For insight into the composition of the N- and O-linked glycoprotein of the S-layer of H. volcanii, a series of compositional analysis techniques were employed, where wild type samples for the WR536 S-layer N- and O-glycans decorating the S-layer protein were assessed for composition by GC-MS. The wild type WR536 sample as extracted and purified by the Eichler group (Section 2.3.1), was digested with pronase enzyme, an enzyme that hydrolyses the peptide backbone into short peptides of only a few amino acids (Section 2.4.3.4). This was done to improve solubility and allow for further steps of hydrolysis of the glycopeptides into monosaccharides for subsequent derivatisation by TMS or alditol acetate methods (Section 2.4.8.6 and Section 2.4.8.7).
TMS and alditol acetate derivatised samples were analysed by GC-MS and run alongside commercial standards using the retention times of elution on the chromatogram as well as the specific mass spectrometric fingerprints of each monosaccharide in order to determine the monosaccharide composition of the glycans present on the S-layer glycoprotein. Notably, hexuronic acid monosaccharides are less easily assessed using the alditol acetate procedure, because acidic sugars are not easily amenable to analysis by GC-MS without additional manipulation, as adequate chromatography cannot be easily achieved for compounds containing a carboxyl group. Considering that the N-glycan of the S-layer glycoprotein of H. volcanii contains hexuronic acids, the digested S-layer glycoprotein was therefore derivatised with a trimethylsilyl reagent prior to TMS analysis by GC-MS (Section 2.4.8.6).
The GC-MS chromatograms for TMS derivatives of the S-layer derived glycans plus standards are shown in Figure 3.2. Comparison of the two chromatograms as well as examination of the mass spectrometric fingerprints of each moiety eluted from the gas chromatography column allowed for full annotation of the chromatogram showing the monosaccharide composition of the S-layer glycans of H. volcanii.
Figure 3.2 – GC-MS chromatogram of TMS derivatised glycans of the H. volcanii S-layer glycoprotein. The glycan composition of the H. volcanii S-layer glycoprotein (top panel) was run alongside commercially obtained standards, at 50nM each (bottom panel), as run on a Perkin Elmer Clarus 500 GC-MS instrument fitted with a RTX-5MS column.
Both preparations were supplemented with 50nM of the internal standard inositol.
The N- and O-glycans of the S-layer glycoprotein, were found to contain mannose (elution time: 12.63, Figure 3.3), galactose (elution time: 13.41 and 13.96), glucose (elution time: 14.42), galacturonic acid (elution time: 14.78 and 14.88, Figure 3.4) and glucuronic acid (elution time: 15.05). The TMS chromatogram peaks and fingerprints were checked for the possible presence of naturally present sugar modifications, such as O-methylation that would be expected to be preserved during sugar release from the S-layer. No naturally occurring modification was observed in the TMS chromatogram.
Figure 3.3 – Mass spectrometric fingerprint of TMS derivatised mannose.
The fingerprint is from the GC-MS chromatogram of the TMS derivatised mannose moiety (top panel) and that of the commercially obtained standard for mannose (bottom panel), as run on a Perkin Elmer Clarus 500 GC-MS instrument fitted
with a RTX-5MS column. The fingerprint is magnified from m/z 231 onwards and is highlighted in red.
Figure 3.4 – Mass spectrometric fingerprint of TMS derivatised galacturonic acid.
The fingerprint is from the GC-MS chromatogram of the TMS derivatised galacturonic acid moiety (top panel) and that of the commercially obtained standard for galacturonic acid (bottom panel), as run on a Perkin Elmer Clarus 500 GC-MS instrument
fitted with a RTX-5MS column. The fingerprint is magnified from m/z 231 onwards and is highlighted in red.
3.2.1
Summary discussion of TMS compositional analysis
The O-glycan disaccharide of the S-layer glycoprotein was previously suggested to be composed of a glucose and galactose disaccharide (Sumper et al., 1990) and this is supported by their presence in the GC- MS chromatogram. However, the H. volcanii S-layer N-glycan also contains two hexose moieties, of which only the final hexose has been identified to be a mannose monosaccharide (Calo et al., 2011, Girrbach et al., 2000, Cohen-Rosenzweig et al., 2012) (Figure 3.2 and Figure 3.3) (Section 3.7.2). The hexose moiety linked to the asparagine residues in the S-layer glycoprotein has not been identified yet, and could therefore be considered to be any of the hexose moieties present in the TMS chromatogram. Furthermore the hexuronic acid moieties present in the N-glycan pentasaccharide of the H. volcanii S-layer glycoprotein were previously suggested to be consistent with glucuronic acid moieties, however, the presence of galacturonic acid moieties in the TMS chromatogram indicates that the N-glycan pentasaccharide contains both glucuronic and galacturonic acid moieties (Figure 3.2 and Figure 3.4) (Magidovich et al., 2010, Yurist- Doutsch et al., 2010).
Unfortunately, no suitable enzymatic or chemical hydrolysis method is available for the selective release of the N- and O-glycans from the S-layer. Therefore, the lack of such separation did not aid in the discrimination of the monosaccharide identifications in the N- and O-glycan moieties. Interestingly, however, the amount of glucuronic acid and galacturonic acid is not substantially less than the mannose, galactose and glucose. The uronic acids are confined to the N-glycans whilst the hexoses are in both the N- and O- glycans. Their comparable abundance suggests that the stoichiometry of the O-glycans is unlikely to be very different from the N-glycans. The proposal that the O-glycan disaccharides occupy multiple sites in the threonine-rich region towards the C-terminal of the H. volcanii S-layer (Mescher and Strominger, 1976, Sumper et al., 1990), therefore does not seem to be supported by the TMS data in Figure 3.2.