Site-specific glycosylation in animal cells. Substitution of glutamine for asparagine 293 in chicken ovalbumin does not allow glycosylation of asparagine 312.

B T Sheares

doi:10.1016/s0021-9258(18)37821-9

Abstract

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.

Highlights

It has been shown previously that chicken ovalbucmelilns is glycosylated at thecorrect site with large amounts of synthesized and secreted in a heterologoucsell system hybrid oligosaccharides,and that thitsype of sugar chain was is glycosylated at the correscite and that the oligosac-unusual and notdetectable on other glycoproteins secreted by charides at thastite, similar to the proteminade in hen mouse L cells
Mouse L cells transfected with the min gene (AAC) was changed to that for Gln (CAA), ovalbumin gene carrying this mutation synthesized and sethereby preventing glycosylation at amino acid 293. creted ovalbumin intothe tissueculture media
Four hundred pl of the cell study ( l ), we showed that ovalbumin secreted by mouse L suspension was inoculated into 10 ml of 2~ LB media containing 70 pg/ml kanamycin sulfate and allowed to shake overnight at 37 “C

Summary

Introduction

It has been shown previously that chicken ovalbucmelilns is glycosylated at thecorrect site with large amounts of synthesized and secreted in a heterologoucsell system hybrid oligosaccharides,and that thitsype of sugar chain was is glycosylated at the correscite and that the oligosac-unusual and notdetectable on other glycoproteins secreted by charides at thastite, similar to the proteminade in hen mouse L cells. Usin2g0-a chain has greater influence on proper site glycosylation and oligosaccharideprocessing of ovalbumin than thespecific cell type.

Results

Conclusion