THE HYDROXY AMINO-ACID IN AN ASN-X-SER THR SEQUON CAN INFLUENCE N-LINKED CORE GLYCOSYLATION EFFICIENCY AND THE LEVEL OF EXPRESSION OF A CELL-SURFACE GLYCOPROTEIN/

N-Linked glycosylation usually occurs at the sequon, Asn-X-Ser/Thr. In this sequon, the side chain of the hydroxy amino acid (Ser or Thr) ma y play a direct catalytic role in the enzymatic transfer of core oligo saccharides to the Asn residue. Using recombinant variants of rabies v irus glycoprotein (RGP), we examined the influence of the hydroxy amin o acid on core glycosylation efficiency. A variant of RGP containing a single Asn-X-Ser sequon at Asn(37) was modified by site directed muta genesis to change the sequon to either Asn-X-Cys or Asn-X-Thr. The imp act of these changes on core glycosylation efficiency was assessed by expressing the variants in a cell-free transcription/translation/glyco sylation system and in transfected tissue culture cells. Substitution of Cys at position 39 blocks glycosylation, whereas substitution of Th r dramatically increases core glycosylation efficiency of Asn(37) in b oth membrane-anchored and secreted forms of RGP. The substitution of T hr for Ser also dramatically enhances the level of expression and cell surface delivery of RGP when the sequon at Asn(37) is the only sequon in the protein. Novel forms of membrane-anchored and secreted RGP whi ch are fully glycosylated at all three sequons were also generated by substitution of Thr at position 39.