REGULATION OF N-LINKED CORE GLYCOSYLATION - USE OF A SITE-DIRECTED MUTAGENESIS APPROACH TO IDENTIFY ASN-XAA-SER THR SEQUONS THAT ARE POOR OLIGOSACCHARIDE ACCEPTORS/

N-linked glycosylation can profoundly affect protein expression and fu nction. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Se r/Thr, where Xaa is any amino acid residue except Pro. However, many A sn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all fo r reasons that are poorly understood. We have used a site-directed mut agenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino a cid residues in sequons influence core-glycosylation efficiency. We re cently demonstrated that certain Xaa amino acids inhibit core glycosyl ation of the sequon, Asn(37)-Xaa-Ser, in rabies virus glycoprotein (RG P). Here we examine the impact of different Xaa residues on core-glyco sylation efficiency when the Ser residue in this sequon is replaced wi th Thr. The core-glycosylation efficiencies of RGP variants with diffe rent Asn(37)-Xaa-Ser/Thr sequons were compared by using a cell-free tr anslation/glycosylation system. Using this approach we confirm that fo ur Asn-Xaa-Ser sequons are poor oligosaccharide accepters: Asn-Trp-Ser , Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr s equons are efficiently glycosylated, even when Xaa = Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-X aa-Thr sequons in other glycoproteins confirms that sequons with Xaa = Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as gl ycosylation sites when introduced into proteins by site-directed mutag enesis.