Distinct Regions of the Haloferax volcanii Dolichol Phosphate-Mannose Synthase AgID Mediate the Assembly and Subsequent Processing of the Lipid-Linked Mannose

Haloferax volcanii AgID is currently the only archaeal dolichol phosphate (DolP)-mannose synthase shown to participate in N-glycosylation. However, the relation between AgID and Pyrococcus furiosus PF0058, the only archaeal DoIP-mannose synthase for which structural information is presently available, was unclear. In this report, similarities between the PF0058 and AgID catalytic domains were revealed. At the same time, AgID includes a transmembrane domain far longer than that of PF0058 or other DolP-mannose syntheses. To determine whether this extension affords AgID functions in addition to generating mannose-charged DolP, a series of volcanii strains expressing truncated versions of AgID was generated. Mass spectrometry revealed that a version of AgID comprising the catalytic domain and only two of the six to nine predicted membrane-spanning domains could mediate mannose addition to DolP. However, in cells expressing this or other truncated versions of AgID, mannose was not transferred from the lipid to the protein-bound tetrasaccharide precursor of the N-linked pentasaccharide normally decorating Hfx. volcanii glycoproteins. These results thus point to AgID as contributing to additional aspects of Hfx. volcanii N-glycosylation beyond charging DolP with mannose. Accordingly, the possibility that AgID, possibly in coordination with AgIR, translocates DolP-mannose across the plasma membrane is discussed. IMPORTANCE In the archaeon Haloferax volcanii, the dolichol phosphate (DolP)mannose synthase AgID charges the lipid DolP with mannose, which is delivered to a protein-bound tetrasaccharide to generate the pentasaccharide decorating glycoproteins in this organism. Structural studies demonstrated the similarity of AgID to Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase with a solved three-dimensional structure. Truncated AgID containing the catalytic domain and only two of the predicted six to nine membrane-spanning regions catalyzed mannose-charging of DolP. Yet, no mannose was delivered to protein-linked tetrasaccharide in cells expressing AgID mutants including only up to five membrane-spanning regions, pointing to a role for the extended C-terminal region in a subsequent step of Hfx. volcanii N-glycosylation, such as DolP-mannose translocation across the plasma membrane.

Automated summary

The archaeon Haloferax volcanii relies on the dolichol phosphate (DolP)mannose synthase AgID to charge the lipid DolP with mannose for glycoprotein synthesis. Structural similarities between AgID and Pyrococcus furiosus PF0058 were revealed, and truncated versions of AgID were generated to study its functional domains. AgID mutants lacking certain membrane-spanning regions still catalyzed mannose-charging of DolP, but failed to deliver mannose to protein-linked tetrasaccharide, suggesting additional functions of AgID in N-glycosylation.