were obtained using an in vitro system with sequon-containing tri- and tetrapeptides acceptors, in addition to larger oligosaccharide acceptors.
Reddy
A
A
Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA.
Gibbs
B S
BS
Liu
Y L
YL
Coward
J K
JK
Changchien
L M
LM
Maley
F
F
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D017931:DNA Primers; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D043324:beta-Fructofuranosidase
D017931:DNA Primers; D010446:Peptide Fragments; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase