Glycobiology vol. 20 no. 9 pp. 1065–1076, 2010
doi:10.1093/glycob/cwq055
Advance Access publication on April 5, 2010
REVIEW
Protein glycosylation in Archaea: Sweet and extreme
2
1,2
2
Department of Life Sciences, Ben Gurion University of the Negev, Beersheva
84105, Israel
Received on March 7, 2010; revised on March 31, 2010; accepted on
March 31, 2010
While each of the three domains of life on Earth possesses
unique traits and relies on characteristic biological strategies, some processes are common to Eukarya, Bacteria and
Archaea. Once believed to be restricted to Eukarya, it is
now clear that Bacteria and Archaea are also capable of
performing N-glycosylation. However, in contrast to Bacteria, where this posttranslational modification is still
considered a rare event, numerous species of Archaea, isolated from a wide range of environments, have been
reported to contain proteins bearing Asn-linked glycan
moieties. Analysis of the chemical composition of the
Asn-linked polysaccharides decorating archaeal proteins
has, moreover, revealed the use of a wider variety of sugar
subunits than seen in either eukaryal or bacterial glycoproteins. Still, although first reported some 30 years ago, little
had been known of the steps or components involved in the
archaeal version of this universal posttranslational modification. Now, with the availability of sufficient numbers of
genome sequences and the development of appropriate experimental tools, molecular analysis of archaeal Nglycosylation pathways has become possible. Accordingly
using halophilic, methanogenic and thermophilic model
species, insight into the biosynthesis and attachment of
N-linked glycans decorating archaeal glycoproteins is
starting to amass. In this review, current understanding
of N-glycosylation in Archaea is described.
Keywords: Archaea /extremophiles / N-glycosylation /
posttranslational modification
Archaea: the third domain of life
The Archaea were first recognized as a distinct domain of life,
unrelated to either Bacteria or Eukarya, in 1977, as a result of
Carl Woese's pioneering use of 16S ribosomal (r)RNA analysis
(Fox et al. 1977; Woese and Fox 1977). At the time, those microorganisms assigned to the archaeal domain were all
extremophiles, i.e. species able to thrive in the face of some
of the most physically challenging conditions on the planet.
Archaea were shown to reside in seemingly ‘harsh’ surround1
To whom correspondence should be addressed: Tel: +972-8646-1343; Fax:
+972-8647-9175; e-mail: [email protected]
ings, such as those characterized by extremes in salinity, pH or
temperature, to live in sulfur-based environments or to produce
methane as a by-product of their anaerobic respiration (cf.
Rothschild and Mancinelli 2001). However, with the subsequent widespread application of 16S rRNA analysis, it
became clear that Archaea are major denizens of ‘normal’ biological niches, including seawater, soil and even our own
intestinal flora (DeLong 1998; Chaban, Ng, et al. 2006). Indeed, it is now recognized that Archaea are major players in
the ecosystem, important for processes as diverse as the Earth's
nitrogen cycle and global warming (Francis et al. 2007; Galperin 2007).
As befitting a distinct form of life, Archaea possess traits
that distinguish them from either Bacteria or Eukarya, in addition to their characteristic rRNA. For example, archaeal
membranes are composed of phospholipids of strikingly different composition than those used elsewhere. Whereas bacterial
and eukaryal phospholipids comprise fatty acyl groups esterlinked to the sn-1,2 positions of glycerol, in Archaea, membrane lipids comprise polyisoprenyl groups ether-linked to
the sn-2,3 positions of a glycerol backbone (Sprott 1992).
On the other hand, Archaea share much in common with both
Bacteria and Eukarya. Like Bacteria, Archaea are single-celled
organisms surrounded by a single membrane and lack internal
organelles. Moreover, in both cases, the genome is organized
as a single, circular chromosome, often divided into operons
(Koonin and Wolf 2008). When, however, replication, DNA
packaging, transcription and other aspects of information processing are considered, Archaea are more reminiscent of !
Eukarya (Sandman and Reeve 2000; Bell and Jackson 2001).
Thus, archaeal biology can be considered a mosaic of archaealspecific, bacterial-like and eukaryal-like traits. As such, the
study of Archaea has not only revealed biological strategies
not seen elsewhere but has also served to reveal links between
bacterial and eukaryal processes previously thought to be unrelated, as well as providing first examples of universal biological
phenomena. With respect to protein glycosylation, Archaea
have provided examples of each of these three scenarios.
Archaeal N-linked glycoproteins: here, there and
everywhere
Since Neuberger reported that a carbohydrate group was an integral part of ovalbumin some 75 years ago (Neuberger 1938),
it was believed that N-glycosylation was a trait restricted to Eukarya. This belief was, however, challenged in 1976 with the
demonstration that the surface (S)-layer glycoprotein of the extreme halophile, Halobacterium salinarum, experiences this
same processing event (Mescher and Strominger 1976a). De-
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2
Doron Calo , Lina Kaminski , and Jerry Eichler
D Calo et al.
tailed analysis showed that the S-layer glycoprotein is modified
by two different Asn-linked oligosaccharides, with Asn-2 being
decorated with a repeating sulfated pentasaccharide, bound via
an N-glycosylamine bond, and 10 other Asn residues being
modified with a sulfated glycan, linked through a glucose residue (Wieland et al. 1980; Wieland et al. 1983; Lechner et al.
1985a; Lechner and Wieland 1989) (Figure 1A). At the time of
first being shown to contain N-linked glycans, Hbt. salinarum
was defined as an obligate halophilic bacterial species and considered an odd member of the bacterial world both because of
its unusual habitat and due to its ability to N-glycosylate protein
targets. However, with the realization that life on Earth comprised three distinct domains, i.e. Eukarya, Bacteria and
Archaea (Woese and Fox 1977), Hbt. salinarum was reassigned
to the archaeal branch of the universal tree of life, with its ability to perform N-glycosylation now offering further support for
the similarity of Archaea to Eukarya and their distinctiveness
from Bacteria, as suggested by the original 16S rRNA analysis
used to distinguish between the three forms of life. Indeed, as
discussed below, the concept of archaeal and eukaryal symmetry gained further support from early investigations into the
archaeal N-glycosylation pathway.
Not long after the Hbt. salinarum S-layer glycoprotein was
identified as the first noneukaryal N-modified glycoprotein,
other similarly modified archaeal polypeptides were described.
These included the Hbt. salinarum flagellin, shown to bear the
same glycan-linked sulfated polysaccharide as the S-layer glycoprotein from this species (Wieland et al. 1985), and the Slayer glycoprotein of Haloferax volcanii, a halophilic species
first isolated from the Dead Sea (Mullakhanbhai and Larsen
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1975; Sumper et al. 1990). Since, N-linked glycans presenting
enormous diversity in their sugar composition have been identified in numerous archaeal proteins derived from a variety of
species isolated from a wide range of environmental niches.
Methanogenic Archaea, possessing the ability to generate
CH4 from CO2 and H2 or other carbon sources and detected
in environments spanning a broad spectrum of temperature, salinity, pressure and pH (Thauer et al. 2008), also express
glycoproteins bearing N-linked glycans. For example, the
Methanothermus fervidus S-layer glycoprotein is modified by
a hexasaccharide composed of a N-acetylgalactosamine (GalNAc) subunit linked to the target asparagine residue, three
mannose residues and finally, two methylated hexoses (mannose or glucose) at the nonreducing end of the glycan
(Figure 1B; Kärcher et al. 1993). In Methanococcus voltae, a
trisaccharide composed of a N-linked N-acetylglucosamine
(GlcNAc), a 2,3-diacetamido-2,3-dideoxy-β-glucuronic acid
group and a terminal 2-acetamido-2-deoxy-β-mannuronic acid
moiety, with the carbonyl group at C-6 forming an amide bond
with the amino group of threonine, was detected on both the Slayer glycoprotein and flagellins (Voisin et al. 2005). In some
strains, this trisaccharide is augmented by an extra 220 or
262 Da entity of unknown identity (Chaban et al. 2009). Finally, the tetrameric glycan N-linked to Methanococcus
maripaludis flagellin is reminiscent of its M. voltae counterpart. In M. maripaludis, the glycan comprises a N-linked
GalNAc, followed in turn by a 2,3-diacetamido-2,3-dideoxyβ-glucuronic acid group, as in the M. voltae glycan, and a 3acetamidino derivative of 2,3-diamino-2,3-dideoxymannuronic
acid amidated with a threonine amino group found in the M.
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Fig. 1. The composition of representative N-linked glycans decorating archaeal glycoproteins. N-Linked glycans from (A) the Hbt. salinarum S-layer glycoprotein,
(B) the M. fervidus S-layer glycoprotein and (C) S. acidocaldarius cytochrome b558/566 are presented. In (A), the upper glycan is found at Hbt. salinarum S-layer
glycoprotein Asn-2, while the lower glycan is found at 10 other sequons. The abbreviations used here but not in the text are: fGal, galactofuranose; GalUA,
galacturonic acid; GlcUA, glucuronic acid; Man, mannose; Me, methyl; Qui, quinovose.
Archaeal N-glycosylation
Table I. N-glycosylation in the three domains of life
Eukarya
Essential property?
Sugar donor
Oligosaccharide-charged lipid carrier
Archaea
Yes
Nucleotide-activated sugars
Dolichol-phosphate-bound glycans
Dolichol pyrophosphate
No
Nucleotide-activated
sugars
Undecaprenol
pyrophosphate
No
Nucleotide-activated sugars?
Dolichol-(pyro)phosphate-bound glycans?
Dolichol pyrophosphate
Dolichol phosphate (Hbt. salinarum, Hfx. volcanii)
ATP independent
Rft1 may be involved in flipping
ATP independent
PglK
Unknown
Unknown
Multimeric
Stt3
Lumenal face of the ER membrane
N-X-S/T; X≠P
Type E
Trimming in ER, elaboration in Golgi
Single subunit
PglB
External surface of cell
D/E-Z-N-X-S/T; Z,X≠P
Type B
None reported
Single subunit
AglB
External surface of cell
N-X-S/T; X≠P (N-X-N/L/V, Hbt. salinarum)
Types A, B and E
None reported
voltae glycan at this position. The M. maripaludis glycan is,
however, capped by the unique terminal sugar, 2-acetamido2,4-dideoxy-5-O-methyl-hexos-5-ulo-1,5-pyranose. This apparently represents the first example of a naturally occurring
diglycoside of an aldulose (Kelly et al. 2009).
Thermophilic and hyperthermophilic Archaea, the latter
growing optimally at temperatures above 80°C, also contain
experimentally confirmed N-modified glycoproteins. Thermoplasma acidophilum, growing optimally at 56°C and pH 2,
lacks a cell wall but is surrounded by a highly glycosylated
membrane protein reported to contain a branched glycan largely based on mannose subunits yet also containing glucose and
galactose moieties, asparagine-linked through GlcNAc (Yang
and Haug 1979a). Cytochrome b558/566 from Sulfolobus acidocaldarius, originally isolated from solfatara regions in
Yellowstone National Park and shown to grow optimally at
75 to 80°C and pH 2 to 3 (Brock et al. 1972), is modified
by a hexasaccharide that includes a glucose subunit, two mannose residues, a 6-sulfoquinovose subunit and two GlcNAc
groups, one of which serves as the linking sugar
(Figure 1C; Hettmann et al. 1998; Zähringer et al. 2000).
Although considered common in glycolipids of chloroplasts
and photosynthetic Bacteria (Imhoff et al. 1982), 6-sulfoquinovose (or 6-deoxy-6-sulfoglucose) had not been previously
detected in a glycoprotein.
For a listing of additional confirmed or proposed N-modified
archaeal glycoproteins, along with the evidence supporting the
claim for glycan modification, the reader is directed to the review by Eichler and Adams (2005).
Not like everybody else
Today, it is clear that organisms from all three domains are capable of performing N-glycosylation (Weerapana and Imperiali
2006; Abu-Qarn, Eichler, et al. 2008). Still, archaeal N-glycosylated proteins can be distinguished from their eukaryal and
bacterial counterparts not only by the extremophilic nature of
their source species or the unusual sugars employed but also in
terms of the amino acid sequence surrounding modified sequons and with respect to how oligosaccharides are N-linked
to target proteins (Table I).
As in Eukarya, archaeal N-glycosylation occurs as NXS/Tbased sequons, in contrast to the more elaborate D/EZNXS/T-
based motif reportedly employed in bacterial (i.e. Campylobacter jejuni) N-glycosylation where X and Z are any residue but
proline (Kowarik et al. 2006; Abu-Qarn and Eichler 2007).
Still, although relatively few sequons in archaeal glycoproteins
have been experimentally verified as being modified, the amino acid composition within and surrounding these sequons can
be distinguished from the comparable positions in eukaryal
glycoproteins (Abu-Qarn and Eichler 2007). For instance, if
the modified Asn is considered as position 0, there is a high
probability of finding aromatic residues at positions −2 and
−1, a small hydrophobic residue (Gly or Val) at the X position
and a larger hydrophobic residue (Ile, Leu, Met, Phe, Trp or
Tyr) at the +1 position in a eukaryal glycoprotein (Ben-Dor
et al. 2004; Petrescu et al. 2004). In contrast, in only few archaeal sequons confirmed as being modified are Phe, Trp or
Tyr detected at positions −2 and −1, while Ser or Thr are readily found at the X position and Ala and Gly predominate at the
+1 position. No example in Archaea of an aromatic residues
being present at the +1 position has been reported. Indeed,
the only time a Tyr was detected at this site was in a sequon
experimentally shown to be nonmodified (Lechner and Sumper
1987). In vitro analysis of oligosaccharyltransferase (OST) activity in the hyperthermophile, Pyrococcus furiosus, revealed
that modification of an Asn-X-Thr-bearing reporter peptide
was more efficient than was processing of the same peptide
presenting an Asn-X-Ser sequon, with either the sequon Thr
or Ser being essential for glycosylation and the presence of
Pro at the X position preventing sequon modification (Igura
et al. 2008). On the other hand, as elaborated below in the section dealing with the archaeal OST, modifications in archaeal
sequon composition are tolerated in Hbt. salinarum, suggesting
that predictive algorithms may have overlooked archaeal glycoproteins bearing similarly modified or even novel Nglycosylation sites (Zeitler et al. 1998).
The linking sugar serving to attach an oligosaccharide to a
modified Asn residue also differs in glycoproteins across the
three domains of life. In bacterial N-glycoproteins characterized
thus far, bacillosamine (2,4-diamino-2,4,6-trideoxyglucopyranose) serves as the linking sugar (Young et al. 2002), whereas
in the vast majority of eukaryal N-glycoproteins, GlcNAc serves
this role (Spiro 1973). Rare instances of eukaryal reliance on
other linking sugars have been reported, such as glucose in
the case of laminin (Schreiner et al. 1994). In Archaea, a va1067
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Flippase
Process
Identity
OST
Oligomeric status
Catalytic subunit
Localization
Sequon recognized
OST type
Modification of N-linked glycan?
Bacteria
D Calo et al.
Early insight into the archaeal N-glycosylation pathway
Encouraged by the conclusion made at the time that N-glycosylation was restricted to Eukarya and Archaea, initial efforts
aimed at defining the pathway responsible for this posttranslational modification in Archaea sought further similarities
between the archaeal pathway and its eukaryal counterpart.
The sugar carrier
As in Eukarya (Burda and Aebi 1999), the archaeal N-linked
oligosaccharide is apparently assembled on a dolichol carrier,
rather than the undecaprenol carrier used in bacterial N-glycosylation (Szymanski and Wren 2005). Accordingly, Archaea
contain both dolichol phosphate and dolichol pyrophosphate
bearing either mono- or polysaccharides. Specifically, Hbt. salinarum has been reported as containing C60-dolichols carrying
glucose, mannose and GlcNAc units, as well as a sulfated tetrasaccharide (Mescher et al. 1976; Lechner et al. 1985a). Hfx.
volcanii contains mannosyl-(β1-4)-galactosyl phosphodolichol, lesser quantities of sulfated or phosphorylated dihexosyl
phosphodolichol and dolichol phosphate bearing a tetrasaccharide comprising mannose, galactose and rhamnose subunits. In
this haloarchaeon, the glycans were all linked to C55- and/or
C60-dolichol moieties (Kuntz et al. 1997). The saccharidecharged dolichol species in Hfx. volcanii are unusual in that
the ω-terminal isoprene unit is saturated and since only monophosphorylated dolichol is observed.
Proof for the involvement of such glycan-charged dolichols
in archaeal protein N-glycosylation has also been provided.
The transfer of radiolabeled glucose from uridine diphosphate
(UDP)-[3H]glucose to glycoproteins was shown to proceed
through a glucose-containing phosphopolyisoprenol intermediate in Hfx. volcanii (Zhu et al. 1995). Treatment with
bacitracin, a compound that interferes with recycling of pyrophosphodolichyl polysaccharide carriers following release of
their bound oligosaccharides (Stone and Strominger 1971),
was able to prevent modification of the Hbt. salinarum S-layer
glycoprotein by the glucose-linked sulfated glycan otherwise
added to 10 sequons of the protein (Mescher and Strominger
1978). In contrast, bacitracin treatment failed to prevent addition of the GlcNAc-linked repeating sulfated pentasaccharide
at Hbt. salinarum S-layer glycoprotein Asn-2, suggesting the
use of dolichol phosphate rather than dolichol pyrophosphate
as the carrier for this glycan (Wieland et al. 1980). Indeed, the
reportedly exclusive use of phosphodolichyl sugar carriers in
Hfx. volcanii N-glycosylation is reflected in the inability of
bacitracin to prevent such protein modification in this species
(Kuntz et al. 1997; Eichler 2001). Further evidence linking dolichol-charged glycans to N-glycosylation comes with the
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observation that in Hbt. salinarum, the glycan moiety of the
pyrophosphodolichol-bound sulfated polysaccharide is also detected on the S-layer glycoprotein and flagellins in this species
(Lechner et al. 1985a; Wieland et al. 1985). The sulfated polysaccharide is methylated in the pyrophosphodolichol-linked
form but not when protein-bound (Lechner et al. 1985b). In
contrast, the hexasaccharide moiety attached to the M. fervidus
S-layer glycoprotein retains the methylation introduced at the
dolicholpyrophosphate carrier level (Hartmann and Konig
1989; Kärcher et al. 1993), as also seems to be the case in
Hfx. volcanii, where methylation of the S-layer glycoproteinbound pentasaccharide is observed (Magidovich et al. 2010).
Facing the outside
When the topology of N-glycosylation was considered, further
parallels between the eukaryal and archaeal pathways were
shown, with evidence obtained pointing to archaeal N-glycosylation as occurring on the outer surface of the cell, the
topological equivalent of the lumenal-facing leaflet of the endoplasmic reticulum (ER) membrane bilayer, the site of Nglycosylation in Eukarya. Localization of archaeal N-glycosylation to the external cell surface is supported by studies
showing the ability of Hbt. salinarum cells to modify cell-impermeable, sequon-bearing hexapeptides with sulfated
oligosaccharides (Lechner et al. 1985a). Although unable to
cross the haloarchaeal plasma membrane (Mescher and Strominger 1978), bacitracin is nonetheless able to interfere with
N-glycosylation in Hbt. salinarum, ultimately preventing the
transfer of sulfated oligosaccharides to the S-layer glycoprotein
(Mescher and Strominger 1978; Wieland et al. 1980). Additional support for archaeal N-glycosylation occurring on the
outer cell surface came with the description of membranebound pyrophosphatases that orient their active site to the exterior (Meyer and Schäfer 1992; Amano et al. 1993). It was
proposed that such enzymes could participate in the archaeal
N-glycosylation process by dephosphorylating dolichol pyrophosphate, presumably following transfer of oligosaccharides
from the lipid carrier to protein targets (Meyer and Schäfer
1992). Accordingly, an externally oriented membrane-bound
pyrophosphatase from the thermoacidophile Sulfolobus tokodaii has been recently shown to be able to hydrolyze
isopentenylpyrophosphate and geranylpyrophosphate (Manabe
et al. 2009). Finally, studies supporting a cotranslational
mode of membrane protein insertion in Archaea also lend
support to protein glycosylation transpiring on the exterior
surface of the cell (Gropp et al. 1992; Dale and Krebs
1999; Ring and Eichler 2004).
Enzymes of glycosylation
In addition to these early efforts aimed at delineating steps and
components of the archaeal N-glycosylation pathway, several
groups also addressed enzymes putatively involved in this
posttranslational modification, including enzymes proposed
to participate in the assembly of the oligosaccharide-charged
phosphodolichol carrier. GlcNAc transferase activity was partially characterized from Hbt. salinarum membranes (Mescher
et al. 1976). Photo-affinity experiments using 5-azido-[32P]
UDP-glucose identified a putative phosphodolichol glucose
synthase in Hfx. volcanii homogenates (Zhu et al. 1995), while
phosphodolichol mannose synthase, susceptible to inhibition
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riety of saccharides have been shown to serve as linking sugar, including glucose, GlcNAc and GalNAc. Indeed, as noted
above, in the Section Archeal N-linked glycoproteins: here,
there and everywhere, two different linking sugars can be
employed within the same Archaeal glycoprotein, as exemplified by the Hbt. salinarum S-layer glycoprotein. The ability
to use different linking sugars in the same glycoprotein is especially intriguing when one considers that Hbt. salinarum
seemingly only encodes a single OST (Magidovich and Eichler 2009; see below).
Archaeal N-glycosylation
by amphomycin, was purified from T. acidophilum (Zhu
and Laine 1996).
Thus, the first two decades of research into archaeal N-glycosylation provided glimpses of selected aspects of the process,
in a variety of model systems. Detailed understanding of the archaeal version of this posttranslational modification would have
to, however, wait until the dawn of the genome era.
Homology shows the way
Sweet and salty: N-glycosylation in Haloferax volcanii
As the major polypeptide in the species and previously shown
to be amenable to the study of other posttranslational modifications (e.g. signal peptide cleavage (Fine et al. 2006) and
protein lipid modification (Konrad and Eichler 2002)), the
Hfx. volcanii S-layer glycoprotein represents an excellent model for addressing archaeal N-glycosylation. At the time of its
description (Sumper et al. 1990; Mengele and Sumper 1992),
it was reported that of the seven putative N-glycosylation sites
present in the S-layer glycoprotein, Asn-13 and Asn-505 were
each modified by a linear chain of β-1-4 linked glucose resi-
Fig. 2. Working models of archaeal N-glycosylation pathways. Currently
identified components of the N-glycosylation pathways of (A) Hfx. volcanii,
(B) M. voltae and (C) M. maripaludis. The abbreviations used are: dolP,
dolichol phosphate; dolPP, dolichol pyrophosphate; NDP, nucleoside
diphosphate; 1P, 1 phosphate. Dolichol, embedded in the schematicallydepicted plasma membrane (drawn as the two parallel horizontal lines), is
represented by a vertical line. The identities of the sugars represented by each
shape are listed next to Asn-linked glycan in each panel. In (B), the optional
introduction of a 220 or 260 Da sugar seen in some M. voltae species (Chaban
et al. 2009) is reflected by the use of parentheses.
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The examination of Archaea at the genome level began in
1996, when the first complete sequence of an archaeon
(corresponding to the fourth genome sequenced overall),
namely that of the methanogen Methanocaldococcus (then
Methanococcus) jannaschii, was published (Bult et al. 1996).
With the additional archaeal genome sequences now available
(today, over 140 archaeal genomes are at various stages of
completion (http://genomesonline.org/index2.htm; Oct.,
2009)), bioinformatics approaches were enlisted in the search
for components of the archaeal N-glycosylation pathway. Analysis of the genome of Archaeoglobus fulgidus, a sulfurmetabolizing organism that grows optimally at 83°C (Klenk
et al. 1997), first revealed the presence of gene clusters containing sequences predicted to encode elements of a Nglycosylation pathway (Burda and Aebi 1999). The major
breakthrough in identifying components of archaeal N-glycosylation pathways soon followed, coming from efforts
combining the identification of archaeal homologues of known
elements of the parallel eukaryal and bacterial pathways, together with gene deletion and subsequent analysis of the
effects of such deletion on the N-glycosylation of reporter glycoproteins. In this manner, Abu-Qarn and Eichler (2006) and
Chaban, Voisin, et al. (2006), respectively, studying N-glycosylation in Hfx. volcanii and M. voltae, simultaneously
identified gene products experimentally verified as participating in this posttranslational modification.
D Calo et al.
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N-glycosylation in the methanogens
Following the discovery that the same novel N-linked trisaccharide is attached to both the S-layer glycoprotein and
flagellins in M. voltae (Voisin et al. 2005), efforts focused on
defining the pathway of N-glycosylation in this obligate anaerobic methanogen. The fact that the same N-linked glycan
decorates both the S-layer glycoprotein and flagellins in M.
voltae (as is also the case in Hbt. salinarum (Wieland et al.
1985)) points to a common pathway for N-glycosylation in a
given species. As with Hfx. volcanii, studies of the N-glycosylation pathway of M. voltae began with the search for
homologues of known eukaryal or bacterial N-glycosylation
genes (Chaban, Voisin, et al. 2006). Accordingly, this strategy
led to the identification of aglA, shown to participate in the addition of the terminal sugar residue of the trisaccharide, namely
N-acetyl mannuronic acid linked to threonine, as well as of the
OST, aglB. Through its ability to complement a conditionally
lethal alg7 mutation in yeast, it was concluded that AglH is a
GlcNAc-1-phosphate transferase, catalyzing the transfer of
UDP-activated GlcNAc to dolichol pyrophosphate and responsible for the first step in the M. voltae N-glycosylation process
(Shams-Eldin et al. 2008). Finally, the second sugar residue of
the trisaccharide, a diacetylated glucuronic acid, is added by
the combined actions of two enzymes, AglC and AglK (Chaban et al. 2009), as reflected in Figure 2B.
The N-linked glycan decorating the flagellins of M. maripaludis corresponds to a tetrasaccharide in which the sugar
residues found at positions two and three are essentially identical to the two outer sugar subunits of the M. voltae N-linked
trisaccharide (Kelly et al. 2009). As such, M. maripaludis
AglA was identified as participating in the addition of the sugar
subunit at position three of the tetrasaccharide, an acetylated
and acetamidino-modified mannuronic acid linked to threonine
(VanDyke et al. 2009). However, given that the linking sugar
of the M. maripaludis glycan is GalNAc, rather than the
GlcNAc employed in M. voltae glycoproteins, the M. maripaludis enzyme involved in adding the diacetylated glucuronic
acid to position two of the tetrasaccharide, AglO, is not homologous to either M. voltae AglC or AglK (VanDyke et al. 2009).
Finally, AglL was assigned as participating in either the attachment of the threonine to the third sugar residue or the addition
of the terminal sugar, the novel 2-acetamido-2,4-dideoxy-5-Omethyl-hexos-5-ulo-1,5-pyranose (VanDyke et al. 2009)
(Figure 2C).
Pass the sugar: AglB, the archaeal OST
OSTs serve to transfer lipid-linked polysaccharides to select
Asn residues of target proteins. While the OST of higher Eukarya exists as a multimeric complex based on the Stt3 subunit
(Zufferey et al. 1995), the OST of Archaea, like that of Bacteria, comprises only a single component, i.e. AglB (Abu-Qarn
and Eichler 2006; Chaban, Voisin, et al. 2006; Igura et al.
2008). AglB was first identified in Hfx. volcanii and M. voltae,
when it was shown that deletion of the encoding gene led to a
loss of N-glycosylation of reporter glycoproteins (Abu-Qarn
and Eichler 2006; Chaban, Voisin, et al. 2006; Abu-Qarn et
al. 2007). Like its eukaryal (i.e. Stt3) and bacterial (i.e. PglB)
counterparts, AglB comprises a multiple membrane-spanning
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dues, while Asn-274 and/or Asn-279 were described as bearing
a glucose-, idose- and galactose-containing polysaccharide.
Later efforts relying on more sophisticated mass spectrometry
tools revealed the Hfx. volcanii S-layer glycoprotein to instead
be modified at Asn-13 and Asn-83 by a pentasaccharide comprising two hexoses, two hexuronic acids and a 190 Da species
(Abu-Qarn et al. 2007), subsequently shown to correspond to a
methyl ester of hexuronic acid (Magidovich et al. 2010). It was
also shown that the sequon at Asn-370 is not modified (AbuQarn et al. 2007).
In addition to redefining the N-linked glycan profile of the
S-layer glycoprotein, these more recent studies also served to
define components of the Hfx. volcanii N-glycosylation pathway. The initial use of bioinformatics tools to identity Hfx.
volcanii homologues of eukaryal and bacterial N-glycosylation
genes (Abu-Qarn and Eichler 2006) led to the identification of
open reading frames, subsequently remained agl (archaeal glycosylation) genes following the nomenclature of Chaban,
Voisin, et al. (2006) and shown to encode for glycosyltransferases (GTs) (i.e. AglD, AglE, AglI and AglJ), an OST (i.e.
AglB) and other sugar-processing enzymes (i.e. AglF) involved
in the assembly and attachment of the pentasaccharide decorating the Hfx. volcanii S-layer glycoprotein (Abu-Qarn et al. 2007;
Abu-Qarn, Giordano, et al. 2008; Yurist-Doutsch et al. 2008).
aglG, encoding a putative GT, was next identified through
it being positioned between the aglB and aglI sequences
(Yurist-Doutsch et al. 2008). Indeed, when the various agl
sequences were mapped to the Hfx. volcanii genome, it was
noted that apart from aglD, all were sequestered to a gene
island stretching from aglJ to aglB (Yurist-Doutsch and Eichler
2009). However, as the current annotation of the Hfx. volcanii
genome did not recognize the aglE sequence as an open reading frame (Abu-Qarn, Giordano, et al. 2008), the agl gene
island was subjected to manual reannotation. In this manner, additional sequences, i.e. aglP, aglQ and aglR, were identified
(Yurist-Doutsch and Eichler 2009), while algM was shown to
lie beyond the original gene island borders (Yurist-Doutsch
et al. 2010).
Although each of the identified Hfx. volcanii Agl proteins
has been assigned a specific role through a combination of
gene deletion and mass spectrometry approaches, only AglF,
AglM and AglP have been characterized biochemically. AglF
was shown to be a glucose-1-phosphate uridyltransferase involved in the biosynthesis of the hexuronic acid found at
position three of the S-layer glycoprotein-linked pentasaccharide (Yurist-Doutsch et al. 2010). AglM, a UDP-glucose
dehydrogenase, was shown to participate in the biogenesis of
the hexuronic acid found at pentasaccharide position two and
likely of the hexuronic acids found at positions three and four,
as well (Yurist-Doutsch et al. 2010). In a combined in vitro reconstitution experiment, AglF and AglM were shown to work
in a coordinated manner to generate UDP-glucuronic acid from
glucose-1-phosphate and uridine triphosphate (UTP) in a
NAD+-dependent manner (Yurist-Doutsch et al. 2010). Finally,
AglP was confirmed to be a S-adenosyl-L-methionine-dependent methyltransferase responsible for the formation of the
methyl ester of hexuronic acid found at position four of the
pentasaccharide (Magidovich et al. 2010). The current working
model of the Hfx. volcanii N-glycosylation pathway is presented in Figure 2A.
Archaeal N-glycosylation
Table II. All three types of OST catalytic centers are found in archaeal AglB proteins
Type B (WWDYG and MI motifs)
Type E (WWDYG and DK motifs)
Euryarchaeota
Archaeoglobus fulgidus
Archaeoglobus profundus
Candidatus Methanoregula boonei
Candidatus Methanosphaerula palustris
Ferroglobus placidus
Haloarcula marismortui
Halobacterium salinarum R1
Halobacterium sp. NRC-1
Haloferax volcanii
Halogeometricum borinquense
Halomicrobium mukohataei
Haloquadratum walsbyi
Halorhabdus utahensis
Halorubrum lacusprofundi
Haloterrigena turkmenica
Methanocella paludicola
Methanococcoides burtonii
Methanocorpusculum labreanum
Methanoculleus marisnigri
Methanosaeta thermophila
Methanosarcina acetivorans
Methanosarcina barkeri
Methanosarcina mazei
Methanospirillum hungatei
Natrialba magadi
Natronomonas pharaonis
Uncultured methanogenic archaeon RC-I
Euryarchaeota
Ferroplasma acidarmanusa
Methanobrevibacter ruminantium
Methanobrevibacter smithii
Methanocaldococcus fervens
Methanocaldococcus infernus
Methanocaldococcus jannaschii
Methanocaldococcus SP FS406-22
Methanocaldococcus vulcanius
Methanococcus aeolicus
Methanococcus maripaludis C5
Methanococcus maripaludis C6
Methanococcus maripaludis C7
Methanococcus maripaludis S2
Methanococcus vannielii
Methanococcus voltae
Methanosphaera stadtmanae
Methanothermobacter thermautotrophicus
Picrophilus torridusa
Thermoplasma volcaniuma
Thermoplasma acidophiluma
Crenarchaeota
Caldivirga maquilingensis
Desulfurococcus kamchatkensis
Hyperthermus butylicus
Ignicoccus hospitalis
Metallosphaera sedula
Pyrobaculum aerophilum
Pyrobaculum arsenaticum
Pyrobaculum calidifontis
Pyrobaculum islandicum
Staphylothermus hellenicus
Staphylothermus marinus
Sulfolobus acidocaldarius
Sulfolobus islandicus
Sulfolobus solfataricus
Sulfolobus tokodaii
Thermofilum pendens
Thermoproteus neutrophilus
Euryarchaeota
Pyrococcus abyssi
Pyrococcus furiosus
Pyrococcus horikoshii
Thermococcus barophilus
Thermococcus gammatolerans
Thermococcus kodakarensis
Thermococcus onnurineus
Thermococcus sibiricus
Thermococcus sp. AM4
Thaumarchaeota
Nitrosopumilus maritimus
Nanoarchaeota
Nanoarchaeum equitans
Korarchaeota
Candidatus Korarchaeum cryptofilum
a
The MI motif corresponds to LxxI/VxxxV in these species and is much farther from the WWDYG motif than in other AglB proteins.
N-terminal domain and a soluble C-terminal domain that includes the WWDYG motif implicated in the catalytic
mechanism of AglB/Stt3/PglB proteins (Wacker et al. 2002;
Yan and Lennarz 2002; Igura et al. 2008). Also as observed with
eukaryal OSTs (Turco et al. 1977; Munoz et al. 1994) and C.
jejuni PglB (Glover et al. 2005; Linton et al. 2005), AglB can
also transfer truncated glycan structures (Chaban, Voisin, et al.
2006; Abu-Qarn et al. 2007), pointing to the relaxed substrate
specificity of the archaeal enzyme. In addition, AglB is apparently also able to transfer not only fully assembled
oligosaccharides but also precursor polysaccharides to target
proteins. Hfx. volcanii AglB is, moreover, unusual in that it
transfers polysaccharides from dolichol phosphate rather
than dolichol pyrophosphate carriers (Kuntz et al. 1997;
Eichler 2001; Abu-Qarn et al. 2007).
Insight into OST action in Archaea, and indeed, across evolution, has been provided by solution of the three-dimensional
structure of the C-terminal soluble domain of P. furiosus AglB
(Igura et al. 2008). Providing the first portrayal of a catalytic
domain of a Stt3/PglB/AglB family member at atomic level
resolution, this study revealed the C-terminal soluble domain
to comprise four structural regions, assembled into a novel architecture. The WWDYG motif, known to participate in Stt3
and PglB function, is found within a β-helix-based central core
region that also includes an 80 residue, antiparallel β-barrellike element. The central core is surrounded by two peripheral
domains, each mainly containing β-strands. Guided by this
structural information, reassessment of the alignment of Stt3/
PglB/AglB family member sequences revealed the presence
of a spatially proximal DxxK motif, proposed to form the active site of the enzyme along with the WWDYG motif of the
protein. Indeed, the Asp and Lys residues of this DxxK motif
were shown to be important for yeast Stt3 activity by site-directed mutagenesis. In the P. furiosus enzyme, the active site
motifs assume unusual conformations, with the first three residues of the WWDYG motif predicted to adopt a rare lefthanded helical conformation and the DxxK motif being part
of an unusually long six-residue helix.
The more recent solution of the C. jejuni PglB structure
(Maita et al. 2010) revealed an impressive degree of folding
similarity to P. furiosus AglB. However, such comparison also
showed that rather than being universal in Stt3/PglB/AglB
family members, the P. furiosus AglB DxxK motif is replaced
by a MxxI motif in the bacterial enzyme. Based on this observation, a reassessment of sequence alignment data extended the
P. furiosus AglB DxxK motif to DxxKxxx[MI], now termed
the DK motif. Likewise, the MxxI motif of C. jejuni PglB
was extended to encompass a MxxIxxx[IVW] motif, now
termed the MI motif. When the distribution of these motifs
across Stt3/PglB/AglB family members in the three domains
of life was addressed, the differential distribution of the MI
and DK motifs, as well as of a variant of the DK motif comprising a DxxMxxx[KI] signature (the DM motif), allowed
evolutionary patterns to be drawn. It was shown that together
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Type A (WWDYG and DM motifs)
D Calo et al.
Putative N-glycosylation pathway components in other
Archaea
Although pathways of N-glycosylation have only been outlined in Hfx. volcanii, M. voltae and M. maripaludis, Nglycosylated proteins have been identified in numerous archaeal species found in a variety of environments (cf. Eichler and
Adams 2005). In the vast majority of these species, nothing is
known of the N-glycosylation process.
As a first step to redressing this situation, the CarbohydrateActive Enzymes (CAZy) database (http://www.cazy.org/fam/
acc_GT.html; Cantarel et al. 2009) was consulted to address
the presence and distribution of predicted GTs in 56 archaeal
genomes (Magidovich and Eichler 2009). In addition to identifying AglB in all but two species considered (i.e. Aeropyrum
pernix and Methanopyrus kandleri), it was also shown that
GTs assigned to the GT2 and GT4 families predominate in
Archaea (Magidovich and Eichler 2009), as reported elsewhere
(Lairson et al. 2008). The fewest GT2 and GT4 glycosyltransferases are detected in hyperthermophilic Archaea. In fact, the
observation that Nanoarchaeum equitans, a hyperthermophile
encoding the smallest genome known (490,885 base pairs; Huber et al. 2002; Waters et al. 2003), contains just a single GT2
and two GT4 glycosyltransferases, while Ignicoccus hospitalis
(the symbiotic host of N. equitans; Paper et al. 2007) encodes
only two GT2 and a single GT4 glycosyltransferases has been
suggested to reflect the GT-A fold-containing inverting GT2
1072
family and the GT-B fold-containing retaining GT4 family as
being the prototypes that eventually gave rise to the array of glycosyltranferases seen today (Lairson et al. 2008). Furthermore,
unlike what is observed in Eukarya and Bacteria, where known
and predicted GTs are respectively assigned to 67 and 54 different GT families, proteins identified as archaeal GTs are
distributed among only 13 GT families.
In Hfx. volcanii, aglB is found within a gene cluster that
includes the agl GTs of this species, with the exception of
aglD, as well as non-GT-encoding agl sequences serving other N-glycosylation-related roles (Yurist-Doutsch and Eichler
2009; Magidovich et al. 2010; Yurist-Doutsch et al. 2010).
In contrast, the agl genes of M. maripaludis S2 are more
widely distributed in the genome, with only aglC being
found in proximity to aglB (VanDyke et al. 2009). In other
Archaea, varying degrees of such aglB-centered clustering of
GT-encoding genes are seen (Magidovich and Eichler 2009).
While substantial gene clustering is observed in halophiles
and many methanogens, this arrangement is seen 3-fold less
often in hyperthermophilic species. The evolutionary significance of this observation remains to be considered.
In addition to the different verified and predicted N-glycosylation pathway components described in halophilic and
methanogenic archaea, additional archaeal sugar-modifying
enzymes have been reported. For instance, the pathway of
UDP-acetamido sugar biosynthesis has been described for two
members of the class Methanococcales, namely M. maripaludis
and M. jannaschii. In each case, the pathway enzymes having
been purified following expression in Escherichia coli and biochemically characterized (Namboori and Graham 2008).
Specifically, M. maripaludis MMP1680 (or M. jannaschii
MJ1420) catalyzes the isomerization and transamination of
fructose-6-P to create α-D-glucosamine-1-phosphate, which is
converted to α-D-glucosamine-1-phosphate by the M. maripaludis MMP1077 (or M. jannaschii MJ1100) phosphomutase.
M. maripaludis MMP1076 (or M. jannaschii MJ1101) then
catalyzes both the acetylation of glucosamine-1-phosphate
and the transfer of the resulting GlcNAc-1-phosphate to UTP
to generate UDP-GlcNAc and pyrophosphate. M. maripaludis
MMP0705 (or M. jannaschii MJ1504) can isomerize UDPGlcNAc to create UDP-N-acetyl-α- D-mannosamine (UDPManNAc), which is oxidized by M. maripaludis MMP0706
(or M. jannaschii MJ0468) to produce UDP-N-acetylmannosaminuronate (UDP-ManNAcA). Given the homologies of
MMP1680 (or MJ1420) to bacterial GlmS, of MMP1077 (or
MJ1100) to GlmM and of MMP1076 (or MJ1101) to GlmU,
these studies point to methanoarchaea as producing acetamido
sugars, e.g. GlcNAc, using the bacterial pathway (MenginLecreulx and van Heijenoort 1994), rather than the Leloir pathway used by Eukarya (Milewski et al. 2006). Still, despite
the fact that acetamido sugars have been detected on the
N-linked glycans of methanoarchaeal glycoproteins (see section
on N-glycosylation in methanogens and Figure 1), the involvement of these enzymes in N-glycosylation remains to be
demonstrated. The same is true of a P. furiosus sugar nucleotidyltransferase with extremely broad sugar and nucleotide
substrate specificity (Mizanur et al. 2004) as well as of phosphohexomutases from Pyrococcus horikoshii (Akutsu et al.
2005), Sulfolobus solfataricus (Ray et al. 2005) and Thermococcus kodakaraensis (Rashid et al. 2004), reported to possess
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with the WWDYG motif, eukaryal Stt3 proteins and archaeal
AglB proteins from members of the archaeal phyla Crenarchaeota (including P. furiosus) and some members of the phylum
Euryarchaeota employ the DK motif to present an E-type catalytic center, as do the sole sequenced members of the archaeal
phyla, Korarchaeota, Nanoarchaeota and Thaumarchaeota.
Along with bacterial PglB proteins, other members of Euryarchaeota, including M. voltae and M. maripaludis, employ
the WWDYG and MI motifs to form the B-type catalytic center. Yet other Euryarchaeota, such as Hfx. volcanii, combine the
WWDYG and DM motifs to create the A-type catalytic center.
Archaeal AglB proteins thus contain catalytic centers of all
three types (Table II). Moreover, in those archaeal species encoding multiple AglB sequences (cf. Magidovich and Eichler
2009), only a single type of catalytic center is found in all of
the predicted AglB proteins of that species.
Bioinformatics-based assignment of multiple versions of
AglB being present within a given archaeal species still requires experimental confirmation that each sequence is in
fact expressed and capable of OST activity. Indeed, justification for the existence of multiple versions of AglB in a
single species is not obvious if one considers the seeming promiscuity of Hbt. salinarum AglB. In this species, it was shown
that replacement of the Ser-4 residue of the S-layer glycoprotein 2Asn- 3Ala- 4Ser sequon with Val, Leu or Asn did not
prevent N-glycosylation at the Asn-2 position (Zeitler et al.
1998). Moreover, the same OST is apparently responsible for
adding both the repeating sulfated pentasaccharide moiety
through a GalNAc link to Asn-2 as well as the sulfated polysaccharide unit attached via a glucose subunit at 10 other Nglycosylation sites of the S-layer glycoprotein (Lechner and
Wieland 1989).
Archaeal N-glycosylation
Roles of N-glycosylation in Archaea
Whereas numerous N-glycosylated proteins have been described in a variety of Archaea living across a wide range of
biological niches, the roles served by this posttranslational
modification remain, for the most part, unexplored. Indeed,
as aglB can be deleted from both Hfx. volcanii and M. voltae
(Abu-Qarn and Eichler 2006; Chaban, Voisin, et al. 2006), it
would seem that N-glycosylation is not essential for the survival of these species, at least under the conditions tested.
Nonetheless, N-glycosylation may contribute to the ability of
Archaea and their proteins to survive or adapt to the harsh environments in which these organisms can thrive.
In comparing the N-linked glycan profiles of two haloarchaeal S-layer glycoproteins (Mengele and Sumper
1992), it was noted that the Hbt. salinarum protein not only
experiences a higher degree of N-glycosylation than does
the same protein in Hfx. volcanii but that the glycans of
former are enriched in sulfated glucuronic acids, in contrast
to the neutral sugars found in the latter. Thus, relative to its
Hfx. volcanii counterpart, the Hbt. salinarum S-layer glycoprotein presents a drastically increased surface charge
density, a property thought to contribute to the stability of
haloarchaeal proteins in the face of molar salt concentrations
(Madern et al. 2000). Accordingly, the Hbt. salinarum Slayer glycoprotein also contains 20% more acidic amino acid residues than does the Hfx. volcanii S-layer glycoprotein
(Lechner and Sumper 1987; Sumper et al. 1990). As a result of these considerations, Hbt. salinarum is able to grow
in higher salt concentrations than does Hfx. volcanii. In Hfx.
volcanii, however, absent or defective N-glycosylation greatly reduced the ability of cells to grow at increasing salt
concentrations (Abu-Qarn et al. 2007). The protection that
an enhanced negative surface charge could also afford in
the face of acidic conditions has been suggested as the reason for N-glycosylation of S. acidocaldarius cytochrome
b558/566 (Hettmann et al. 1998; Zähringer et al. 2000). Moreover, it was proposed that much of the protein surface is
shielded from the ∼pH 2 environment in which these cells
exist by the high degree of N-glycosylation it experiences
(Zähringer et al. 2000). It has also been postulated that
the N-linked glycan of the M. fervidus S-layer glycoprotein
is involved in the stabilization of this surface protein at high
temperatures (Kärcher et al. 1993). Available evidence also
points to Archaea as being able to modulate the N-glycosylation profile of target proteins. For instance, glycosylation
of Methanospirillum hungatei flagellins was reported to only
occur in low phosphate media (Southam et al. 1990). In
Hfx. volcanii, the transcription of the agl genes involved
in N-glycosylation occurs in a coordinated yet differential
manner in the face of different growth paradigms, pointing
to this posttranslational modification as serving an adaptive
role (Yurist-Doutsch et al. 2008; Yurist-Doutsch et al. 2010).
In addition to possibility affording advantages in the face
of environmental challenges, archaeal N-glycosylation has
been cited as providing structural support. This is best exemplified in Hbt. salinarum, where bacitracin treatment
transformed rod-shaped cells into spheres (Mescher and
Strominger 1976b). In cell wall-lacking T. acidophilum,
the glycan moieties of the major membrane glycoprotein
coating the cell surface have been suggested to either trap
water molecules or encourage interaction between cell surface proteins. In either case, protein glycosylation would
contribute to cell surface rigidity (Yang and Haug 1979b).
N-glycosylation of archaeal proteins has also been implicated in protein assembly and function. Defective Hfx.
volcanii N-glycosylation resulted in an unstructured S-layer,
comprised solely of the S-layer glycoprotein (Sumper et al.
1990), while the absence of N-glycosylation compromised Slayer stability (Abu-Qarn et al. 2007). Missing or partial M.
voltae flagellin N-glycosylation led to a lack or reduction in
flagella numbers, concomitant with motility defects (Chaban,
Voisin, et al. 2006). Likewise, bacitracin-mediated interference
with Methanococcus deltae flagellin glycosylation resulted in a
loss of flagellation (Kalmokoff et al. 1992). In mutant Hbt. salinarum cells over-producing under-glycosylated flagellins,
increased levels of flagella were detected in the growth medium, implying that proper flagellin glycosylation is necessary
for effective flagellar incorporation into the plasma membrane
(Wieland et al. 1985). In addition, glycosylation appears to
play a role in stabilization against proteolysis, as reflected by
the loss of Hfx. volcanii S-layer glycoprotein resistance to
added protease in mutants lacking many of the Agl proteins
involved in assembling and attaching the pentasaccharide
N-linked to the protein (Yurist-Doutsch et al. 2008; YuristDoutsch et al. 2010). Finally, N-glycosylation has been proposed to modulate the interaction of binding proteins with
the cell membrane or envelope of S. acidocaldarius (Albers
et al. 2004), while in Methanothermus sociabilis, the concept
of N-glycosylation playing a role in cell aggregation has been
raised (Kärcher et al. 1993).
Archaeal O-glycosylation
In addition to N-glycosylation, archaeal proteins can also experience O-glycosylation. In the cases of both the Hbt. salinarum
and the Hfx. volcanii S-layer glycoproteins, Thr-rich regions
adjacent to the predicted membrane-spanning domain of the
protein are modified with galactose–glucose disaccharides
(Mescher and Strominger 1976a; Sumper et al. 1990). Unfortunately, virtually nothing is known of the archaeal Oglycosylation pathway at present.
Concluding remarks
Although initially described over 30 years, only recently
have major advances in delineating the archaeal pathway
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both phosphoglucomutase and phosphomannomutase activities.
In contrast, deletion of the gene encoding the putative M. maripaludis acetyltransferase MMP0350 impaired flagellin Nglycosylation (VanDyke et al. 2008), a process known to involve
acetylated sugars (Kelly et al. 2009).
Finally, in Pyrolobus fumarii, growing optimally at 106°C
(Blöchl et al. 1997), novel UDP-sugars, including UDP-βG l c N A c - 3 - N A c a n d U D P -β - G l c N A c 3 N A c - ( 4 - 1 ) - βGlcpNAc3NAc, have been reported (Gonçalves et al. 2008).
Nothing, however, is known of their biosynthesis, whether
they are employed in N-glycosylation or, indeed, whether P.
fumarii even performs this posttranslational modification.
D Calo et al.
Funding
Research in the Eichler laboratory is supported by the Israel
Science Foundation (grant 30/07) and the US Army
Research Office (grant W911NF-07-1-0260). L.K. is the
recipient of a Negev-Zin Associates Scholarship.
Conflict of interest statement
None declared.
Abbreviations
ER, endoplasmic reticulum; GalNAc, N-acetylgalactosamine;
GlcNAc, N-acetylglucosamine; GT, glycosyltransferase;
OST, oligosaccharyltransferase; rRNA, ribosomal RNA;
S-layer, surface layer; UDP, uridine diphosphate; UTP, uridine triphosphate.
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