The multiple evolutionary origins of the eukaryotic N-glycosylation pathway

Abstract

Background: The N-glycosylation is an essential protein modification taking place in the membranes of the endoplasmic reticulum (ER) in eukaryotes and the plasma membranes in archaea. It shares mechanistic similarities based on the use of polyisoprenol lipid carriers with other glycosylation pathways involved in the synthesis of bacterial cell wall components (e.g. peptidoglycan and teichoic acids). Here, a phylogenomic analysis was carried out to examine the validity of rival hypotheses suggesting alternative archaeal or bacterial origins to the eukaryotic N-glycosylation pathway.

Results: The comparison of several polyisoprenol-based glycosylation pathways from the three domains of life shows that most of the implicated proteins belong to a limited number of superfamilies. The N-glycosylation pathway enzymes are ancestral to the eukaryotes, but their origins are mixed: Alg7, Dpm and maybe also one gene of the glycosyltransferase 1 (GT1) superfamily and Stt3 have proteoarchaeal (TACK superphylum) origins; alg2/alg11 may have resulted from the duplication of the original GT1 gene; the lumen glycosyltransferases were probably co-opted and multiplied through several gene duplications during eukaryogenesis; Alg13/Alg14 are more similar to their bacterial homologues; and Alg1, Alg5 and a putative flippase have unknown origins.

Conclusions: The origin of the eukaryotic N-glycosylation pathway is not unique and less straightforward than previously thought: some basic components likely have proteoarchaeal origins, but the pathway was extensively developed before the eukaryotic diversification through multiple gene duplications, protein co-options, neofunctionalizations and even possible horizontal gene transfers from bacteria. These results may have important implications for our understanding of the ER evolution and eukaryogenesis.

Reviewers: This article was reviewed by Pr. Patrick Forterre and Dr. Sergei Mekhedov (nominated by Editorial Board member Michael Galperin).

Keywords: Archaea; Bacteria; Eukaryogenesis; Eukaryotes; Glycosyltransferase; N-glycosylation; Polyisoprenol; Prokaryotic cell walls.

Fig. 1

Basic components of the polyisoprenol-based machineries. a Typical polyisoprenol lipid carriers in the three domains of life. b Topology (membrane orientation) of characteristic N-glycosylation pathways in the three domains of life and bacterial peptidoglycan biosynthesis. The lipid carrier is embedded in a cell membrane (ER membrane in eukaryotes, plasma membranes in archaea and bacteria), first facing the cytoplasmic side, then flipped to the opposite side (i.e. ER lumen in eukaryotes, periplasm in prokaryotes). Monosaccharides are attached one by one to the lipid carriers by specific glycosyltransferases, although in the eukaryotic N-glycosylation each kind of sugar is only represented once per compartment, for simplicity. The monosaccharides are nucleotide-activated in the cytoplasmic side or translocated to the ER lumen by separate lipid carriers

Fig. 2

Polyprenol-based glycosylation pathways (and GPI biosynthesis) colored according to detected homology groups. Horizontal lines represent ER membranes in the eukaryotes, plasma membranes in prokaryotes. Horizontal rectangles represent cytoplasmic glycosyltransferases (GTs) if they are below the membrane or lumen/periplasmic GTs if they are above the membrane. Vertical rectangles depict flippases or translocation mechanisms. Ovals represent the oligosaccharide transferases from the lipid carrier to the acceptor molecule. Diamonds portray proteins that are neither GTs nor translocases (e.g. acetyl or ethanolamine transferases in GPI biosynthesis). Extra shapes in the eukaryotic oligosaccharide transferases reflect the fact that these are complexes with many subunits. The cytoplasmic GTs depicted after a transfer to the acceptor molecule (e.g. Dpm1 and Alg5 in eukaryotic N-glycosylation) represent polyisoprenol-P-monosaccharide synthases tranfering single mannoses or glucoses to a lipid carrier to supply these sugars to the opposite side of the membrane. Proteins are colored according to the homology group to which they belong (as defined in Methods). Plain symbols represent proteins that were detected using the procedure described in Methods, whilst empty white shapes show more distant relationships that required bibliographic or extra analyses to be established. Empty transparent shapes represent the lack of detection of any homologues in the dataset

Fig. 3

Bayesian phylogeny of the HPT homologues. a Schematic phylogenetic tree including the bacterial sequences (see Additional file 2 for details). b Specific phylogeny of the archaeal/eukaryotic clade. The tree was reconstructed using 124 representative sequences and 203 conserved sites. Multifurcations correspond to branches with Bayesian posterior probabilities <0.5. Numbers at nodes indicate Bayesian posterior probabilities higher than 0.5. Bootstrap values from maximum likelihood analyses are reported on basal and major nodes. Colors on leaves represent the affiliation of sequences to a domain of life: archaea (blue), bacteria (orange) and eukaryotes (purple)

Fig. 4

Bayesian phylogeny of the Alg13/catalytic MurG domain. a Schematic phylogenetic tree including the bacterial MurG homologues (see Additional file 3 for details). b Specific phylogeny excluding the bacterial MurG clade. The tree is unrooted and was reconstructed using 132 representative sequences and 116 conserved sites. Multifurcations correspond to branches with Bayesian posterior probabilities <0.5. Numbers at nodes indicate Bayesian posterior probabilities higher than 0.5. Bootstrap values from maximum likelihood analyses are reported on basal and major nodes. Colors on leaves represent the affiliation of sequences to a domain of life: archaea (blue), bacteria (orange) and eukaryotes (purple)

Fig. 5

Bayesian phylogeny of the closest relatives to the eukaryotic Alg2/Alg11 homologues. The tree is unrooted and was reconstructed using 211 representative sequences and 194 conserved sites. Multifurcations correspond to branches with Bayesian posterior probabilities <0.5. Numbers at nodes indicate Bayesian posterior probabilities higher than 0.5. Bootstrap values from maximum likelihood analyses are reported on basal and major nodes. Colors on leaves represent the affiliation of sequences to a domain of life: archaea (blue), bacteria (orange) and eukaryotes (purple)

Fig. 6

Bayesian phylogeny of the catalytic N-OST subunit (Stt3/AglB/PglB) homologues. The tree is unrooted and was reconstructed using 163 representative sequences and 268 conserved sites. Multifurcations correspond to branches with Bayesian posterior probabilities <0.5. Numbers at nodes indicate Bayesian posterior probabilities higher than 0.5. Bootstrap values from maximum likelihood analyses are reported on basal and major nodes. Colors on leaves represent the affiliation of sequences to a domain of life: archaea (blue), bacteria (orange) and eukaryotes (purple)

Fig. 7

Origins of the eukaryotic N-glycosylation proteins and presence of related superfamilies in the last common ancestors of each domain of life and the cenancestor (summary)