Project description:Motility in the Archaea domain is facilitated by a unique motility structure termed the archaellum. N-glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. Here, we report the structure of the archaellin glycan from Methanothermococcus thermolithotrophicus, a methanogen which grows optimally at 65°C. Four archaellin genes (flaB1-4) have previously been identified. In gel digestion and LC-MS analysis revealed the identity of the upper band as FlaB1 and the lower band as FlaB3. Examination of the protein sequences for the four archaellins indicated multiple possible N-linked glycosylation sites in each. We observed using mass spectrometry that Mtc. thermolithotrophicus archaellins is posttranslationally modified at multiple sites with an N-linked branched oligosaccharide composed of 7 sugars (1414 Da). NMR analysis of the purified glycan determined the structure to be α-D-glycero-D-manno-Hep3OMe6OMe-(1-3)-[α-GalNAcA3OMe-(1-2)-]-β-Man-(1-4)-[-GalA3OMe4OAc6CMe-(1-4)--GalA-(1-2)-]-α-GalAN-(1-3)-β-GalNAc-Asn. A detailed investigation by HILIC-MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan suggesting a common N-glycosylation pathway.
