Complementation of an aglB Mutant of Methanococcus maripaludis with Heterologous Oligosaccharyltransferases

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 Δ aglB 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 Δ aglB 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 Δ aglB strain were unsuccessful.