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Identification of a novel N-linked glycan on the archaellins and S-layer protein of the thermophilic methanogen, Methanothermococcus thermolithotrophicus

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DOIResolve DOI: https://doi.org/10.1074/jbc.RA120.012790
AuthorSearch for: 1; Search for: 1ORCID identifier: https://orcid.org/0000-0002-5364-1376; Search for: 1; Search for: 1; Search for: 1; Search for: ORCID identifier: https://orcid.org/0000-0001-8512-0180; Search for: ORCID identifier: https://orcid.org/0000-0001-9263-7604; Search for:
Affiliation
  1. National Research Council Canada. Human Health Therapeutics
FunderSearch for: Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada
FormatText, Article
Subjectglycan; post-translational modification; archaellin; S-layer protein; archaeaglycobiology; glycoproteinmass spectrometry (MS); nuclear magnetic resonance (NMR); thermophile
Abstract
Motility in archaea is facilitated by a unique structure termed the archaellum. N-Glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. The identity of some of these N-glycans has been determined, but archaea exhibit extensive variation in their glycans, meaning that further investigations can shed light not only on the specific details of archaellin structure and function, but also on archaeal glycobiology in general. Here we describe the structural characterization of the N-linked glycan modifications on the archaellins and S-layer protein of Methanothermococcus thermolithotrophicus, a methanogen that grows optimally at 65 °C. SDS-PAGE and MS analysis revealed that the sheared archaella are composed principally of two of the four predicted archaellins, FlaB1 and FlaB3, which are modified with a branched, heptameric glycan at all N-linked sequons except for the site closest to the N termini of both proteins. 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)-[β-GalA3OMe4OAc6CMe-(1–4)-α-GalA-(1–2)-]-α-GalAN-(1–3)-β-GalNAc-Asn. A detailed investigation by hydrophilic interaction liquid ion chromatography–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. The M. thermolithotrophicus archaellin N-linked glycan is larger and more complex than those previously identified on the archaellins of related mesophilic methanogens, Methanococcus voltae and Methanococcus maripaludis. This could indicate that the nature of the glycan modification may have a role to play in maintaining stability at elevated temperatures.
Publication date
PublisherElsevier
American Society for Biochemistry and Molecular Biology
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  • © 2020 American Society for Biochemistry and Molecular Biology
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  • Journal of Biological Chemistry 295, no. 43 (23 October 2020): 14618–14629.
LanguageEnglish
Peer reviewedYes
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Record identifiere1a6e027-a0a8-480f-b9de-d2dd948a95c6
Record created2022-03-28
Record modified2026-03-18

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