Evolutionary considerations of the oligosaccharyltransferase AglB and other aspects of N-glycosylation across Archaea.

Various biological markers in members of the TACK and Asgard archaeal super-phyla show Eukarya-like traits. These include the oligosaccharyltransferase, responsible for transferring glycans from the lipid carrier upon which they are assembled onto selected asparagine residues of target proteins during N-glycosylation. In Archaea, oligosaccharyltransferase activity is catalyzed by AglB. To gain deeper insight into AglB and N-glycosylation across archaeal phylogeny, bioinformatics approaches were employed to address variability in AglB sequence motifs involved in enzyme activity, construct a phylogenetic tree based on AglB sequences, search for archaeal homologues of non-catalytic subunits of the multimeric eukaryal oligosaccharyltransferase complex and predict the presence of aglB-based clusters of glycosylation-related genes in the Euryarchaeota and the DPANN, TACK and Asgard super-phyla. In addition, site-directed mutagenesis and mass spectrometry were employed to study the natural variability in the WWDXG motif central to oligosaccharyltransferase activity seen in archaeal AglB. The results clearly distinguish AglB from members of the DPANN super-phylum and the Euryarchaeota from the same enzyme in members of the TACK and Asgard super-phyla, which showed considerable similarity to its eukaryal homologue Stt3. The results thus support the evolutionary proximity of Eukarya and the TACK and Asgard archaea.