Haloferax volcanii AglD is currently the only archaeal dolichol phosphate (DolP)-mannose synthase shown to participate in N-glycosylation. However, the relation between AglD and Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase for which structural information is presently available, was unclear. In this report, similarities between the PF0058 and AglD catalytic domains were revealed. At the same time, AglD includes a transmembrane domain far longer than that of PF0058 or other DolP-mannose synthases. To determine whether this extension affords AglD functions in addition to generating mannose-charged DolP, a series of Hfx. volcanii strains expressing truncated versions of AglD was generated. Mass spectrometry revealed that a version of AglD 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 AglD, 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 AglD as contributing to additional aspects of Hfx. volcanii N-glycosylation beyond charging DolP with mannose. Accordingly, the possibility that AglD, possibly in coordination with AglR, translocates DolP-mannose across the plasma membrane is discussed. IMPORTANCE In the archaeon Haloferax volcanii, the dolichol phosphate (DolP)mannose synthase AglD 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 AglD to Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase with a solved three-dimensional structure. Truncated AglD 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 AglD 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.
- Dolichol phosphate-mannose synthase
- Haloferax volcanii
ASJC Scopus subject areas
- Molecular Biology