Distinct glycan-charged phosphodolichol carriers are required for the assembly of the pentasaccharide N-linked to the Haloferax volcanii S-layer glycoprotein

Abstract

In Archaea, dolichol phosphates have been implicated as glycan carriers in the N-glycosylation pathway, much like their eukaryal counterparts. To clarify this relation, highly sensitive liquid chromatography/mass spectrometry was employed to detect and characterize glycan-charged phosphodolichols in the haloarchaeon Haloferax volcanii. It is reported that Hfx. volcanii contains a series of C(55) and C(60) dolichol phosphates presenting saturated isoprene subunits at the α and ω positions and sequentially modified with the first, second, third and methylated fourth sugar subunits comprising the first four subunits of the pentasaccharide N-linked to the S-layer glycoprotein, a reporter of N-glycosylation. Moreover, when this glycan-charged phosphodolichol pool was examined in cells deleted of agl genes encoding glycosyltransferases participating in N-glycosylation and previously assigned roles in adding pentasaccharide residues one to four, the composition of the lipid-linked glycans was perturbed in the identical manner as was S-layer glycoprotein N-glycosylation in these mutants. In contrast, the fifth sugar of the pentasaccharide, identified as mannose in this study, is added to a distinct dolichol phosphate carrier. This represents the first evidence that in Archaea, as in Eukarya, the oligosaccharides N-linked to glycoproteins are sequentially assembled from glycans originating from distinct phosphodolichol carriers.

Fig 1

Normal phase LC/MS identification of dolichol phosphate from the total lipid extract of Hfx. volcanii. A. Total ion chromatogram of the NP-LC/MS analysis in the negative ion mode. B. The [M-H]⁻ ions of C₅₅ and C₆₀ dolichol phosphate detected at m/z 849.695 and 917.757, indicated by C₅₅ and C₆₀, respectively. The mass spectrum shown is averaged from spectra acquired during the 20–21 min window, indicated by the shaded area in A. C. MS/MS of the [M-H]⁻ ion of C₆₀ dolichol phosphate. The inset shows the predicted chemical structure of dolichol phosphate (according to Kuntz et al., 1997) and the MS/MS fragmentation scheme.

Fig 2

Normal phase LC/MS/MS identification of mono-, di-, tri-, and tetrasacchride-charged dolichol phosphate from the total Hfx. volcanii lipid extract. The left panels show the [M-H]⁻ ions of (A) hexose-modified, (B) hexuronic acid-hexose-modified and (C) dihexuronic-hexose-modified C₅₅ and C₆₀ phosphodolichol. The left panel of (D) shows the doubly charged [M-2H]^2- ions of methyl ester of hexuronic acid-dihexuronic acid-hexose-modified C₅₅ and C₆₀ phosphodolichol, detected at m/z 766.43 and 810.46, respectively. RT refers to retention time. The right panels show the MS/MS spectra of the [M-H]⁻ ion of (A) hexose-modified, (B) hexuronic acid-hexose-modified and (C) dihexuronic acid-hexose-modified C₆₀ phosphodolichol. The right panel of (D) shows the MS/MS spectrum of doubly charged [M-2H]^2- ions of methyl ester of hexuronic acid-dihexuronic acid-hexose-modified C₆₀ phosphodolichol. The inset in each right panel shows the chemical structure of the glycan-charged C₆₀ phosphodolichol and the MS/MS fragmentation scheme of the [M-H]⁻ ion (or the [M-2H]^2- ions in (D)). The arrows indicating ×20 and ×50 reflect magnification of the ion peaks in the corresponding region of the m/z values on the graph.

Fig 3

LC/MS profiling of glycan-charged phosphodolichols in the parent and agl mutant strains. The presence or absence of mono-, di-, tri- and tetra-sacchride-modified phosphodolichols in each strain was revealed by generating extracted ion chromatograms. The [M-H]⁻ ion at m/z 1079.8, the [M-2H]^2- ion at m/z 627.4, the [M-3H]^3- ion at m/z 476.6, and the [M-3H]^3- ion at m/z 540.0 were selected for monitoring the mono-, di-, tri-, and tetracchride-modified phosphodolichols. Each of these 4 ions represents the highest-abundance charge state observed by ESI/MS of the individual glycan-modified C₆₀ phosphodolichol species. Above each peak, schematic representation of the linked glycan is shown. The full circles correspond to hexose, the full squares correspond to hexuronic acid and the open square corresponds to the methyl ester of hexuronic acid. Note that the monosaccharide-modified phosphodolichol pool comprises several species.

Fig 4

The absence of AglD eliminates mannose-modified phosphodolichol. Normal phase LC extracted ion chromatograms (EIC) of the dolichylphosphate-hexose [M-H]⁻ ion at m/z 1079.8 from the parent strain (upper panel) and the ΔaglD (middle panel) are shown. The peaks at different retention times reflect the existence of three different dolichylphosphate-hexose species (Kaminski et al., 2010). The 16.92 min peak is eliminated in the mutant, as compared with the parent strain, suggesting AglD to be specific for the formation of the third monosaccharide-modified phosphodolichol species. The monosaccharide-modified phosphodolichol peak affected by the absence of AglD is retained at the position of a mannose-modified phosphodolichol standard (16.99 min; lower panel). The identities of the two other monosaccharide-modified phosphodolichols with retention times of 14.77 and 16.06 min, respectively, remain to be determined. While the results shown address C₅₅ dolichol phosphate, similar results were obtained with C₆₀ dolichol phosphate (not shown).

Fig 5

The working model of the Hfx. volcanii N-glycosylation pathway. Select Asn residues of the Hfx. volcanii S-layer glycoprotein are modified by a pentasaccharide comprising a hexose, 2 hexuronic acids, a methyl ester of hexuronic acid and a terminal mannose residue. Based on the findings of the present study and earlier reports (16,17,23–25,33), a working model of the Hfx. volcanii N-glycosylation pathway is provided. AglJ, AglG, AglI, AglE and AglD are assigned roles in either modifying dolichol phosphates or adding sugars to dolichol phosphate-bound sugars. AglB serves as the oligosaccharyltransferase, while AglF, AglP and AglM serve various sugar processing roles. At present, the flippase(s) responsible for delivering the lipid-charged glycans across the plasma membrane remain to be defined and are indicated by question marks. dolP, dolichol phosphate; NDP, nucleoside diphosphate.