N-glycosylation in the thermoacidophilic archaeon Sulfolobus acidocaldarius involves a short dolichol pyrophosphate carrier

Abstract

N-glycosylation is a post-translational modification that occurs across evolution. In the thermoacidophilic archaea Sulfolobus acidocaldarius, glycoproteins are modified by an N-linked tribranched hexasaccharide reminiscent of the N-glycans assembled in Eukarya. Previously, hexose-bearing dolichol phosphate was detected in a S. acidocaldarius Bligh-Dyer lipid extract. Here, we used a specialized protocol for extracting lipid-linked oligosaccharides to detect a dolichol pyrophosphate bearing the intact hexasaccharide, as well as its biosynthetic intermediates. Furthermore, evidence for N-glycosylation of two S. acidocaldarius proteins by the same hexasaccharide and its derivatives was collected. These findings thus provide novel insight into archaeal N-glycosylation.

Keywords: N-glycosylation; Sulfolobus acidocaldarius; archaea; dolichol; thermoacidophile.

Fig 1

Glycan-charged phosphorylated dolichol species in S. acidocaldarius. LC-ESI MS analysis of a S. acidocaldarius lipid extract revealed the presence of (A) C₄₅ DolP modified by a hexose and (B) C₄₅ DolPP modified by a hexasaccharide comprising N-acetylhexosamine₂-hexose₃-sulfated quinovose (QuiS). C. MS/MS spectrum of [M-2H]²⁻ at m/z 958.4. D. Fragmentation scheme for the observed product ions in C. The depicted DolPP-linked glycan structure is based on the previously reported protein N-linked glycan [24,25]. The arrows indicating x20 reflect magnification of the ion peaks in the corresponding region of the m/z values on the graph. Where the position of a double bond is only speculated, a dotted line is drawn. Diamonds represent N-acetylhexosamines, full circles represent hexoses, open circles represent quinovose and S represents sulfation.

Fig 2

Glycan-charged S. acidocaldarius DolPP species. (A) C₄₅ DolP modified by a pentasaccharide comprising N-acetylhexosamine₂-hexose₂-sulfated quinovose. (B) C₄₅ DolP modified by a tetrasaccharide comprising N-acetylhexosamine₂-hexose-sulfated quinovose. Diamonds represent N-acetylhexosamines, full circles represent hexoses, open circles represent quinovose and S represents sulfation.

Fig 3

N-glycosylation of S. acidocaldarius SlaB. A. LC-ESI MS analysis of a tryptic fragment of SlaB that includes Asn-278 revealed the peptide (inset) and its modification by a hexasaccharide comprising N-acetylhexosamine₂-hexose₃-sulfated quinovose. [M+2H]²⁺ ion peaks are presented. B. MS/MS profile of the [M+2H]²⁺ base peak of the peptide observed at m/z 1621.72. The inset schematically represents the fragmentation scheme. The N represents Asn-278, diamonds represent N-acetylhexosamines, full circles represent hexoses, open circles represent quinovose and S represents sulfation. The arrows indicating x10 reflect magnification of the ion peaks in the corresponding region of the m/z values on the graph.

Fig 4

N-glycosylation of S. acidocaldarius FlaB. A. LC-ESI MS analysis of a tryptic fragment of FlaB that includes Asn-195 revealed the peptide (inset) and its modification by a hexasaccharide comprising N-acetylhexosamine₂-hexose₃-sulfated quinovose. [M+3H]³⁺ ion peaks are presented. B. MS/MS profile of the ¹³C₁-containing isotopic [M+2H]²⁺ peak at m/z 1890.291. The inset schematically represents the fragmentation scheme. The N represents Asn-195, diamonds represent N-acetylhexosamines, full circles represent hexoses, open circles represent quinovose and S represents sulfation. The arrows indicating x5 and x10 reflect magnification of the ion peaks in the corresponding region of the m/z values on the graph.