Exploring protein N-glycosylation in ammonia-oxidizing Nitrososphaerota archaea through glycoproteomic analysis

Abstract

Ammonia-oxidizing archaea of the phylum Nitrososphaerota, formerly known as Thaumarchaeota, are globally distributed and play critical roles in the nitrogen and carbon cycles, particularly in environments with low ammonia concentrations. Like most archaea, Nitrososphaerota cells are enveloped by S-layer proteins, implicated in concentrating ammonium ions. These proteins are typically modified post-translationally by N-glycans, which often play significant roles in various biological processes, including protein function regulation, protection from phages, and environmental adaptation. Nevertheless, the glycobiological characteristics of Nitrososphaerota remain largely unexplored. Here, we investigated the glycoproteome of ammonia-oxidizing Nitrososphaerota, specifically focusing on the terrestrial Nitrososphaera viennensis and the marine Nitrosopumilus piranensis. Both species exhibited similar protein arrays throughout their growth phases, including those associated with N-glycosylation. Ns. viennensis consistently exhibited N-glycosylation predominantly on an S-layer protein and multicopper oxidase domain-containing proteins throughout all growth phases, with a marked increase during and after the late exponential phase. The glycan, characterized as a novel hexasaccharide with a chitobiose core, is hypothesized to play a role in nitrogen storage due to its probable nitrogen-rich composition, modifying asparagine residues within the conserved triplet sequence (Asn-X-Ser or -Thr). In contrast, Np. piranensis also showed a high abundance of S-layer protein but displayed no apparent N-glycosylation on any protein, suggesting variability in cell surface physical properties between these archaea. Despite similarities in their proteomes and energy metabolism, these two archaea exhibited significant differences in post-translational modification of proteins, revealing previously unrecognized diversity that may have implications for understanding their adaptive transitions to diverse environments.

Importance: Autotrophic ammonia-oxidizing archaea of the phylum Nitrososphaerota, formerly known as Thaumarchaeota, are notoriously difficult to culture yet play important roles in the global nitrogen and carbon cycles. Inhabiting environments with extremely low ammonia concentrations, these archaea are expected to conserve ammonia strictly for energy production. However, using advanced liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance techniques, we discovered that one of these archaea decorates its cell surface proteins with the most nitrogen-rich glycan identified to date, suggesting a previously unrecognized function of protein glycosylation in nitrogen storage. This newly identified N-glycan, with a chitobiose core similar to those in Thermoproteota and eukaryotes, not only deepens our understanding of archaeal evolution but also underscores the molecular adaptations enabling these archaea to thrive in diverse environments.

Keywords: Nitrososphaerota; S-layer; Thaumarchaeota; ammonia-oxidizing archaea; glycan.

Fig 1

Modification mass distribution of glycopeptides in 0.1 Da increments, showing detected peptide-spectrum matches (PSMs) with modifications of m/z values ranging from 300 to 2,000 Da, derived from open searching data for Ns. viennensis (a) and Np. piranensis (b). Predominant m/z values are shown.

Fig 2

Heat maps visualizing the distribution of peptide-spectrum matches (PSMs) or glycopeptide spectral matches within the S-layer proteins of Ns. viennensis (a) and Np. piranensis (b). The numbers on the X-axis indicate amino acid positions starting from the N-terminus. The color intensity represents the frequency of peptide detection: an increased red intensity indicates higher PSM counts, on a scale from 0 to 800. Glycosylation sites where glycan modification was confirmed are highlighted in red, and those without confirmed glycosylation are shown in blue.

Fig 3

Electron-transfer/higher-energy collision dissociation MS/MS spectrum showing the N-glycan structure on the S-layer of Ns. viennensis. The spectrum displays the fragmentation pattern of the glycopeptide, including specific mass shifts corresponding to glycan or peptide constituents. The inset provides a diagrammatic representation of the glycopeptide, indicating probable glycan constituents attached at a specific asparagine residue.

Fig 4

Structural diversity of N-linked glycans in archaea, showing examples from Pyrodictium abyssi (54), Sulfolobus acidocaldarius (52), Metallosphaera hakonensis (56), Pyrobaculum calidifontis (51), Nanobdella aerobiophila (56), and Microcaldus variisymbioticus (56).

Fig 5

NMR spectra of the glycopeptide from Ns. viennensis. (a) The anomeric region of ¹H-¹³C HSQC and (b) ¹H-¹³C heteronuclear multiple bond correlation (black) and ¹H-¹³C HSQC (red) spectra showing intraresidue scalar connectivities of the 287.11 Da units.