THE AMINO-ACID FOLLOWING AN ASN-X-SER THR SEQUON IS AN IMPORTANT DETERMINANT OF N-LINKED CORE GLYCOSYLATION EFFICIENCY/

Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosy lation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr sequons where X not equal Pro. However, many Asn-X-Ser/ Thr sequons are not glycosylated or are glycosylated inefficiently. In efficient glycosylation at one or more Asn-X-Ser/Thr sequons in a prot ein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of express ion, stability, antigenicity, or function. The signals which control t he efficiency of N-linked glycosylation at individual Asn residues hav e not been fully defined. In this report, we use a site-directed mutag enesis approach to investigate the influence of the amino acid at the position following a sequon (the Y position, Asn-X-Ser/Thr-Y). Variant s of rabies virus glycoprotein containing a single Asn-X-Ser/Thr sequo n at Asn(37) were generated. Variants were designed with each of the t wenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of e ach variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.