Available online at www.sciencedirect.com
Not just for Eukarya anymore: protein glycosylation in Bacteria
and Archaea
Mehtap Abu-Qarn1
, Jerry Eichler1
and Nathan Sharon2
Of the many post-translational modifications proteins can
undergo, glycosylation is the most prevalent and the most
diverse. Today, it is clear that both N-glycosylation and
O-glycosylation, once believed to be restricted to eukaryotes,
also transpire in Bacteria and Archaea. Indeed, prokaryotic
glycoproteins rely on a wider variety of monosaccharide
constituents than do those of eukaryotes. In recent years,
substantial progress in describing the enzymes involved in
bacterial and archaeal glycosylation pathways has been made.
It is becoming clear that enhanced knowledge of bacterial
glycosylation enzymes may be of therapeutic value, while the
demonstrated ability to introduce bacterial glycosylation genes
into Escherichia coli represents a major step forward in glycoengineering.
A better understanding of archaeal protein
glycosylation provides insight into this post-translational
modification across evolution as well as protein processing
under extreme conditions. Here, we discuss new structural and
biosynthetic findings related to prokaryotic protein
glycosylation, until recently a neglected topic.
Addresses
1
Department of Life Sciences, Ben Gurion University, Beersheva 84105,
Israel
2
Department of Biological Chemistry, Weizmann Institute of Science,
Rehovot 76100, Israel
Corresponding author: Eichler, Jerry (jeichler@bgu.ac.il) and Sharon,
Nathan (nathan.sharon@weizmann.ac.il)
Current Opinion in Structural Biology 2008, 18:544–550
This review comes from a themed issue on
Carbohydrates and glycoconjugates
Edited by Daan van Aalten and Natalie Strynadka
Available online 26th August 2008
0959-440X/$ – see front matter
# 2008 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.sbi.2008.06.010
Introduction
Long believed to be restricted to Eukarya, it is now clear
that both Bacteria and Archaea are capable of glycosylation
(for reviews, see [1,2,3
]). In fact, glycosylation in
prokaryotes appears to lead to a greater diversity of glycan
compositions and structures than that found in eukaryotic
cells. Currently, heightened attention is focused on
protein glycosylation in Bacteria, primarily because of
the increasing frequency with which this post-translational
modification is seen in pathogenic species [4
].
Accordingly, research conducted in several laboratories
over the past decade has served to decipher the
N-glycosylation pathway of the intestinal pathogen Campylobacter
jejuni, making it the first bacterium for which
such a complete pathway has been described [3
]. In
addition, O-glycosylation has been shown to occur in
several other bacterial pathogens, such as Neisseria gonorhoeae
and Helicobacter pylori [4
], with glycosylation-defective
mutants displaying attenuated virulence-associated
properties. As such, pathways of protein glycosylation in
these pathogens, especially those responsible for the
biosynthesis of rare glycoprotein sugars not present in
humans, may offer novel therapeutic targets. Moreover,
the demonstration that the C. jejuni pgl gene cluster,
responsible for N-glycosylation in this organism, can be
expressed in Escherichia coli, and the fact that the C. jejuni
oligosaccharyltransferase (OST) is of relaxed specificity,
raise the hope that protein N-glycosylation will eventually
be added to the menu of biological and biotechnological
processes performed by this workhorse of molecular
biology [5
,6
].
While significant strides are being made in understanding
bacterial glycosylation, with, for instance, some 40 S-layer
glycoprotein structures having been fully or partially
elucidated (for a recent example, see [7]), much less is
known of the steps involved in this post-translational
modification in Archaea. This is despite the fact that
the first noneukaryal N-glycosylated protein was discovered
over three decades ago in the haloarchaea Halobacterium
salinarum [8] and that N-glycosylated proteins are
more prevalent in Archaea than in Bacteria [1,2]. Nonetheless,
studies emerging from several groups have
started to make inroads into identifying and characterizing
components involved in the archaeal version of Nglycosylation.
By contrast, virtually nothing is known of
the archaeal version of O-glycosylation.
Bacterial protein glycosylation
Bacteria are capable of forming both protein-attached Nglycans
and O-glycans. Such glycans can include rare
monosaccharides, often serving as carbohydrate–peptide
linking groups (Figure 1), in addition to more common
sugars present in eukaryotic glycoproteins, such as glucose,
galactose, N-acetylgalactosamine, and xylose.
N-Glycosylation — the Campylobacter Pgl proteins
Following the first isolation of an Asn-attached heptasacccharide
from C. jejuni and its structural characterization
[9
] (Table 1), the pathway responsible for its
biosynthesis has been delineated. The pgl gene cluster
Current Opinion in Structural Biology 2008, 18:544–550 www.sciencedirect.com
encodes, among others, five putative glycosyltransferases
(i.e. PglA, PglC, PglH, PglI, and PglJ) involved in the
assembly of the heptasaccharide on a lipid carrier and
PglB, responsible for transfer, en bloc, of the glycan from
the carrier to protein (Figure 2) in a pathway similar to
that employed in eukaryotes [10,11]. The same gene
cluster also encodes PglF, a UDP-N-acetylglucosamine
C-6 dehydratase [12], PglE, a C-4 aminotransferase
[13,14], and PglD, an N-acetyltransferase [15]
(Figure 3), responsible for transforming UDP-GlcNAc
into di-N-acetylbacillosamine, the carbohydrate–peptide
linking monosaccharide, as postulated over 40 years ago
[16
]. The in vitro biosynthesis of the complete lipidlinked
C. jejuni heptasaccharide from UDP derivatives of
galactose and N-acetylglucosamine by coupling the
required enzymes, followed by the transfer of the glycan
to a target protein, is a particularly impressive achievement
[12,15]. Structural insight into the reactions of
bacterial N-glycosylation is now beginning to emerge
with the recent solution of the crystal structure of PglD,
solved in complex with acetyl-CoA as cosubstrate [17].
The central enzyme of the Pgl system is the OST, PglB.
This 82 kDa integral membrane protein shares significant
primary sequence similarity with STT3, an essential
component of the nine-member OST complex of yeast
and crucial for the proper functioning of all eukaryotic
cells [5
]. In the case of N-glycosylation by the C. jejuni
OST, the eukaryotic sequon, N-X-S/T is N-terminally
extended to D/E-Z-N-X-S/T, where Z and X can be any
amino acid except proline [18]. Although DQNAT is the
optimal bacterial acceptor sequence [19], not all sequons
are glycosylated, as elsewhere.
O-Glycosylation — a target for therapeutics?
Bacterial O-glycosylation also makes use of several unusual
sugars. The pili of Neisseria meningitides [20] and Neisseria
gonorrhoeae [21] contain serine O-linked glycans, the first of
which was reported 13 years ago as Gal-b1,3-Gal-a1,3-2,
4-diacetamido-2,4,6-trideoxyhexose (DATDH) [20].
Despite the passage of time, the stereochemistry of
Bacterial and archaeal protein glycosylation Abu-Qarn, Eichler and Sharon 545
Figure 1
Rare monosaccharide constituents of archaeal and bacterial
glycoproteins: di-N-acetylbacillosamine, N-acetylfucosamine,
pseudaminic acid, legionaminic acid, 6-Thr-2-acetamido-2-deoxy-bmannuronic
acid, and 2,3-diacetamido-2,3-dideoxy-b-glucuronic acid.
Table 1
Select protein-linked glycans in Bacteria and Archaea
Organism Glycan structure Reference
Bacteria
N-linked
Campylobacter jejuni GalNAc-a1,4-GalNAc-a1,4-(Glc-b1,3)-GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,3-BacAc2-Asn [9
]
O-linked
Neisseria gonorrhoeae Gal-b1,4-Gal-a1,3-DATH-Ser [21]
Pseudomonas aeruginosa 1244 5N(3-OH)But7NFmPse-a2,4-Xyl-b1-3FucNAc-b-Ser [24]
Archaea
N-linked
Halobacterium salinarum [GalNAc-3-1-(3fGal)GalA-4-1-(6GalA3OCH3)GlcNAc-4-](10–15)-1-GalNAc-Asn [46]
(OSO3)GlcA-[b1,4-GlcA(OSO3)]2-b1,4-Glc-Asn [37]
Methanococcus voltae ManNAcA6Thr-b1,4-GlcNAc3NAcA-b1,3-GlcNAc-Asn [36]
Linking sugars are shown in bold. Abbreviations used: BacA2, di-N-acetylbacillosamine; DATH, 2,4-diacetamido-2,4,6-trideoxyhexose; FmPse,
formyl-pseudaminic acid; FucNAc, N-acetylfucosamine; Gal, galactose; GalA, galacturonic acid; GalNAc, N-acetylgalactosamine; Glc, glucose;
GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; GlcNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-glucuronic acid; ManNAcA, N-acetylmannuronic
acid; Xyl, xylose.
www.sciencedirect.com Current Opinion in Structural Biology 2008, 18:544–550
DATDH has yet to be unambiguously determined,
though it is believed to be that of bacillosamine. Neisseria
spp. type IV pilin has been shown to be glycosylated at
Ser63 by a derivative of DATDH in which the position 4
acetamide is replaced by a glyceramide, CH2OH–
CH2OHCONH [22]. By contrast, pili of Pseudomonas
aeruginosa contain O-linked pseudaminic acid (Pse)
(Figure 1), an analog of sialic acid [23], as do flagellin
proteins of other Gram-negative pathogens, such as H.
pylori [24] and Campylobacter coli [25], the latter of which
also contains another rare monosaccharide sharing the
same stereochemistry as sialic acid, that is, legionaminic
acid (Figure 1) [26]. In P. aeruginosa strain 1244, pilin
proteins contain a glycan of unusual structure, namely a
trisaccharide composed of hydroxybutyryl-formyl-pseudaminic
acid a2,4-linked to xylose, in turn b1,4-linked to
N-acetylfucosamine (Table 1) [22]. The latter sugar is
attached to the hydroxyl of the pilin terminal Ser, the
carboxyl of which must be unsubstituted [27]. Mention
should also be made of the threonine-linked oligomers of
a-arabinofuranosides found in type IV pilins of P. aeruginosa
[28].
546 Carbohydrates and glycoconjugates
Figure 2
Models of N-linked and O-linked glycosylation in Bacteria and Archaea. Upper panel: N-glycosylation in Campylobacter jejuni. Middle panel: Oglycosylation
of Neisseria gonorrhoeae pilin. Lower panel: N-glycosylation of the Haloferax volcanii S-layer glycoprotein. Bac, di-Nacetylbacillosamine;
DATDH, Gal-b3-Gal-a3-2,4-diacetamido-2,4,6-trideoxyhexose; Dol-P, dolichyl phosphate; GalNAc, N-acetylgalactosamine; Glc,
glucose; GlcNAc, N-acetylgucosamine; Hex, hexose; HexUA, hexuronic acid; UMP, uridine monophosphate; Und-PP, undecaprenyl-pyrophosphate;
190 Da, uncharacterized 190 Da saccharide. The top two panels are reproduced, with permission, from [34
].
Current Opinion in Structural Biology 2008, 18:544–550 www.sciencedirect.com
Pilin and flagellin glycosylation is, in many cases, necessary
for proper assembly of the corresponding filaments,
for bacterial motility, for colonization, and hence, for
virulence. As such, deciphering bacterial O-glycosylation
pathways will open novel avenues for combating bacterial
diseases. Till date, the pathways of Pse [13,29–31] and
legionaminic acid [25,32] biosynthesis have been defined
(Figure 3). Interestingly enough, the immediate precursor
of legionaminic acid is di-N-acetylbacillosamine. In
their active forms, both Pse and legionaminic acid are
attached to CMP, just like N-acetylneuraminic acid
(Figure 2). Remarkably, the conversion of UDP-GlcNAc
to CMP-Pse was achieved in a single reaction combining
the six enzymes involved in its biosynthesis [30]. This
represents the first complete in vitro enzymatic synthesis
of a sialic acid like sugar and sets the groundwork for
future small-molecule inhibitor screening and design.
Indeed, CMP-Pse was found to be a potent inhibitor
of PseB, the first enzyme of the Pse pathway in
Campylobacter coli and H. pylori [33]. This observation
led to the conclusions that Pse levels are feedbackregulated
within the bacterial cell and that PseB represents
a key control point for Pse production, underscoring the
importance of this enzyme as a therapeutic target.
The three-dimensional structure of another central component
in Pse biosynthesis, that is, the aminotransferase
PseC (HP0366), has been determined in complex with
pyridoxal phosphate alone and in combination with the
UDP-4-amino-4,6-dideoxy-L-AltNAc (N-acetylaltrosamine)
intermediate [31]. This is the first time the structure
of a nucleotide–sugar aminotransferase has been
cocrystallized with its natural ligand, revealing the
enzyme to form a homodimer, where each monomer
contributes to the active site.
Quite surprisingly, the bacterial pilin O-glycosylation and
N-glycosylation pathways resemble each other (Figure 2).
The N. meningitides pgl gene cluster, responsible for the Olinked
glycosylation of pilin in this species, displays
significant homology to the pgl cluster of C. jejuni. On
the basis of the results of combined reverse genetics and
mass spectrometry, a neisserial pilin glycosylation pathway
paralleling that proposed for C. jejuni N-linked
protein glycosylation has been proposed [34
]. Like
the N-linked oligosaccharide of C. jejuni, the O-linked
oligosaccharide of N. gonorhoaea is also preassembled
through the sequential addition of nucleotide-activated
monosaccharides onto a lipid carrier (Figure 2). This is
extremely unusual, since all other O-glycans studied till
date are assembled via sequential addition of their monosaccharide
constituents directly onto a protein acceptor.
Finally, as with the C. jejuni N-glycosylation pathway, the
O-glycosylation systems of P. aeruginosa 1244 and N.
meningitidis MC58 have been expressed in E. coli [35
].
In both cases, assembled oligosaccharides are transferred,
Bacterial and archaeal protein glycosylation Abu-Qarn, Eichler and Sharon 547
Figure 3
Pathways for the biosynthesis of (a) di-N-acetylbacillosamine, N-acetylfucosamine, and N,N0
-diacetyllegionaminic acid [16
,25,32] and (b)
pseudaminic acid [29].
www.sciencedirect.com Current Opinion in Structural Biology 2008, 18:544–550
en bloc, from a lipid carrier by the action of the OSTs PilO
(in P. aeruginosa) or PglL (in N. meningitidis). Although
both PilO and PglL show relaxed glycan specificity, the
former activity is restricted to short oligosaccharides. By
contrast, PglL is able to transfer diverse oligosaccharides
and polysaccharides. Such functional characterization
supports the concept that despite their low sequence
similarity, PilO and PglL belong to a new family of ‘OOTAses’
that transfer oligosaccharides from lipid carriers
to hydroxylated amino acids in proteins [35
]. To date, no
such activity has been identified in eukaryotes.
Archaeal N-glycosylation
Post-translational modification in extreme conditions
In contrast to bacterial N-glycosylation, which is still
considered a relatively rare event, N-glycosylation of
archaeal proteins is more widespread. Although the sugar
content of several archaeal glycoproteins has been known
for several years, more detailed structural information on a
limited number of glycans has recently become available.
In the methanoarchaea Methanococcocus voltae, mass spectrometry,
together with NMR analysis, has revealed the
structure of the glycan entity decorating 15 of the 17
sequons distributed among the 4 flagellin proteins of this
species (FlaA, FlaB1, FlaB2, and FlaB3), as well as the Slayer
glycoprotein, as a novel trisaccharide, 6-O-threonylManNAcb1,4GlcNAc3NAcAb1,3GlcNAc
[36] (Table 1).
The similar modification of both flagellins and the S-layer
glycoprotein points to a common N-glycosylation mechanism
in these cells. Mass spectrometry has also provided
initial characterization of the pentasaccharide decorating
at least two of the seven putative sequons of the S-layer
glycoprotein in the haloarchaea Haloferax volcanii. Instead
of the linear string of b1,4-linked glucose residues originally
reported as decorating the protein [37], it now appears
that the protein is modified by a pentasaccharide comprising
two hexoses, two hexuronic acids, and a 190 Da
species [38]. Recent in vitro studies using either isolated
membranes or affinity-purified OST from the hyperthermophile
Pyrococcus furiosus have examined the glycan
moiety transferred to a fluorescently tagged, sequonpresenting
hexapeptide [39,40
]. Mass spectrometry
revealed the Asn-attached oligosaccharide to be composed
of two N-acetylhexosamines, two hexoses, one
hexuronic acid, and two pentoses, with GalNAc serving
as the protein-linking sugar [40
].
Over the past two years, in parallel to efforts aimed at
describing the structure of N-linked glycan structures,
major strides have been made in identifying archaeal
genes whose products are involved in the protein Nglycosylation
process. In H. volcanii and M. voltae, several
agl (archaeal glycosylation) genes have been cloned and
their products functionally characterized via combined
gene deletion and mass spectrometry approaches
[41
,42
]. In M. voltae, AglA serves to add the terminal
sugar subunit of the N-linked trisaccharide, namely a
modified mannuronic acid with a covalently attached
threonine residue (Table 1) [42
], while AglH, a homolog
of the yeast Alg7 protein, is involved in addition of the
Asn-bound linking sugar, GlcNAc [43]. In H. volcanii,
AglD, AglE, AglF, AglG, and AglI participate in the
addition of the second to fifth sugar subunits of the still
incompletely characterized pentasaccharide linked to the
S-layer glycoprotein (Figure 2) [38,44,45].
In both M. voltae and H. volcanii, aglB encodes the sole
component of the archaeal OST. Indeed, the archaeal
OST, comprising a single subunit homologous to the core
STT3 subunit of the multimeric eukaryotic complex,
offers a simplified model system for understanding
the mechanism of the enzyme. Accordingly, Kohda and
548 Carbohydrates and glycoconjugates
Figure 4
The 3D structure of the soluble carboxyl terminal domain of Pyrococcus furiosus AglB/STT3. Stereo-view of the carboxyl terminal soluble domain of
AglB/STT3 (residues 471–967). The core domain is shown in blue, the insertion domain is shown in green, and the peripheral domains are shown in red
and orange. The WWDYG catalytic motif is shown in magenta, while the C638–C658 disulfide bond is shown as yellow sticks. The yellow sphere
represents a bound metal ion. Reprinted by permission from Macmillan Publishers Ltd: [40
], copyright 2008.
Current Opinion in Structural Biology 2008, 18:544–550 www.sciencedirect.com
colleagues recently solved the crystal structure of the P.
furiosus STT3/AglB C-terminal domain to 2.7 A˚ , for the
first time providing structural insight into OST activity
[40
] (Figure 4). This soluble domain assumes a compact,
globular structure that can be divided into four
regions, based on tertiary folds. The central core domain,
largely consisting of a-helices, includes the WWDYG
motif implicated in the catalytic activity of the enzyme.
The insert domain corresponds to a 10-stranded antiparallel
b-barrel structure found in the central core domain
and appears to be unique to P. furiosus and its close
relatives. Two peripheral domains, composed mostly of
b-strands, surround the central core. Multiple alignments
of P. furiosus STT3/AglB and its homologs from Archaea,
Bacteria,andEukaryarevealedthepresenceofa conserved
DxxK motif. Examination of the 3D structure of P. furiosus
Stt3/AglB reveals the Asp and Lys residues of this motif are
found on the same side of a long helix, positioned in
proximity to the catalytic WWDYG motif. It was
suggested, therefore, that the DxxK sequence represents
a new motif that also participates in the activity of the
enzyme, probably interacting with the pyrophosphate
moiety of the lipid carrier on which N-linked oligosaccharides
are assembled and transported. Subsequent sitedirectedmutagenesisof
these residuesin the yeast enzyme
has provided experimental support for this hypothesis.
Conclusions
Researchers are only now beginning to appreciate the
variety of prokaryotes capable of protein glycosylation
and the wide range of monosaccharides found in the glycan
moieties of prokaryotic glycoproteins. At the same time,
prokaryotic protein glycosylation pathways are being
delineated. Continued investigation into the bacterial
N-glycosylationandO-glycosylationprocesseswilladvance
glyco-engineering efforts as well as the development of
new antibacterial agents. A more comprehensive understanding
of N-glycosylation and O-glycosylation in Archaea
also carries enormous applied potential, given the possible
links between glycosylation of archaeal proteins and their
ability to withstand diverse physical challenges. Clearly,
the future looks sweet for the field of bacterial and archaeal
protein glycosylation.
Acknowledgements
The authors wish to thank Martin Young and Susan Logan for their
insightful comments. The Eichler laboratory is supported by the Israel
Science Foundation (grant 30/07), the US Air Force Office for Scientific
Research (grant FA9550-07-10057), and the US Army Research Office
(grant W911NF-07-1-0260).
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550 Carbohydrates and glycoconjugates
Current Opinion in Structural Biology 2008, 18:544–550 www.sciencedirect.com