Estrogen influences dolichyl phosphate distribution among glycolipid pools in mouse uteri

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Abstract
The steroid hormone 17.beta.-estradiol dramatically induces uterine N-linked glycoprotein assembly [Dutt, A., Tang, J.-P., Welply, J. K., and Carson, D. D. (1986) Endocrinology (Baltimore) 118, 661-673]. To determine the role that dolichyl phosphate availability plays in this induction, we studied the effects of estrogen priming on the content of dolichyl phosphate and the distribution of dolichyl phosphate among various glycolipids in uteri. Dolichol-linked saccharides were metabolically labeled to equilibrium with either [3H]glucosamine or [3H]mannose and extracted from primary explants of uterine tissue. The amount of dolichol-linked saccharide was calculated from the specific radioactivity determined for the corresponding sugar nucleotides extracted from the tissues. The major dolichol-linked saccharides identified were mannosylphosphoryldolichol (MPD), oligosaccharylpyrophosphoryldolichol (OSL), and N,N''-diacetylchitobiosylpyrophosphoryldolichol (CBL). Estrogen increased the levels of MPD and OSL 4-fold; however, CBL levels did not change. After 3 days of treatment, the levels of these glycolipids were very similar to those in uteri from pregnant mice. Remarkably, MPD constituted 90-95% of dolichol-linked saccharides detected under all conditions. The tissue contents of total dolichyl phosphate and alkali-labile dolichyl phosphate, presumably MPD, were estimated by liquid chromatography. The levels of alkali-labile dolichyl phosphate determined in this way were in good agreement with the values estimated for MPD by metabolic labeling; moreover, alkali-labile dolichyl phosphate constituted 50-98% of the total dolichyl phosphate pool. The variations in MPD content depended upon the steroid hormone influence, most notably that of estrogen. Owing to our inability to metabolically label glucosylphosphoryldolichol (GPD) effectively, it was not possible to determine the tissue levels of GPD; however, conditions were optimized for the in vitro assay of GPD synthase in crude microsomal fractions. The specific activity of GPD synthase was similar under all conditions studied. Consequently, it was concluded that fluctuation in GPD synthase activity was an unlikely mode of regulation of oligosaccharide assembly or glycoprotein synthesis. These studies provide the first determination of the levels of dolichol-linked saccharides in tissues and how these levels change during hormonal induction of glycoproteon assembly. Coupled with our earlier studies, the present work demonstrates that among a number of key points of N-linked oligosaccharide assembly and transfer only synthesis of MPD increases coordinately with the increases observed in lipid- and protein-linked oligosaccharide assembly that occurs in vivo in response to estrogen. We suggest that control of MPD levels is an important regulatory aspect of N-linked glycoprotein assembly in this system.