Site-specific glycosylation in animal cells. Substitution of glutamine for asparagine 293 in chicken ovalbumin does not allow glycosylation of asparagine 312.
It has been shown previously that chicken ovalbumin synthesized and secreted in a heterologous cell system is glycosylated at the correct site and that the oligosaccharides at that site, similar to the protein made in hen oviduct, are predominantly of the hybrid type (Sheares, B. T., and Robbins, P. W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1993-1997). This site-specific glycosylation of Asn293, but not Asn312, suggested a prominent role for the nascent protein chain rather than the specific cell type in directing the proper attachment of oligosaccharide chains. In the present study, the effect of glycosylation at Asn293 on the glycosylation of Asn312 has been investigated. Using a 20-base oligodeoxynucleotide primer containing a 2-base mismatch, the codon for Asn293 in the chicken ovalbumin gene (AAC) was changed to that for Gln (CAA), thereby preventing glycosylation at amino acid 293. Constructions containing this mutation were transfected into mouse L (tk-) cells which were subsequently labeled with [35S]methionine. Ovalbumin secreted by these cells was recovered by immunoaffinity chromatography and analyzed for the presence of an oligosaccharide attached at Asn312. Treatment of the material with peptide:N-glycosidase F demonstrated that ovalbumin molecules containing Gln substituted for Asn293 were not glycosylated. This further supports our earlier hypothesis that the nascent protein chain is responsible for directing site-specific glycosylation of ovalbumin, and that the presence of an oligosaccharide chain at the first site has no influence on glycosylation at the second site.
Sheares
B T
BT
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D001483:Base Sequence; D002645:Chickens; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000378:metabolism*; D006031:Glycosylation; D007739:L Cells; D051379:Mice; D009154:Mutation; D009693:Nucleic Acid Hybridization; D009844:Oligosaccharides / Q000378:metabolism; D010047:Ovalbumin / Q000235:genetics / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010957:Plasmids; D014162:Transfection; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D009844:Oligosaccharides; D005973:Glutamine; D001216:Asparagine; D010047:Ovalbumin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase