Regulation of dolichyl phosphate-mediated protein glycosylation: Estrogen effects on glucosyl transfers in oviduct membranes

Abstract

Regulation of Glc transfer from UDP-Glc via Glc-P-Dolichol to form Glc 3-Man 9-oligosaccharide-lipid has been studied during estrogen-induced chick oviduct differentiation. The process was studied as two distinct reactions: transfer of Glc from UDP-Glc to Dol-P, forming Glc-P-Dol; and transfer of Glc from Glc-P-Dol to Man 9-OL (oligosaccharide-lipid), forming a series of glucosylated oligosaccharide-lipids. Kinetic analysis of [ 14C]Glc transfer from UDP-[ 14C]Glc to endogenous Dol-P shows that Dol-P is limiting in membrane preparations and that, concomitant with estrogen-induced differentiation, there is an increase in Dol-P available for Glc transfers. There is also greater glucosyl transferase activity present in membranes from mature hens and estrogenized chicks than in membranes from immature chicks. In order to study the second phase of glucosylation, transfer to the oligosaccharide, it was necessary to develop an assay in which membranes could be reacted with exogenously added substrates, [ 14C]Glc-P-Dol and [ 3H]Man 9-OL. This reaction is dependent on detergent (0.02% NP-40 was used) and is stimulated by EDTA. The apparent K m for Glc-P-Dol was about 1.5 μ m. A series of double-labeled oligosaccharides having sizes consistent with Glc 1-, Glc 2-, and Glc 3-Man 9-OL were formed. Chemical and enzymatic analysis of [ 14C]Glc oligosaccharides formed by incubation with the exogenous substrates, or by incubation with UDP-[ 14C]Glc and endogenous acceptors, indicated that the glucosylated oligosaccharides were similar. Assays of membranes from estrogenized chicks, mature hens, and hormone-withdrawn chicks showed increased glucosyl transferase activity upon hormone treatment. Similar assays in the absence of exogenous Man 9-OL indicated that hormone treatment was also accompanied by increased levels of endogenous oligosaccharide-lipid acceptors.