N-Linked Glycans Are Assembled on Highly Reduced Dolichol Phosphate Carriers in the Hyperthermophilic Archaea Pyrococcus furiosus.

Michelle M Chang,Ziqiang Guan,Jerry Eichler,Barbara Imperiali

doi:10.1371/journal.pone.0130482

Abstract

In all three domains of life, N-glycosylation begins with the assembly of glycans on phosphorylated polyisoprenoid carriers. Like eukaryotes, archaea also utilize phosphorylated dolichol for this role, yet whereas the assembled oligosaccharide is transferred to target proteins from dolichol pyrophosphate in eukaryotes, archaeal N-linked glycans characterized to date are derived from a dolichol monophosphate carrier, apart from a single example. In this study, glycan-charged dolichol phosphate from the hyperthermophile Pyrococcus furiosus was identified and structurally characterized. Normal and reverse phase liquid chromatography-electrospray ionization mass spectrometry revealed the existence of dolichol phosphate charged with the heptasaccharide recently described in in vitro studies of N-glycosylation on this species. As with other described archaeal dolichol phosphates, the α- and ω-terminal isoprene subunits of the P. furiosus lipid are saturated, in contrast to eukaryal phosphodolichols that present only a saturated α-position isoprene subunit. Interestingly, an additional 1-4 of the 12-14 isoprene subunits comprising P. furiosus dolichol phosphate are saturated, making this lipid not only the longest archaeal dolichol phosphate described to date but also the most highly saturated.

Highlights

The polyisoprenols are a family of hydrophobic polymers comprising up to more than 100 isoprene subunits bearing a terminal hydroxyl group at the α-terminus of the molecule [1,2,3]
In several archaea, including Hbt. salinarum [14] and Sulfolobus acidocaldarius [12], dolichol phosphate (DolP) is charged with the same glycans as those N-linked to glycoproteins in these organisms
The same profile included 13C1-containing isotopic [M-2H]2- peaks of m/z 914.551, 948.575 and 979.583, corresponding to C60, C65 and C70 DolP attached to the pentasaccharide precursor of the complete heptasaccharide, as well as [M-2H]2- peaks of m/z 980.585, 1014.089 and 1048.16, corresponding to C60, C65 and C70 DolP attached to the hexasaccharide precursor of the same heptasaccharide (Fig 1A, inset)

Summary

Introduction

The polyisoprenols are a family of hydrophobic polymers comprising up to more than 100 isoprene subunits bearing a terminal hydroxyl group at the α-terminus of the molecule [1,2,3]. The two main groups of polyisoprenoids, polyprenols and dolichols, can be distinguished by the presence of an unsaturated (polyprenols) or saturated (dolichols) α-isoprene subunit. While various biological roles have been suggested for the polyisoprenoid alcohols, these remain to be clearly delineated [1,2,3,4,5]. On the other hand, phosphorylated polyprenols and dolichols are well.

Methods

Results

Conclusion