Complementation of an aglB Mutant of Methanococcus maripaludis with Heterologous Oligosaccharyltransferases

Abstract

The oligosaccharyltransferase is the signature enzyme for N-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in Methanococcus maripaludis deleted for aglB, using archaellin FlaB2 as the reporter protein since all archaellins in Mc. maripaludis are modified at multiple sites by an N-linked tetrasaccharide and this modification is required for archaellation. In the Mc. maripaludis ΔaglB strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from Methanococcus voltae and Methanothermococcus thermolithotrophicus functionally replaced the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from Mc. voltae has a relaxed specificity for the linking sugar of the transferred glycan since while the N-linked glycan present in Mc. voltae is similar to that of Mc. maripaludis, the Mc. voltae glycan uses N-acetylglucosamine as the linking sugar. In Mc. maripaludis that role is held by N-acetylgalactosamine. This study also identifies aglB from Mtc. thermolithotrophicus for the first time by its activity. Attempts to use AglB from Methanocaldococcus jannaschii, Haloferax volcanii or Sulfolobus acidocaldarius to functionally replace the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain were unsuccessful.

Comparative Analysis of Surface Layer Glycoproteins and Genes Involved in Protein Glycosylation in the Genus Haloferax

“…AglB orthologues from different haloarchaea were shown to be able to functionally complement the H. volcanii enzyme and successfully transfer the tetrasaccharide precursor of the N -linked pentasaccharide decorating the H. volcanii S-layer glycoprotein from the dolichol phosphate carrier to target asparagine residues [ 59 ] and inter-species complementation was also successfully shown in methanogens [ 60 ]. This implies considerable substrate flexibility, which can theoretically allow an aglB homolog acquired by horizontal gene transfer (HGT) to replace an ancestral orthologue, in a process known as xenologous gene displacement [ 61 ]—the most frequent outcome of HGT in haloarchaea [ 51 ].…”