Mechanisms and principles of N-linked protein glycosylation
Highlights
► Protein N-linked glycosylation is a homologous process found both in eukaryotes and in prokaryotes. ► Prokaryotic N-glycan precursors are heterogeneous, whereas eukaryotes produce a conserved lipid-linked oligosaccharide structure. ► The N-X-S/T sequon is a unique acceptor sequence conserved in N-glycosylation. ► The specificity of oligosaccharyltransferase defines the complexity of the glycoproteome.
Introduction
Asparagine (N)-linked glycosylation of proteins is a fundamental and extensive post-translational modification that results in the covalent attachment of an oligosaccharide onto asparagine residues of polypeptide chains. This protein modification is found both in eukaryotes and in prokaryotes. Although a number of variations occur, studies in model organisms representing all three domains of life reveal that three homologous processes represent the core of N-glycosylation:
- 1.
A glycan is assembled from nucleotide-activated building blocks on a lipid anchor through the stepwise incorporation of monosaccharides by various glycosyltransferases. The lipid-linked oligosaccharide (LLO) is then re-oriented from the cytosolic to the luminal side of the eukaryotic ER membrane or of the plasma membrane in prokaryotes, where it serves as donor for glycosylation. The LLO can be further extended in many eukaryotes after flipping to the luminal side of the ER.
- 2.
Proteins with the consensus sequence for glycosylation (N-X-S/T) when translocated to the ER lumen (or to the periplasm) serve as acceptors.
- 3.
The oligosaccharyltransferase (OST) catalyzes the en bloc transfer of the oligosaccharide to the asparagine side chain of the acceptor polypeptides.
After being covalently linked to proteins, the N-glycan can be modified in eukaryotes. This sequential processing is coupled to the secretory pathway and results in a species-specific or even cell type-specific diversity of N-linked glycans.
Recently, an alternative type of N-glycosylation has been identified in a few bacteria [1••, 2, 3••, 4]. This novel N-glycosylation pathway takes place in the cytoplasm and it is characterized by the addition of monosaccharides from nucleotide activated donors to asparagine side chains. Notably, cytosolic N-glycosylation is performed by an enzyme structurally different from OST, but displaying the same acceptor site specificity N-X-S/T, as in the case for the conventional N-glycosylation process.
This review describes the basic structural concepts of N-glycosylation and places them in a phylogenetic framework. Recent studies have uncovered a substantial diversity among archaeal and bacterial N-glycans, while at the same time making available novel features of OST. The detection of an alternative pathway for N-glycosylation taking place in the cytoplasm of bacteria represents an unexpected turn in the field, and raises questions about the driving forces leading to the N-X-S/T sequon as a general acceptor for a post-translational modification of proteins.
Section snippets
Structure of N-linked glycan precursors: diverse in prokaryotes, conserved in eukaryotes
The oligosaccharide substrate for N-glycosylation is assembled at the membrane of the endoplasmic reticulum in eukaryotes and at the plasma membrane in prokaryotes. Phosphorylated polyisoprenoid lipids of different chain length and saturation (dolicol or undecaprenol) are invariably used as a molecular dock for oligosaccharide biosynthesis at membranes. The use of a lipid as a carrier for the activated oligosaccharide has several consequences. On the one hand, it leads to an increased local
The N-X-S/T sequon: a unique acceptor sequence conserved in N-glycosylation
The acceptor substrate of N-glycosylation is an asparagine residue present within the consensus sequence N-X-S/T [18]. Site occupancy analysis of different model glycoproteins shows a preference for N-X-T sites over N-X-S [19, 20], and reveals that proline is not tolerated in the second position. Based on these findings, it has been hypothesized that a specific conformation of the acceptor peptide is required to increase the nucleophilicity of the amide group of the asparagine [21, 22]. These
OST specificity defines the complexity of the glycoproteome
The view that there is a selective advantage of a ‘general’ N-glycosylation system is further supported by a phylogenetic analysis of the central enzyme of the pathway. The Stt3 protein accounts alone for OST activity in some organisms. In fact, bacterial and archaeal Stt3 homologs (called PglB or AglB) glycosylate proteins and peptides [32, 33, 34•, 35]. Similarly, some protists express Stt3 proteins that are self-sufficient for oligosaccharide transfer [36, 37]. By contrast, in complex
A functional view of eukaryotic N-glycan structure
The biosynthetic route of the eukaryotic LLO is divided into two branches (Figure 3). The first part resembles that of archaea, as it occurs at the cytosolic side and glycosyltransferases build the Man5-GlcNAc2 glycan by using nucleotide activated monosaccharides. After translocation of LLO to the ER lumen, glycosyltransferases of a different class complete the glycan structure by utilizing dolichyl phosphate-activated monosaccharides. Notably, these glycosyltransferases seem to share a common
Conclusions and perspectives
The model that we propose allows for a phylogenetic interpretation of the N-glycosylation process. This view is based on a structural framework characterized in different model systems. The better we understand the different mechanisms of N-linked glycosylation at a molecular level, the clearer we can formulate the underlying principles. For instance, determining the catalytic mechanism of OST and NGT will be important to recognize how these two enzymes activate the poorly reactive amide group.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
Research in the Aebi laboratory is supported by the Swiss National Science Foundation and the ETH Zurich.
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