Abstract
Protein glycosylation constitutes a critical post-translational modification that supports a vast number of biological functions in living organisms across all domains of life. A seemingly boundless number of enzymes, glycosyltransferases, are involved in the biosynthesis of these protein-linked glycans. Few glycan-biosynthetic glycosyltransferases have been characterized in vitro, mainly due to the majority being integral membrane proteins and the paucity of relevant acceptor substrates. The crenarchaeote Pyrobaculum calidifontis belongs to the TACK superphylum of archaea (Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota) that has been proposed as an eukaryotic ancestor. In archaea, N-glycans are mainly found on cell envelope surface-layer proteins, archaeal flagellins and pili. Archaeal N-glycans are distinct from those of eukaryotes, but one noteworthy exception is the high-mannose N-glycan produced by P. calidifontis, which is similar in sugar composition to the eukaryotic counterpart. Here, we present the characterization and crystal structure of the first member of a crenarchaeal membrane glycosyltransferase, PcManGT. We show that the enzyme is a GDP-, dolichylphosphate-, and manganese-dependent mannosyltransferase. The membrane domain of PcManGT includes three transmembrane helices that topologically coincide with “half” of the six-transmembrane helix cellulose-binding tunnel in Rhodobacter spheroides cellulose synthase BcsA. Conceivably, this “half tunnel” would be suitable for binding the dolichylphosphate-linked acceptor substrate. The PcManGT gene (Pcal_0472) is located in a large gene cluster comprising 14 genes of which 6 genes code for glycosyltransferases, and we hypothesize that this cluster may constitute a crenarchaeal N-glycosylation (PNG) gene cluster.
Highlights
Across all three domains of life, protein glycosylation constitutes a critical post-translational modification that supports a vast number of biological functions in living organisms, such as protein stability, sorting, cell–cell recognition and signaling, transport, and dynamic adaptation to changing environments [1,2,3,4]
The diffraction capacity of PcManGT crystals could be improved from 4 to 2.6 Å resolution by crystallization in meso using bicelles, which are open disk-like structures that offer an environment that is more akin to natural, native membranes
The α-1,6mannosyl branch is extended by four α-1,2-linked mannose units and a terminal α-1,3-linked mannose, and the α-1,3-mannosyl branch is capped by one α1,2-linked mannose unit
Summary
Across all three domains of life, protein glycosylation constitutes a critical post-translational modification that supports a vast number of biological functions in living organisms, such as protein stability, sorting, cell–cell recognition and signaling, transport, and dynamic adaptation to changing environments [1,2,3,4]. About two-thirds of all eukaryotic proteins are predicted to be glycosylated, and the majority of these carry asparagine-linked (N)glycans [5]. While all three domains of life perform protein glycosylation, the archaeal glycosylation machineries appear more closely related to the eukaryotic systems [6], albeit highly blended with distinct traits of both bacteria and eukarya [7]. N-glycosylation is more common in eukarya and archaea than in bacteria [6]. All N-glycans are assembled on a membranebound lipid carrier. Eukaryotes use dolichyldiphosphate (Dol-PP) as lipid carrier, archaea use either Dol-PP or Dol-P, and bacteria use undecaprenol monophosphate. The Dol-PP-linked heptasaccharide core (GlcNAc)2-(Man) of the lipidlinked oligosaccharide (LLO) is assembled at the cytoplasmic face of the ER membrane, and creativecommons.org/licenses/by/4.0/)
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