Mechanisms and principles of N-linked protein glycosylation

https://doi.org/10.1016/j.sbi.2011.08.005Get rights and content

N-linked glycosylation, a protein modification system present in all domains of life, is characterized by a high structural diversity of N-linked glycans found among different species and by a large number of proteins that are glycosylated. Based on structural, functional, and phylogenetic approaches, this review discusses the highly conserved processes that are at the basis of this unique general protein modification system.

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.

References (57)

  • R. Oriol et al.

    Common origin and evolution of glycosyltransferases using Dol-P-monosaccharides as donor substrate

    Mol Biol Evol

    (2002)
  • M. Aebi et al.

    N-glycan structures: recognition and processing in the ER

    Trends Biochem Sci

    (2009)
  • K.J. Choi et al.

    The Actinobacillus pleuropneumoniae HMW1C-like glycosyltransferase mediates N-linked glycosylation of the Haemophilus influenzae HMW1 adhesin

    PLoS ONE

    (2010)
  • S. Grass et al.

    The Haemophilus influenzae HMW1 adhesin is glycosylated in a process that requires HMW1C and phosphoglucomutase, an enzyme involved in lipooligosaccharide biosynthesis

    Mol Microbiol

    (2003)
  • S. Grass et al.

    The Haemophilus influenzae HMW1C protein is a glycosyltransferase that transfers hexose residues to asparagine sites in the HMW1 adhesin

    PLoS Pathog

    (2010)
  • J. Gross et al.

    The Haemophilus influenzae HMW1 adhesin is a glycoprotein with an unusual N-linked carbohydrate modification

    J Biol Chem

    (2008)
  • P. Burda et al.

    The dolichol pathway of N-linked glycosylation

    Biochim Biophys Acta

    (1999)
  • G.P. Zhou et al.

    Characterization by NMR and molecular modeling of the binding of polyisoprenols and polyisoprenyl recognition sequence peptides: 3D structure of the complexes reveals sites of specific interactions

    Glycobiology

    (2003)
  • N.M. Young et al.

    Structure of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni

    J Biol Chem

    (2002)
  • F. Schwarz et al.

    Relaxed acceptor site specificity of bacterial oligosaccharyltransferase in vivo

    Glycobiology

    (2011)
  • A.J. Jervis et al.

    Characterization of N-linked protein glycosylation in Helicobacter pullorum

    J Bacteriol

    (2010)
  • B. Chaban et al.

    Identification of genes involved in the biosynthesis and attachment of Methanococcus voltae N-linked glycans: insight into N-linked glycosylation pathways in Archaea

    Mol Microbiol

    (2006)
  • J. Kelly et al.

    A novel N-linked flagellar glycan from Methanococcus maripaludis

    Carbohydr Res

    (2009)
  • J. Lechner et al.

    Structure and biosynthesis of prokaryotic glycoproteins

    Annu Rev Biochem

    (1989)
  • D.J. Kelleher et al.

    Dolichol-linked oligosaccharide selection by the oligosaccharyltransferase in protist and fungal organisms

    J Cell Biol

    (2007)
  • R.D. Marshall

    The nature and metabolism of the carbohydrate-peptide linkages of glycoproteins

    Biochem Soc Symp

    (1974)
  • Y. Gavel et al.

    Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering

    Protein Eng

    (1990)
  • M.M. Chen et al.

    From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni

    Biochemistry

    (2007)
  • Cited by (520)

    View all citing articles on Scopus
    View full text