Glycobiology Advance Access originally published online on March 1, 2006
Glycobiology 2006 16(6):91R-101R; doi:10.1093/glycob/cwj099
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REVIEW |
Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139
1 To whom correspondence should be addressed; e-mail: imper{at}mit.edu
accepted on February 27, 2006
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Abstract |
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 Top Abstract IntroductionEukaryotic glycosylationProkaryotic glycosylationConclusionConflict of interest statementAcknowledgmentsReferences |
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Asparagine-linked protein glycosylation is a prevalent protein modification reaction in eukaryotic systems. This process involves the co-translational transfer of a pre-assembled tetradecasaccharide from a dolichyl-pyrophosphate donor to the asparagine side chain of nascent proteins at the endoplasmic reticulum (ER) membrane. Recently, the first such system of N-linked glycosylation was discovered in the Gram-negative bacterium, Campylobacter jejuni. Glycosylation in this organism involves the transfer of a heptasaccharide from an undecaprenyl-pyrophosphate donor to the asparagine side chain of proteins at the bacterial periplasmic membrane. Here we provide a detailed comparison of the machinery involved in the N-linked glycosylation systems of eukaryotic organisms, exemplified by the yeast Saccharomyces cerevisiae, with that of the bacterial system in C. jejuni. The two systems display significant similarities and the relative simplicity of the bacterial glycosylation process could provide a model system that can be used to decipher the complex eukaryotic glycosylation machinery.
Key words: Campylobacter jejuni / dolichol pathway / oligosaccharyl transferase / pgl gene cluster / protein glycosylation
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Introduction |
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TopAbstractIntroductionEukaryotic glycosylationProkaryotic glycosylationConclusionConflict of interest statementAcknowledgmentsReferences |
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Glycosylation is a complex, co- or post-translational protein modification that serves to expand the diversity of the proteome. A vast array of carbohydrate units, together with a variety of glycan-protein linkages, have been identified in glycoproteins originating from eukaryotic, archaeal and bacterial organisms (Spiro, 2002
). Eukaryotic glycoproteins have been implicated in a multitude of cellular processes including the immune response, intracellular targeting, intercellular recognition, and protein folding and stability (Varki, 1993). The biological role of prokaryotic glycoproteins requires further exploration, but it is evident that glycosylation plays a vital role in pathogenicity and host invasion (Upreti et al., 2003).
This review focuses on N-linked protein glycosylation, which is characterized by a ß-glycosylamide linkage to asparagine. This system has been extensively investigated in eukaryotes, particularly in the yeast, Saccharomyces cerevisiae, and has been reviewed in detail (Kellenberger et al., 1997; Burda and Aebi, 1999; Kelleher and Gilmore, 2005). Recently, a system of N-linked glycosylation was discovered in a Gram-negative bacterium, Campylobacter jejuni, which was the first observation of this protein modification in the bacterial domain (Szymanski et al., 1999; Szymanski and Wren, 2005). Although the ß-glycosylamide-linkage to protein is conserved, the structure of the glycan that is transferred is strikingly different between eukaryotic and bacterial systems (Burda and Aebi, 1999; Young et al., 2002). The biosynthetic machinery responsible for this elaborate protein modification follows a similar overall progression, whereby an oligosaccharide is assembled in a step-wise fashion on a polyisoprenyl-pyrophosphate carrier and then ultimately transferred to protein. Here, we provide a detailed comparison of the N-linked glycosylation machinery of eukaryotic organisms, exemplified by S. cerevisiae, with the parallel process in the Gram-negative bacterium, C. jejuni.
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Eukaryotic glycosylation |
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TopAbstractIntroductionEukaryotic glycosylationProkaryotic glycosylationConclusionConflict of interest statementAcknowledgmentsReferences |
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Overview
N-linked glycosylation in eukaryotes occurs at the Asn-Xaa-Ser/Thr sequon and is a co-translational process that is catalyzed by oligosaccharyl transferase (OT), a protein complex localized in the lumen of the ER. Proteins destined for the secretory pathway possess a signal sequence that is recognized by the signal recognition particle (SRP) (Figure 1) (Halic and Beckmann, 2005
; Shan and Walter, 2005). The SRP directs the growing polypeptide chain to the translocon machinery, whereby transport across the ER membrane occurs (Nikonov and Kreibich, 2003; Chavan et al., 2005). The S. cerevisiae signal peptidase complex, containing the essential Sec11p protein, then cleaves the signal sequence (Bohni et al., 1988), which moves the polypeptide to the compartment where OT-mediated glycosylation takes place. Since the protein is still being translated by the ribosome during this process, global folding and tertiary structure of the protein are not important determinants in the recognition events leading to glycosylation. However, there is evidence that the local secondary structure around the site of glycosylation may be a vital determinant in this enzymatic process, where glycosylation induces a conformational switch from an Asx-turn to a ß-turn structure (O’Connor and Imperiali, 1997, 1998).
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OT transfers a tetradecasaccharide "core" unit (Glc3Man9GlcNAc2) to the polypeptide chain. After OT-catalyzed glycosylation, the two terminal glucose residues are trimmed by glucosidase I and II, and the protein enters a folding cycle mediated by two ER-resident lectins with chaperone function, calnexin (membrane-bound) and calreticulin (soluble) in conjunction with a co-chaperone, Erp57 (a thiol-oxidoreductase) (Figure 2). After removal of the third glucose residue by glucosidase II, misfolded proteins are reglucosylated by UDP-glucose:glycoprotein glucosyltransferase (UGGT) and re-enter the calnexin/calreticulin cycle. For a detailed description of the protein quality control pathway in eukaryotes, refer to recent reviews in the field (Helenius and Aebi, 2001, 2004; Roth, 2002; Trombetta, 2003). Eventually, non-native conformations are recognized by mannosidase-like lectins (Mnl1p, EDEM) to initiate the ER-associated degradation (ERAD) pathway (Meusser et al., 2005). Fully folded proteins undergo 
1,2-mannose cleavage by an ER-resident mannosidase and the resulting Man8GlcNAc2-containing glycoproteins are transported to the Golgi. N-glycan processing in the Golgi involves the action of several glycosidases (Herscovics, 1999) and glycosyltransferases that result in further glycan trimming and elaboration. Glycan processing in yeast affords a variety of highly branched, mannosylated oligosaccharides. In mammalian systems, glycosyltransferases in the Golgi facilitate the addition of a plethora of diverse monosaccharide units to the glycoprotein such as GlcNAc, galactose, N-acetylneuraminic acid, fucose, and GalNAc sugars (Helenius and Aebi, 2001; Roth, 2002; Wildt and Gerngross, 2005).
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The dolichol pathway
The glycan that is transferred to the asparagine side chain of a nascent protein is a tetradecasaccharide (Glc3Man9Glc NAc2ß1,N-Asn) that is highly conserved in higher order eukaryotic systems (Figure 3). This tetradecasaccharide is assembled at the ER membrane on a dolichyl-pyrophosphate carrier by a series of glycosyltransferases, in a process known as the dolichol pathway (Figure 4) (Burda and Aebi, 1999). The dolichols constitute a family of -saturated, (S)-polyisoprenyl-phosphates containing 14–17 isoprene units that are biosynthesized from farnesyl-pyrophosphate on the cytoplasmic face of the ER (Schenk et al., 2001). The biosynthesis of the oligosaccharide donor begins on the cytoplasmic face of the ER with Alg7, an N-acetylglucosamine-phosphate transferase that elaborates Dol-P to Dol-PP-GlcNAc (Kukuruzinska and Robbins, 1987). Recent bioinformatics studies followed by mutational analyses have determined that the addition of the second 1,4-GlcNAc residue is catalyzed by a hetero-oligomeric protein classified as Alg13/14 (Bickel et al., 2005; Chantret et al., 2005; Gao et al., 2005). The role of Alg1, which catalyzes the first mannosylation step, has been extensively characterized in vitro due to the advantageous formation of the chemically challenging ß1,4-mannosidic linkage, which may be useful in chemoenzymatic syntheses (Wilson et al., 1995; Watt et al., 1997). Two mannosyltransferases, Alg2 and Alg11, are implicated in the remaining cytosolic glycosylations in the dolichol pathway (Yamazaki et al., 1998; Cipollo et al., 2001; Thiel et al., 2003), yet their precise roles in this process remain to be rigorously validated. Biochemical and genetic assays have shown that Alg1 interacts with both Alg2 and Alg11, suggesting a role for protein-complex formation in maintaining the fidelity of the early steps in the dolichol pathway (Gao et al., 2004). Once the heptasaccharide is assembled, it is then flipped to the lumenal face of the ER membrane by the ATP-independent, bi-directional, membrane-spanning flippase, Rft1p (Helenius et al., 2002).
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The remaining biosynthetic steps occur on the lumenal face of the ER membrane, where the glycan donors are the dolichyl-phosphate-linked Dol-P-Man and Dol-P-Glc. Four mannosyltransferases and three glucosyltransferases are involved in the completion of the dolichyl-pyrophosphate-linked tetradecasaccharide. All of the "lumenal" glycosyltransferases in the dolichol pathway have now been identified and are all highly hydrophobic, basic proteins (MW 65–75 kDa) that include multiple transmembrane domains with small hydrophilic loops (Burda and Aebi, 1999). Recently, the bi-functional nature of Alg9 was demonstrated, whereby Alg9 catalyzes the addition of both the seventh and the ninth mannose residues (both 1,2-mannosyl linkages) (Frank and Aebi, 2005). Once the entire tetradecasaccharide has been assembled in the lumen of the ER, it is utilized by OT, which catalyzes transfer of the oligosaccharide to protein.
OT
The OT complex is a multimeric, membrane-associated enzyme that is localized in the membrane of the lumen of the ER, with the active site disposed to the lumenal compartment (Imperiali and Hendrickson, 1995; Yan and Lennarz, 1999; Dempski and Imperiali, 2002; Kelleher and Gilmore, 2005). This enzyme complex has been most extensively investigated in the yeast S. cerevisiae and comprises at least eight membrane-bound protein subunits (Figure 5) that exist in three sub-complexes, Ost1p-Ost5p, Ost2p-Swp1p-Wbp1p, and Stt3p-Ost4p-Ost3p/Ost6p. Genetic-knockout experiments have revealed that five of these subunits (Ost2p, Ost1p, Stt3p, Swp1p, and Wbp1p) are absolutely essential for yeast viability (Heesen et al., 1993; Zufferey et al., 1995; Yan and Lennarz, 1999; Dempski and Imperiali, 2004). For a recent review on the details of the protein subunits involved in the OT complex, refer to Kelleher and Gilmore (Kelleher and Gilmore, 2005).
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Stt3p is a highly conserved transmembrane protein that is found in all eukaryotic organisms. Site-directed mutagenesis combined with photoactivated-crosslinking experiments show that Stt3p is directly involved in the catalytic process (Yan and Lennarz, 2002; Nilsson et al., 2003). The S. cerevisiae Stt3p comprises 11–13 transmembrane segments and a hydrophilic C-terminal domain. A highly conserved WWDYG amino acid sequence is present in all homologs of Stt3p in organisms that contain a system for N-linked glycosylation. A bacterial protein from C. jejuni, PglB, is homologous to Stt3p, and N-linked glycosylation in this organism is abolished with the loss of PglB (Wacker et al., 2002; Young et al., 2002). Mutations within this conserved WWDYG motif result in the loss of glycosylation activity, suggesting that this sequence includes residues that are essential for catalysis in PglB and Stt3p (Wacker et al., 2002; Nita-Lazar et al., 2005).
N-linked glycosylation in eukaryotic systems is an extremely complex process involving a multitude of enzymes acting in concert to biosynthesize the dolichyl-pyrophosphate-linked tetradecasaccharide and facilitate the ultimate transfer of the glycosyl moiety to protein. All of the proteins involved in this process are highly hydrophobic, membrane-associated proteins, which complicate detailed in vitro biochemical and biophysical characterization. In order to understand this complex process of N-linked protein glycosylation, it would be ideal to have access to a parallel system that is more amenable to biochemical characterization. The recent discovery of a system of N-linked protein glycosylation in the Gram-negative bacterium, C. jejuni, has provided us with a parallel, yet simpler, system that is more suitable for in-depth biochemical characterization and can potentially shed light on the more complex eukaryotic process.
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Prokaryotic glycosylation |
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TopAbstractIntroductionEukaryotic glycosylationProkaryotic glycosylationConclusionConflict of interest statementAcknowledgmentsReferences |
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Overview
Investigations into glycosylation systems in eukaryotic organisms have prevailed since the late 1930s, yet for many decades it was assumed that bacteria and archaea were devoid of this important protein modification (Messner, 2004
). The discovery of surface layer (S-layer) glycoproteins in the Gram-negative halophile, Halobacterium salinarium, was the first such system to be found outside of the eukaryotic domain (Mescher and Strominger, 1976). These S-layer glycoproteins in archaea have the unique feature of assembling into two-dimensional crystalline arrays on the cell wall of halobacteria and are characterized by a variety of glycans and a diverse array of linkages to protein (Schaffer and Messner, 2004). Since this initial report of S-layer glycoproteins in halobacteria, several characterizations of similar glycosylated proteins in the bacterial domain have also surfaced (Messner, 1997). These glycoproteins are integrated into cell-surface appendages, such as pili and flagella (Power et al., 2000; Messner, 2004). The pili of pathogenic bacteria, such as Neisseria meningitides (Stimson et al., 1995) and Neisseria gonorrhoeae (Hegge et al., 2004) contain O-linked glycans that involve unusual sugars such as 2,4-diacetamido-2,4,6-trideoxyhexose (DATDH) and pili of Pseudomonas aeruginosa contain analogs of O-linked pseudaminic acid (a nine-carbon sugar that resembles sialic acid) (Castric et al., 2001). The flagella of Gram-negative bacteria such as C. jejuni (Thibault et al., 2001) and Helicobacter pylori (Schirm et al., 2003) have also been shown to include O-linked pseudaminic acid analogs.
The first system of N-linked protein glycosylation to be discovered in Gram-negative bacteria comes from C. jejuni (Szymanski, Logan et al., 2003; Szymanski and Wren, 2005), a human-gut mucosal pathogen that is implicated in gastroenteritis. Campylobacter enteritis is characterized by acute abdominal pain and inflammatory diarrhea (Ketley, 1997), hence, understanding the pathogenicity of C. jejuni could potentially lead to better prevention and infection-control strategies. The sequencing of the C. jejuni genome, together with detailed genetic maps, has facilitated genetic characterization of various strains of this organism (Taylor et al., 1992; Karlyshev et al., 1998; Parkhill et al., 2000; Fouts et al., 2005).
In 1999, it was discovered that C. jejuni contains a gene locus that is involved in the biosynthesis of a number of highly immunogenic glycoproteins (Szymanski et al., 1999). This cluster was termed the "pgl gene cluster" and contained the genes pglA to pglG, which demonstrate significant homology to enzymes involved in bacterial lipopolysaccharide (LPS) and capsular polysaccharide (CPS) biosynthesis. Mutagenesis of key residues in this cluster resulted in no discernible effect on CPS or LPS levels but caused a dramatic reduction in the immunoreactivity of numerous C. jejuni proteins, suggesting that these proteins functioned independently from the known LPS/CPS biosynthetic pathways.
The highly immunogenic C. jejuni proteins affected by mutations in the pgl gene cluster bind strongly to the soybean agglutinin (SBA) lectin, which is known to bind terminal GalNAc residues. This observation allowed the identification of PEB3 and CgpA, two highly immunoreactive glycoproteins in C. jejuni (Linton et al., 2002). The glycan attached to these proteins was not affected by ß-elimination, which generally removes O-linked glycans, thus suggesting a linkage via a glycosyl amide to an asparagine residue. Additionally, MS/MS collision-induced dissociation of the glycopeptide confirmed that the oligosaccharide was N-linked (Young et al., 2002). Through the action of specific exoglycosidases, the oligosaccharide was shown to include one or more -linked GalNAc residues (Linton et al., 2002). The PEB3 glycoprotein was partially purified and analyzed by mass spectrometry to reveal a modification via an Asn-linked glycan with a mass of 1406 Da. Using nano-NMR techniques on the pronase-digested glycopeptides, the structure was determined to be the heptasaccharide, GlcGalNAc5Bacß1,N-Asn where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose) (Figure 6) (Young et al., 2002). Furthermore, this heptasaccharide structure was shown to be conserved throughout all C. jejuni and C. coli strains examined (Szymanski, St Michael et al., 2003).
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The C. jejuni heptasaccharide is structurally very different from the tetradecasaccharide transferred in eukaryotic N-linked glycosylation. Bacteria utilize a wide variety of amino- and deoxy-sugars that are not found in eukaryotic systems (Maki and Renkonen, 2004). This feature is exemplified by the N-linked glycan in C. jejuni that incorporates bacillosamine, a diacetamido-trideoxy-sugar found in several bacterial strains such as Neisseria. The first bacillosamine derivative was originally discovered in Bacillus subtilis (Sharon and Jeanloz, 1960), and since then several syntheses of bacillosamine have been reported (Liav et al., 1973; Bundle and Josephson, 1980). Recently a synthetic route to undecaprenyl-pyrophosphate-linked bacillosamine (Und-PP-Bac) was described and this synthetic compound was utilized as a tool to investigate the enzymes in the pgl gene cluster in vitro (Weerapana et al., 2005).
The Pgl pathway
Computational analysis of the pgl gene cluster (Figure 7) suggested that the locus encodes five putative glycosyltransferases (PglA, PglC, PglH, PglI, and PglJ), and three enzymes involved in sugar biosynthesis (PglD, PglE, and PglF). The PglB protein demonstrates significant homology to the Stt3p subunit of the yeast OT complex and WlaB is a putative ATP-binding cassette (ABC) transport protein (Linton et al., 2002). The gne gene encodes a bifunctional UDP-Glc/GlcNAc 4-epimerase that converts UDP-Glc and UDP-GlcNAc to UDP-Gal and UDP-GalNAc. This gene is not exclusively involved in N-linked glycosylation, and is also required for LPS and CPS biosynthesis (Bernatchez et al., 2005).
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This pgl gene cluster in C. jejuni displays significant homology to a cluster found in the genome of N. meningitides, which is known to be responsible for the O-linked glycosylation of pilin (Figure 8). Pilin glycosylation involves an O-modified serine with a Gal-ß1,3-Gal-1,3-DATDH modification (Stimson et al., 1995). The stereochemistry of the DATDH sugar in pilin glycosylation has not been unambiguously determined, but is most likely to be bacillosamine. Bioinformatic analysis of the pgl gene cluster in C. jejuni was greatly facilitated by the fact that several homologous genes in the N. meningitides cluster were already functionally annotated (Power et al., 2000). The genes involved in pilin glycosylation in N. meningitides are also denoted "pgl" similar to the C. jejuni gene cluster; however, the two classification systems are completely independent. Analogs of the sugar modifying enzymes, PglF, PglE, and PglD, are present in the N. meningitides cluster (Nm PglD, PglC, and PglB) and are attributed to the biosynthesis of bacillosamine. Recently, the PglF and PglE proteins from Campylobacter were studied in vitro and annotated as a dehydratase and amino transferase, respectively (Schoenhofen et al., 2006). Nm pglB encodes a bi-functional protein demonstrating both glycosyltransferase and acetyltransferase activity. The pglC gene in C. jejuni encodes a protein that is homologous to the N-terminal portion of the Nm PglB protein and is thought to be responsible for transferring the first sugar phosphate onto a polyisoprene-phosphate carrier (Power et al., 2000). The pglA gene in C. jejuni is homologous to Nm pglA, which is responsible for the Gal1,3-Bac linkage. The other putative glycosyltransferase genes in C. jejuni are pglH, pglI, and pglJ, but the bioinformatics data are insufficient to assign GalNAc1,4- or Glcß1,3-transferase function to these genes (Young et al., 2002). There is no ortholog of the C. jejuni PglB protein in N. meningitides.
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In critical work by Aebi and coworkers, the pgl gene cluster was functionally transferred to E. coli, and two C. jejuni periplasmic proteins, AcrA and PEB3, were shown to be glycosylated in this modified E. coli system (Wacker et al., 2002). This suggests that the pgl cluster contains all of the genes necessary for the biosynthesis of the polyisoprenyl-pyrophosphate-linked heptasaccharide and its eventual transfer to protein. It is postulated that the prokaryotic oligosaccharide is assembled onto a polyisoprenyl-pyrophosphate in a manner similar to the assembly of dolichyl-pyrophosphate-linked oligosaccharide in eukaryotes. The polyisoprene used is undecaprenol (also known as bactoprenol), and contains 11 isoprene units, where the -isoprene unit is unsaturated, in contrast to the -saturated nature of the corresponding unit in the dolichols (Wacker et al., 2002). Analysis of Campylobacter isolates using the SBA lectin, which binds GalNAc residues, resulted in the isolation of up to 38 proteins that were identified as potentially containing this N-linked glycan (Young et al., 2002). These glycoproteins are predominantly annotated as periplasmic proteins, which suggest that the glycosylation machinery is specific for periplasmic substrates. An AcrA mutant that lacks the periplasmic signal sequence is not glycosylated, further supporting the identification of the periplasm as the site of modification (Nita-Lazar et al., 2005).
Through mutational studies of the pgl gene cluster in E. coli, the exact roles of various pgl genes were explored using structural analysis of the glycan transferred to protein (Linton et al., 2005). As predicted by bioinformatics analysis, the pglA, pglJ, pglH, and pglI genes were shown to encode specific glycosyltransferases responsible for sequential addition of monosaccharides to form the ultimate heptasaccharide donor. The pglA mutant showed transfer of monosaccharide to protein, verifying the earlier observation that PglA transfers the 1,3-GalNAc to bacillosamine. The pglJ mutant showed transfer of disaccharide, suggesting that PglJ is responsible for the first 1,4-GalNAc linkage to afford the trisaccharide. The pglH mutant showed transfer of a trisaccharide to protein, suggesting a role for PglH in the transfer of the second 1,4-GalNAc sugar. Finally, the pglI mutant showed transfer of a linear hexasaccharide, suggesting its role as a glucosyltransferase, adding the final branching ß1,3-glucose residue (Figure 9). While this study provided crucial information on the role of several Pgl glycosyltransferases, it did not provide information on the identity of the transferases responsible for the addition of the two terminal 1,4-GalNAc residues. Therefore, the suggested scenarios were that PglH added all three terminal GalNAc residues or that PglH and PglJ acted alternately, adding two GalNAc residues each, to form the hexasaccharide.
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To further corroborate the mutagenesis data, the role of each of the Pgl enzymes was unambiguously validated through in vitro biochemical analysis, using chemically synthesized Und-PP-Bac and purified Pgl glycosyltransferases (Glover, Weerapana, Numao et al., 2005; Weerapana et al., 2005). These data provided further evidence to support the bioinformatics and mutational analyses above and also demonstrated that PglH is a sugar polymerase, adding three 1,4-linked GalNAc residues to the undecaprenyl-pyrophosphate-linked trisaccharide. Reconstitution of the sequence of enzymatic steps in vitro also provided valuable insight into how these enzymes function together at a membrane interface (Glover, Weerapana, and Imperiali, 2005).
PglB: the OT of C. jejuni
The pglB gene shares significant homology with the STT3 gene that codes for the largest subunit of the yeast, S. cerevisiae, OT complex (Wacker et al., 2002; Yan and Lennarz, 2002). PglB contains a highly conserved amino acid motif WWDYG that is present in all putative OT homologs. This conserved sequence is located on the hydrophilic C-terminal portion of PglB. In a pglB mutant strain, PEB3 and AcrA, both known glycoproteins from C. jejuni, were found to be unglycosylated (Wacker et al., 2002, Young et al., 2002). When functionally reconstituted in E. coli, the pgl cluster containing a mutation in the 457WWDYG462 motif of PglB (W458A, D459A) resulted in unglycosylated protein (Wacker et al., 2002; Nita-Lazar et al., 2005). These results suggest the direct involvement of PglB in the glycosylation process, whereby PglB facilitates the transfer of the heptasaccharide onto the side chain of asparagine.
There are significant similarities between the Pgl pathway and the biosynthesis of the O-antigen LPS, where sequential addition of glycans results in an isoprenyl pyrophosphate-bound oligosaccharide that is transferred to the Lipid A core (Raetz and Whitfield, 2002). When the O-antigen ligase in E. coli was replaced with PglB, various O-antigen glycans were transferred to acceptor proteins (Figure 10) (Feldman et al., 2005). This behavior illustrates the substrate flexibility of PglB, which can accept a diverse array of undecaprenyl-linked oligosaccharide substrates.
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All of the glycosylated proteins identified in C. jejuni were shown to contain the Asn-Xaa-Ser/Thr sequon (Linton et al., 2002). Other proteins in the genome also contain this sequon but do not appear to be glycosylated. Hence, similar to the eukaryotic system, it appears that the Asn-Xaa-Ser/Thr sequon is a necessary but not absolute determinant of glycosylation. Detailed investigation of the glycosylation sequon illustrated that, similar to the eukaryotic process, proline is not accepted as the Xaa amino acid, hence indicating the importance of peptide conformation in the glycosylation process (Nita-Lazar et al., 2005). In vitro studies using an E. coli cell membrane fraction expressing PglB showed that, similar to the yeast OT system, PglB can accept a truncated peptide substrate (KDFNVSKA-NH2) in place of a full-length protein. Initial studies indicate that the recognition sequence for PglB may require determinants in addition to the canonical tripeptide substrate (Bz-NLT-NHMe) for yeast OT (Glover, Weerapana, Numao et al., 2005).
The glycosyl modifications synthesized by the pgl genes are highly immunogenic (Szymanski et al., 1999). Mutations in pglB and pglE resulted in a significant reduction in adherence to, and invasion of, INT407 cells in vitro, and a reduced ability to colonize the intestinal tract of mice, suggesting a role for these N-linked glycans in Campylobacter virulence (Szymanski et al., 2002). The immunogenic nature of N-linked glycans was further demonstrated by a pglH mutant strain that displayed reduced adherence to, and invasion of, human epithelial cells and affected the colonization of chicks (Karlyshev, Everest et al., 2004). Recently it was demonstrated that the N-linked glycan in C. jejuni plays a direct role in complex protein assembly. VirB10 is an N-linked glycoprotein that is present in the type IV secretion system (T4SS) of C. jejuni. Lack of VirB10 glycosylation results in C. jejuni cells containing a competence defect due to lack of protein complexation (Larsen et al., 2004). Interestingly, the closest homolog of the VirB10 glycoprotein is found in Wolinella succinogenes, which is the only other bacterium currently known to contain a putative N-linked glycosylation system similar to the pgl system.
Due to the essential role played by the C. jejuni N-glycans in bacterial adherence and pathogenicity, PglB and the Pgl pathway as a whole appear to be interesting potential targets for antibacterial therapeutics. The extensive work done on the synthesis of inhibitors for the eukaryotic OT system (Kellenberger et al., 1997) can now be applied toward the design of potent inhibitors of PglB. The periplasmic location of PglB also makes it a much more accessible target than the eukaryotic OT complex that is located in the ER lumen.
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Conclusion |
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TopAbstractIntroductionEukaryotic glycosylationProkaryotic glycosylationConclusionConflict of interest statementAcknowledgmentsReferences |
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There are several striking similarities as well as differences in the N-linked glycosylation processes of eukaryotic and prokaryotic systems. A summary of the characteristics of each of the systems is provided in Table I.
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The process of protein translocation and glycosylation is a well-characterized eukaryotic phenomenon. Significant work has been done on the role of the translocon, the signal peptidase and OT in this protein modification process. The exact machinery involved in bacterial N-linked glycosylation, however, is poorly defined. The glycosylation process is thought to occur in the periplasm of bacteria, which is the functional equivalent of the ER in eukaryotes. Currently, there is no conclusive evidence to demonstrate either the co-translational or post-translational nature of N-linked glycosylation in the bacterial periplasm. If the C. jejuni machinery functions post-translationally, on fully folded proteins, it could potentially be one of the most significant differences between the eukaryotic and prokaryotic processes of N-linked glycosylation.
The calnexin/calreticulin cycle exemplifies the intricate mechanisms by which eukaryotic cells maintain protein quality control to prevent the release of misfolded proteins into the extra-cellular milieu. Such a complex system of glycoprotein folding has not been demonstrated in the bacterial system, and the processes by which these organisms maintain glycoprotein quality is a prevailing question. Additionally, the glycan structures displayed on all N-linked glycoproteins of C. jejuni are identical, lacking the immense diversity of eukaryotic N-linked glycoproteins. This characteristic is due to the lack of the glycan trimming/elaboration steps that occur post-glycosylation in the Golgi of eukaryotic cells. Since bacterial cells lack the extensive compartmentalization present in eukaryotic cells, there is no functional equivalent of the Golgi, where such elaboration steps can take place.
The bacterial Pgl pathway shares striking similarities with the eukaryotic dolichol pathway. The oligosaccharide substrate for the OT is built up sequentially on a polyisoprenyl-pyrophosphate carrier (undecaprenol in C. jejuni, dolichol in S. cerevisiae) by a series of glycosyltransferases that utilize nucleotide-activated sugar donors or dolichol-phosphate-activated sugar donors. This sequence of biosynthetic transformations occurs in the periplasmic membrane of C. jejuni. Interestingly, in both systems, the glycan is built up to a heptasaccharide structure on the cytoplasmic face and then flipped to the other side of the membrane, either the ER lumen or the periplasm, by a flippase. The flippase in the C. jejuni system, WlaB, has been annotated to be an ABC transporter, whereas the Rft1p protein in the dolichol pathway is non-ATP dependent. In the dolichol pathway, this heptasaccharide is further elaborated to the tetradecasaccharide, whereas in C. jejuni, no further elaboration occurs. Both pathways contain at least one enzyme that catalyzes the transfer of multiple glycans (PglH in C. jejuni, Alg9 in yeast). One striking difference is that the ALG genes in yeast encode highly hydrophobic proteins, which all include at least one transmembrane domain. Although the Pgl glycosyltransferases function on similar isoprene-bound intermediates, they contain no predicted transmembrane domains. This renders the Pgl enzymes significantly more amenable to detailed biochemical analysis. Both pathways are examples of multistep enzymatic transformations that occur at a membrane interface. Studies devoted to understanding the interactions that occur amongst the Pgl glycosyltransferases at the membrane interface can provide clues on how the corresponding Alg enzymes maintain the fidelity of the dolichol pathway.
The OT complex in S. cerevisiae and PglB in C. jejuni both catalyze a similar reaction, the transfer of an oligosaccharide from a polyisoprenyl-pyrophosphate-linked glycan to an asparagine side chain. Yet, the S. cerevisiae system requires at least 8 proteins to efficiently catalyze this process, whereas the bacterial system appears to use only a single protein. In the yeast system, OT is required to interact with multiple other protein complexes such as the translocon and the signal peptidase for efficient co-translational glycosylation. It is hypothesized that some of the OT subunits play a role in these interactions. A predominant area of current research is to purify PglB to homogeneity in order to discern whether it is solely responsible for catalysis. Regardless, the simplicity of the PglB-mediated process provides us a great opportunity to investigate the mechanism of this intriguing enzymatic reaction in more depth.
The OT complex in S. cerevisiae shows a high degree of specificity with regards to the dolichyl-pyrophosphate-linked glycan substrate and accepts very few truncated and non-native structures (Karaoglu et al., 2001; Tai and Imperiali, 2001). PglB, on the other hand, appears to display much greater glycan flexibility by accepting various O-antigen structures as well as glycans of varying length and structure (Feldman et al., 2005; Glover, Weerapana, Numao et al., 2005). The glycans are, however, limited to those containing a C-2 N-acetamido-group on the proximal sugar, which suggests a role of the N-acetamido group in the catalytic mechanism of both OT and PglB. This substrate promiscuity of PglB suggests great promise for the potential of using the bacterial glycosylation system in engineering humanized glycoproteins.
Glycosylated proteins in both the yeast and Campylobacter systems contain the Asn-Xaa-Ser/Thr consensus sequence. In both systems, glycosylation is abolished when the Xaa amino acid is proline, suggesting that local conformation plays an important role in the glycosylation reaction. Although the minimum recognition motif for the eukaryotic system is a simple -Asn-Xaa-Ser/Thr- tripeptide sequence, initial studies indicate that the aspartate residue in the -Asp-Xaa-Asn-Xaa-Ser/Thr- sequence found in Campylobacter glycoproteins is important for recognition by PglB.
Due to the similarities between the dolichol (Alg) pathway and the Pgl pathway, as well as the parallels between the Stt3p-catalyzed glycosylation with the PglB reaction, the eukaryotic and prokaryotic systems are greatly intertwined. Our knowledge accumulated over decades of research devoted to understanding eukaryotic N-linked glycosylation can now be applied to the recently discovered prokaryotic system. Hopefully, the reduced complexity of the C. jejuni glycosylation process will allow for detailed biochemical and biophysical characterization that is currently virtually impossible with the eukaryotic system. Hence, the knowledge that can be gained from understanding the prokaryotic process will be invaluable in shedding light on the mechanism and function of the eukaryotic glycosylation system.
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Conflict of interest statement |
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TopAbstractIntroductionEukaryotic glycosylationProkaryotic glycosylationConclusionConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionEukaryotic glycosylationProkaryotic glycosylationConclusionConflict of interest statementAcknowledgmentsReferences |
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The authors acknowledge the NIH (GM39334) for support of their research on eukaryotic and prokaryotic asparagine-linked glycosylation.
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Abbreviations |
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CPS, capsular polysaccharide; DATDH, 2,4-diacetamido-2,4,6-trideoxyhexose; ER, endoplasmic reticulum; LPS, lipopolysaccharide; OT, oligosaccharyl transferase
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References |
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TopAbstractIntroductionEukaryotic glycosylationProkaryotic glycosylationConclusionConflict of interest statementAcknowledgmentsReferences |
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