Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation

Abstract

The glycosylation of asparagine residues is the predominant protein modification in all three domains of life. An oligosaccharide chain is preassembled on a lipid-phospho carrier and transferred onto asparagine residues by the action of a membrane-bound enzyme, oligosaccharyltransferase. The oligosaccharide donor for the oligosaccharyl transfer reaction is dolichol-diphosphate-oligosaccharide in Eukaryota and polyprenol-diphosphate-oligosaccharide in Eubacteria. The donor in some archaeal species was reportedly dolichol-monophosphate-oligosaccharide. Thus, the difference in the number of phosphate groups aroused interest in whether the use of the dolichol-monophosphate type donors is widespread in the domain Archaea. Currently, all of the archaeal species with identified oligosaccharide donors have belonged to the phylum Euryarchaeota. Here, we analyzed the donor structures of two species belonging to the phylum Crenarchaeota, Pyrobaculum calidifontis and Sulfolobus solfataricus, in addition to two species from the Euryarchaeota, Pyrococcus furiosus and Archaeoglobus fulgidus The electrospray ionization tandem mass spectrometry analyses confirmed that the two euryarchaeal oligosaccharide donors were the dolichol-monophosphate type and newly revealed that the two crenarchaeal oligosaccharide donors were the dolichol-diphosphate type. This novel finding is consistent with the hypothesis that the ancestor of Eukaryota is rooted within the TACK (Thaum-, Aig-, Cren-, and Korarchaeota) superphylum, which includes Crenarchaea. Our comprehensive study also revealed that one archaeal species could contain two distinct oligosaccharide donors for the oligosaccharyl transfer reaction. The A. fulgidus cells contained two oligosaccharide donors bearing oligosaccharide moieties with different backbone structures, and the S. solfataricus cells contained two oligosaccharide donors bearing stereochemically different dolichol chains.

Keywords: AglB; N-linked glycosylation; archaea; crenarchaeota; dolichol; euryarchaeota; evolution; lipid-linked oligosaccharide; mass spectrometry (MS); oligosaccharyltransferase.

FIGURE 1.

ESI-MS/MS analysis of the glycopeptide produced in the P. calidifontis membrane fractions. The Triton X-100-solubilized membrane fractions of the P. calidifontis cells were incubated with an acceptor peptide, Ac-AAYNVTKRK(TAMRA)-OH. The purified glycopeptide product was analyzed by direct infusion ESI-MS/MS, in the positive ion mode. The triply charged monoisotopic precursor ion is marked by the vertical arrow. The inset shows the predicted chemical structure of the glycopeptide and the MS/MS fragmentation scheme. The branching structure of the N-glycan was determined by NMR analysis (unpublished data). The expected m/z values were observed within 0.02 of the theoretical mass values for triply charged fragment ions.

FIGURE 2.

Normal phase LC-ESI-MS analyses of LLOs from four archaeal species. The total lipid extracts of P. furiosus (A), A. fulgidus (B), P. calidifontis (C), and S. solfataricus (D) were analyzed. The TIC of the ESI-MS analyses, and the TOF-MS spectra averaged from the mass scans acquired during the time period indicated by the horizontal thick bars in the TICs are shown. The symbol S in the TICs denotes the number of monosaccharide residues. The fluorescence images of the SDS-PAGE gels in the TICs are the results of the oligosaccharyl transfer assay. The bands in the gel images are the glycopeptides produced, and their intensities are proportional to the amounts of LLO in the NPLC fractions. Note that a contrast-enhanced fluorescence image of the SDS-PAGE gel is provided in D to shown the faint glycopeptide band corresponding to the S6-B peak.

FIGURE 3.

Expansion of the selected TOF-MS ion peaks and comparison with simulated MS spectra. Shown are the raw and simulated ion peaks of P. furiosus (A), A. fulgidus (B), P. calidifontis (C), and S. solfataricus (D). The most intense ion peaks in the TOF-MS spectra in Fig. 2 were selected, and the expansions of the ion peaks are displayed in the left column. The chemical structure of the ion peaks is represented as Si:Pj:Ck:σl, where i is the number of sugar residues, j is the number of phosphate groups, k is the number of carbon atoms of the dolichol moiety, and l is the number of saturated isoprene units. The ions used in the MS/MS analyses (Fig. 4) are indicated by triangles with their m/z values. The simulated MS spectra are shown in the right column, to account for the mixed states of the dolichol species with different degrees of isoprene saturation. The R² values of the peak intensity correlation between the raw and simulated mass spectra were calculated. Note that the saturation number includes the saturation of the double bond in the α-isoprene unit, which is the signature for the definition of dolichol.

FIGURE 4.

ESI-MS/MS spectra of the oligosaccharide-charged Dol-P and Dol-PP molecules. The NPLC fractions containing the LLOs from P. furiosus (A), A. fulgidus (B), P. calidifontis (C), and S. solfataricus (D) were collected and analyzed by direct infusion ESI-MS/MS in the negative ion mode. The precursor ions are marked by the vertical arrows. The insets show the predicted chemical structures of the oligosaccharide-charged Dol-P and Dol-PP molecules and their MS/MS fragmentation schemes. The expected m/z values were observed within 0.05 of the theoretical mass values.

FIGURE 5.

Analysis of the lipid-phosphate products by normal phase LC and ESI-MS/MS. A–C, the XICs of the NPLC, the ESI-MS/MS spectra, and the fragmentation schemes of the [M-H]⁻ ions of the Dol-P (P1:C-60:σ4) generated by the A. fulgidus AglB-L (A), the Dol-P (P1:C-50:σ5) generated by the P. calidifontis membrane fractions (B), and the Dol-P (P1:C-45:σ5) generated by the S. solfataricus membrane fractions (C). The fragmentation patterns in the MS/MS spectra indicated that the lipid part is dolichol.

FIGURE 6.

Normal phase LC XIC profiles for the estimation of the compositions of the LLO species. The original data are shown in Fig. 2. Ions with different numbers of monosaccharide residues (S-5–S-11) or different numbers of carbons in the dolichol moiety (C-30–C-65) were selected to show the XIC profiles of the LLO species from P. furiosus (A), A. fulgidus (B), P. calidifontis (C), and S. solfataricus (D). The peak intensities were used to estimate the compositions of the LLO species in terms of the number of monosaccharide residues and the length of the dolichol chain.

FIGURE 7.

Chemical structures of the archaeal LLOs that serve as oligosaccharide donors for the oligosaccharyl transfer reaction. The compositions of the number of sugar residues (S), the number of carbon atoms in dolichol (C), and the number of isoprene saturations (σ) are shown in pie charts. Note that the stereochemical features drawn in this figure are not entirely correct. First, the trans-cis arrangement of the double bonds in the dolichol chain was assumed to be α-polycis-trans₂-ω for euryarchaeal species and α-polycis-trans₃-ω for crenarchaeal species (18, 46). Second, the positions of the saturated isoprene units were assumed to be located from the ω-terminus, except for the α-isoprene unit. Finally, an unidentified atypical stereochemical feature should exist in the dolichol chain of the S. solfataricus LLO-B.

FIGURE 8.

Evolution of LLOs that serve as oligosaccharide donors for the N-oligosaccharyl transfer reaction. The size of the colored circles is proportional to the structural diversity of the oligosaccharide parts in LLOs. The small colored circle in the large white circle indicates that not all Eubacteria have an N-glycosylation system. Pren, polyprenol; Dol, dolichol; P, monophosphate; PP, diphosphate; OS, oligosaccharide.