| Abstrakt: |
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 Asn93 is 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. [ABSTRACT FROM PUBLISHER] |
