Increased transfer of oligosaccharide from oligosaccharide pyrophosphoryl dolichol to protein acceptors upon estrogen-induced chick oviduct differentiation.

B.N Singh,J.J Lucas

doi:10.1016/s0021-9258(18)43226-7

Abstract

In an earlier report (Lucas, J. J., and Levin, E. (1977 J. Biol. Chem. 252, 4330-4336), we showed that immature chick oviduct membranes could not transfer [14C]mannose from GDP-[14C]Man to endogenous oligosaccharide pyrophosphoryl dolichol or protein. Estrogen treatment of chicks however, stimulated [14C]mannose transfer to those acceptors. In order to determine whether the oligosaccharide transfer responsible for N-glycosylation of oviduct proteins is present in immature oviduct membranes exogenous carboxymethylated alpha-lactalbumin and radiolabeled oligosaccharide-lipid were added to oviduct membranes. Because purity of the oligosaccharide-lipid is essential for consistent transfer of oligosaccharide to carboxymethylated alpha-lactalbumin a high pressure liquid chromatography procedure has been developed for oligosaccharide-lipid purification. The data obtained indicate that immature chick oviduct membranes do have an enzyme capable of oligosaccharide transfer and that the activity is enhanced 3- to 4-fold by estrogen treatment of chicks. A Glc-oligosaccharide is transferred approximately 3-fold more effectively than the nonglycosylated Man-oligosaccharide by membrane preparations from immature chicks, estrogen-treated chicks, and mature hens.

Highlights

The involvement of dolichyl mono- and pyrophosphoryllinked saccharides in N-glycosylation of eukaryotic proteins is well established [1,2,3,4].The terminal reaction in the glycosylation of asparagine residues is the transfer of an oligosaccharide from oligosaccharide pyrophosphoryldolichol to aprotein acceptor
We have been concerned with regulation of dolichyl phosphate-mediated protein glycosylation during chick oviduct differentiation
Because of the assay employed in that study it was not possible to determine whetherthis intriguing result is due to induction of endogenous acceptor proteins, due to induction of oligosaccharide-lipid synthesis, or due to enhancement of oligosaccharide transferase activity

Summary

Introduction

The involvement of dolichyl mono- and pyrophosphoryllinked saccharides in N-glycosylation of eukaryotic proteins is well established [1,2,3,4].The terminal reaction in the glycosylation of asparagine residues is the transfer of an oligosaccharide from oligosaccharide pyrophosphoryldolichol to aprotein acceptor. By incubating oviduct membrane preparationswith CM-aLac’ and exogenous radioactive oligosaccharide-lipid the presence of the transferase in membranes prepared from immature chicks can be assessed. Samples were loaded on the column in Incubation mixtures contained 500 pgof hen oviduct membrane protein and varying amounts of ['4C]Man-oligosaccharide-lipid.

Results

Conclusion