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

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 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 which grows optimally at 65°C. SDS-PAGE and mass spectrometry 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 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. The Mtc. 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.

New insights into the architecture and dynamics of archaella

“…Glycans were modelled in Coot using the sugar unit identity and connectivity reported for the glycan structure of M. thermolithotrophicus. This structure was recently reported as α- 31 . Restraints dictionaries for non-standard sugars were prepared using JLIGAND 55 .…”

“…Since no information on sugar connectivity and identity is available for N-glycans of M. villosus, we modelled the branched heptameric glycan from the related archaeal species Methanocaldococcus thermolithotrophicus 31 in order to aid model building and refinement.…”

“…Archaellins that are thought to form the bulk of the filament are referred to as major. Biochemical and genetic experiments suggest that in Methanococcus voltae 29 , Methanococcus maripaludis 30 , Methanothermococcus thermolithotrophicus 31 , Halobacterium salinarum 32 , and Halorubrum lacusprofundi DL18 33 the archaellum filament consists of two major archaellins, hypothesised to be equally distributed along the filament. Nevertheless, the three high-resolution structures of archaella that are available to date show homopolymeric filaments consisting of only single (major) archaellin 21,34,35 .…”

Comprehensive glycoproteomics shines new light on the complexity and extent of glycosylation in archaea

“…In archaea, glycosylation of SLG, type IV pili, and archaella has been confirmed for various species, including non-haloarchaea like Methanococcus maripaludis and Sulfolobus acidocaldarius [14][15][16][17]. Efforts to identify additional archaeal glycoproteins are limited to the characterization of purified proteins [18], the lectin affinity-based enrichment of glycoproteins [19], and periodic acid-Schiff staining of proteins (as summarized in the ProGlycProt database [11]).…”

Glycoproteomics ofHaloferax volcaniireveals an extensive glycoproteome and concurrence of differentN-glycosylation pathways

“…In archaea, glycosylation of SLG, type IV pili and archaella has been confirmed for various species, including non-haloarchaea like Methanococcus maripaludis and Sulfolobus acidocaldarius (Guan et al, 2016;Jarrell et al, 2014;Kelly et al, 2020;Meyer and Albers, 2013). Efforts to identify additional archaeal glycoproteins are limited to the characterization of purified proteins (Vinogradov et al, 2012), the lectin affinity based enrichment of glycoproteins (Palmieri et al, 2013) and periodic acid-Schiff staining of proteins (as summarized in the ProGlycProt database (Choudhary et al, 2019)).…”