The S-layer glycoprotein of the crenarchaeote Sulfolobus acidocaldarius is glycosylated at multiple sites with chitobiose-linked N-glycans

Abstract

Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L(1004)-Q(1395). Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30-40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc(1)Man(2)GlcNAc(2) plus 6-sulfoquinovose (QuiS), consistent with the tribranched hexasaccharide previously reported in the cytochrome b(558/566) of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

Figure 1

(a) Structure of the 6-sulfoquinovose-containing tribranched hexasaccharide identified in cytochrome b_558/566 [7]. (b) This shows a symbolic representation of the glycan which is used in the annotations of Figures 4–6.

Figure 2

Polypeptide sequence of the S. acidocaldarius S-layer. The N-terminal signal sequence has been omitted. Consensus sequences for N-glycosylation are shaded and the predicted products of tryptic digestion are shown by underlining.

Figure 3

SDS PAGE gel of the S. acidocaldarius S-layer showing the band that was cut out for in-gel digestion.

Figure 4

Summed mass spectra recorded between 46 and 52 min from an on-line nanoLC-ES-MS/MS analysis of an in-gel tryptic digest of the S-layer. The quadruply charged signals at m/z 1014.07, 1111.59 and 1151.09 are molecular ions of glycopeptides of sequence GAGVVEFLLTAYPYTGNITFAPPWFIAENVVK carrying the glycans shown in the annotations. Unassigned signals are molecular ions of peptides from elsewhere in the S-layer.

Figure 5

MS/MS of m/z 1151.09 (a) and m/z 1014.07 (b) (see mass spectrum in Figure 4). Note the diagnostic fragment ions for HexNAc in both spectra and for HexNAc-QuiS in (a). The peptide sequence ions are identical in (a) and (b), and their assignments are shown in (c).

Figure 6

Summed mass spectra of T24 recorded between 48.5 and 50.6 min from an on-line nanoLC-ES-MS/MS analysis of an in-gel tryptic digest of the S-layer. The triply charged signals at m/z 1256.38, 1378.09, 1507.28, and 1561.45 are molecular ions of glycopeptides of sequence LLNLNVQQLNNSILSVTYHDYVTGETLTATTK carrying the glycans shown in the annotations. Unassigned signals are molecular ions of peptides from elsewhere in the S-layer.

Figure 7

Sequence of the C-terminal domain of the S-layer which was mapped in the glycoproteomics experiments. Consensus sites (in bold) which were shown to be glycosylated are underlined.