N-linked core-glycosylation efficiency is influenced by the x and hydroxy amino acids in asn-x-ser/thr sequons

N-linked glycosylat ion can profoundly affect the expression and function of eukaryotic proteins. The sequon, Asn-X-Ser/Thr-Y, is generally required for relinked glycosylation. Pro at the X or Y position generally blocks glycosylation. We tested the impact of the X and hydroxy (Ser/Thr) amino acids by comparing the coreglycosylation of rabies virus glycoprotein variants with a single sequon at Asn37. Mutagenesis was used to vary the X amino acid of the sequon with either Ser or Thr at the hydroxy position. The core-glycosylation efficiency of the sequon in each variant was quant if ied in a cell-free translation/glycosylation system. We find that Trp, Asp, Glu, or Leu at the X posit ion of Asn-X-Ser sequon s markedly inhibits glycosylation, whereas Asn-X-Ser sequons with other X amino acids are efficiently glycosylated. In contrast, Asn-X-Thr sequons are efficiently glycosylated, even when X=Trp, Asp, Glu, or Leu. The generalizability of these findings is supported by a comparison of sequons in other glycoproteins. That analysis confirms that Asn-X-Ser sequons with X=Trp, Asp, Glu, or Leu are rarely glycosylated, whereas most Asn-X-Thr sequons with these X amino acids are glycosylated provided that YPro. These findings further define the signals which control N-linked glycosylat ion, and fac ilitate predict ions of the glycosylation patterns of proteins from their amino acid sequences. Supported by the National Blood Foundation.