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PROGRESS Extreme sweetness: protein glycosylation in archaea Jerry Eichler Abstract | Although N‑glycosylation was first reported in archaea almost 40 years ago, detailed insights into this process have become possible only recently, with the availability of complete genome sequences for almost 200 archaeal species and the development of appropriate molecular tools. As a result of these advances, recent efforts have not only succeeded in delineating the pathways involved in archaeal N‑glycosylation, but also begun to reveal how such post-translational protein modification helps archaea to survive in some of the harshest environments on the planet. The advent of high-throughput sequencing has had an enormous impact on many areas of the biological sciences, but perhaps nowhere more so than in microbiology. At the same time, it has become clear that deciphering the genome sequence of a given microorganism cannot in itself explain the complexity and variability of the associated proteome. Instead, several processes are responsible for the diversity seen at the protein level, including alternative RNA splicing, modulation of the rate at which different proteins are synthesized and/ or degraded, and distinct processing leading to any of a long list of possible post-translational modifications, including phosphorylation, acetylation, methylation and glycosylation. Of the various post-translational modifications that a protein can undergo, N‑glycosylation is the most complex 1–3. First described by Neuberger in 1938 (REF. 4), N‑glycosylation — that is, the covalent linkage of a glycan moiety to selected Asn residues in a target protein — was long held to be a trait exclusive to eukaryotes, in which proteins residing in or passing through the secretory pathway en route to the cell surface or beyond the confines of the cell can be thus modified. This dogma was dispelled in 1976, when the surface layer (S-layer) glycoprotein of Halobacterium salinarum, a halophilic archaeon, was identified as the first noneukaryotic glycoprotein5. Today, it is clear that bacteria, like archaea, are capable of N‑glycosylation (as well as O‑glycosylation, in which sugars are added to Ser or Thr residues in target proteins)6–8. Until recently, our knowledge of archaeal N‑glycosylation was limited, compared to our relatively detailed understanding of this process in eukaryotes and bacteria. In the past few years, however, genome sequencing efforts have allowed for comparative studies, and the growing number of species amenable to genetic manipulation has allowed the development of gene deletion and induced protein overexpression methods, as well as biochemical tools suitable for use with archaeal proteins that require defined surroundings, such as high salt concentrations. As a direct result of these advancements, many of the details of the archaeal version of N‑glycosylation have been elucidated. In this Progress article, I discuss recent advances describing the diversity in archaeal N‑linked glycan content and the multiple pathways of archaeal N‑glycosylation that are used to achieve such diversity. I also consider how N‑glycosylation helps archaea cope with the extreme environments that they inhabit. The diverse world of N‑linked glycans In eukaryotes, the process of N‑glycosylation is best understood in Saccharomyces cerevisiae and in vertebrates1–3. In these organisms, seven nucleotide-activated sugars are sequentially added to dolichol pyrophosphate (the polyprenol that serves as the lipid glycan carrier in eukaryotic N‑glycosylation) on the NATURE REVIEWS | MICROBIOLOGY cytoplasmic face of the ER membrane. The lipid-linked heptasaccharide is then trans located to face the ER lumen, where it is further modified by the addition of seven more sugars, derived from individual dolichol phosphates that are charged on the cytoplasmic face of the ER and flipped to face the lumen. The resulting 14‑subunit branched oligosaccharide is added en bloc to a target protein and subsequently undergoes trimming and remodelling through the addition of other sugars, such as sialic acids, as the glycoprotein travels through the ER and Golgi; differential trimming and remodel ling of this oligosaccharide yields one of a set of tissue-, development stage- or proteinspecific N‑linked glycans. In plants and lower eukaryotes (for example, invertebrates and protists)9, variants of this oligomer — using the same N‑acetylglucosamine (GlcNAc)- and mannose-based core but lacking or including additional sugar residues from a limited roster at various positions — serve to expand the repertoire of N‑linked glycans. In bacteria, N‑glycosylation is thought to be restricted to deltaproteobacteria and epsilonproteobacteria6, and to date the pathway involved has been analysed only in Campylobacter jejuni 6,10. In this species, a heptasaccharide is assembled by the ordered addition of seven nucleotide-activated sugars onto undecaprenol pyrophosphate, which is found in the cytoplasm-facing leaflet of the plasma membrane. After translocation of the lipid-linked heptasaccharide across the membrane, the glycan is delivered to select Asn residues of the modified protein without undergoing further processing. An examination of the structures of N‑linked glycans from almost 30 different Campylobacter spp., along with Wolinella succinogenes, Helicobacter pullorum and Helicobacter winghamensis, revealed some variability in glycan content and in the identity of the Asn-linking sugar, with di-N-acetylbacillosamine and N‑acetylhexosamines fulfilling this role11,12. This variety, however, pales in comparison to that found in the archaeal N‑linked glycans. Although the process of archaeal N‑glycosylation is generally reminiscent of its eukaryotic and bacterial counterparts VOLUME 11 | MARCH 2013 | 151 © 2013 Macmillan Publishers Limited. All rights reserved PROGRESS Table 1 | N‑glycosylation across the three domains of life Feature Eukarya Bacteria Archaea Carrier molecules Dolichol phosphate Dolichol pyrophosphate Undecaprenol pyrophosphate Dolichol phosphate Dolichol pyrophosphate Degree of lipid saturation Monosaturated Not saturated Disaturated Polysaturated Flippase or associated protein RFT1 PglK AglR N‑glycan diversity Limited Limited Extensive Linking sugars GlcNAc diNacBac HexNAc Variable Multibranched N‑glycan Yes No Yes N‑glycan modification Yes No Yes Distinct N-glycans on the same protein No No Yes Composition Multimer or single subunit Single subunit Single subunit Catalytic subunit Stt3 PglB AglB Catalytic motifs* WWDYG DXXKXXX(M/I) WWDYG MXXIXXX(I/V/W) WWDYG DXXKXXX(M/I) or MXXIXXX(I/V/W) or EXnKXXX(M/I/P) Sequons recognized‡ NX(S/T) NX(S/T) (D/E)XNX(S/T) NX(S/T) NX(N/V/L) Lipid glycan carrier N‑linked glycan Oligosaccharyltransferase Agl, archaeal glycosylation; diNAcBac, di-N-acetylbacillosamine; HexNAc, N‑acetylhexosamine; GlcNAc, N-acetylglucosamine; Pgl, protein glycosylation. *X represents any amino acid. ‡X represents any amino acid except Pro. (TABLE 1), analysis of the 13 archaeal N‑linked glycans that have been characterized to date (FIG. 1) reveals a diversity in size, architecture and composition (even in the composition of the different glycans that can be N‑linked to the same protein) that is not paralleled in the other two domains. Indeed, when one considers the limited number of archaeal species for which the N‑linked glycans have been elucidated, it is likely that the diversity reported thus far reflects only the tip of the iceberg 13. It follows that such diversity would reflect processing by an equal number of different archaeal N‑glycosylation pathways, in marked contrast to the relatively conserved systems that are present in the other two domains. Recent studies of halophilic, methanogenic and thermophilic archaea are discussed below and show that this does indeed seem to be the case. Halophiles: two routes to the same glycan The simple growth requirements of halo archaea, coupled with the large set of genetic and biochemical tools available for working with these organisms and their proteins, despite the high salt requirements of these species14, means that they currently offer the most detailed insights into archaeal N‑glycosylation. In Haloferax volcanii, a series of Agl (archaeal glycosylation) proteins constitute the N‑glycosylation pathway used to assemble the pentasaccharide that modifies glycoproteins in this species; this pentasaccharide is composed of a hexose, two hexuronic acids, a methyl ester of hexuronic acid and a mannose7,15 (FIGS 1,2a). The pathway begins with nucleotideactivated versions of the first four N‑linked pentasaccharide subunits being sequentially added to a common dolichol phosphate via the ordered actions of the glycosyltransferases AglJ, AglG, AglI and AglE16–19. At the same time, AglD adds the final mannose subunit of the pentasaccharide to a unique dolichol phosphate20. Despite serving a similar function, Hfx. volcanii dolichol phosphate can be distinguished from its eukaryotic counterpart because the eukaryotic molecule is saturated only at the α-isoprene, whereas the haloarchaeal molecule is saturated at both the α-isoprene and the ω-isoprene16,21. Dolichol phosphate loaded with the tetrasaccharide (and its precursors) is translocated across the haloarchaeal plasma membrane in an as‑yet-unknown manner, at which point the glycan moiety is transferred to a select Asn residue in a target protein by the oligosaccharyltransferase (OST), AglB18. Dolichol phosphate charged with the final mannose 152 | MARCH 2013 | VOLUME 11 is flipped across the membrane in a process involving AglR16,22. AglS then delivers the terminal mannose subunit to the proteinbound tetrasaccharide23. In addition, AglF (a glucose-1‑phosphate uridyltransferase), AglP (a S-adenosylmethionine-dependent methyltransferase) and AglM (a UDPglucose dehydrogenase) also contribute to the pathway 24,25. Like Hfx. volcanii, Haloarcula marismortui originates from the Dead Sea and is surrounded by a protein shell based on an S‑layer glycoprotein that is modified by a similar N‑linked pentasaccharide to that found on Hfx. volcanii glycoproteins26. Despite this and the phylogenetic proximity of the two species, each has a distinct strategy for glycan assembly. Rather than recruiting two different glycan-charged dolichol phosphate carriers, as occurs in Hfx. volcanii, Har. marismortui assembles the entire N‑linked glycan on a single dolichol phosphate carrier 26. Thus, N‑glycosylation in Hfx. volcanii, involving multiple lipid glycan carriers in a multistep protein-processing event, is reminiscent of the eukaryotic pathway 1,2, whereas the same post-translational modification process in Har. marismortui is similar to the bacterial pathway, as exemplified by C. jejuni, in which the complete www.nature.com/reviews/micro © 2013 Macmillan Publishers Limited. All rights reserved PROGRESS Archaeoglobus fulgidus Halobacterium salinarum N – O3 – O3 – O3 OS OS N – H3 O3 OC OS N 10–15 N H Methanococcus maripaludis CO 3 Methanococcus voltae T T T T N N – O3 OS OS OS N Haloferax volcanii – O3 N N Methanothermus fervidus Sulfolobus acidocaldarius – O3 H3 H3 OC OC OS N N Pyrococcus furiosus Thermoplasma acidophilum N N Hexose N-acetylhexosamine Hexuronic acid Deoxy-hexose Galactose N-acetylgalactosamine Galacturonic acid Galactofuranose Glucose N-acetylglucosamine Glucuronic acid Di-N-acetylglucuronic acid Mannose N-acetylmannuronic acid 3-acetamidino-N-acetylmannuronic acid (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose Quinovose 6-deoxy-6-C-sulpho-D-galactose α-D-glycero-D-galacto-heptose Rhamnose Pentose 220 kDa or 262 kDa moiety N Asn T Thr Figure 1 | The diversity of N‑linked glycans decorating archaeal glycoproteins. The composition Reviews | Microbiology of the characterized N‑linked glycans discussed in the text is presented,Nature along with the composition of the N-linked glycans from Methanothermus fervidus62 and Thermoplasma acidophilum63. The Asn residue of the target protein (to which the glycan is added) is shown for each glycan. N‑linked glycan is assembled on a single lipid carrier and then delivered to the protein target as a complete unit 6,10. N‑glycosylation and the methanogens Methanogens have also provided substantial insights into the process of archaeal N‑glycosylation. The first archaeal genes involved in N‑glycosylation were reported in an isolate of Methanococcus voltae str. PS27. In this strain, glycoproteins are modified by a N‑linked trisaccharide28, although in a different isolate of the same strain this tri saccharide is extended by an additional 220 kDa or 262 kDa moiety that is apparently carbohydrate in nature29 (FIG. 1). Today, many of the steps in M. voltae N‑glycosylation are known (FIG. 2b). AglH charges the phospho dolichol with GlcNAc, the first sugar of the N‑linked glycan. This role for AglH is supported by the ability of the aglH gene to complement a conditionally lethal mutation in S. cerevisiae alg7, the gene encoding the enzyme responsible for adding GlcNAc to the dolichol phosphate carrier in eukaryotic N-glycosylation30. To date, this is the only example of a component of an archaeal N‑glycosylation pathway that can function across domain lines. AglC and AglK contribute to the biosynthesis or addition of the second sugar subunit, a di‑N‑acetyl glucuronic acid, and AglA is involved in adding the final sugar, N-acetylmannuronic acid, of the N‑linked trimer 29. Finally, as in other archaea, AglB is the OST that delivers the presumably lipid-bound glycan to an Asn residue in the target protein27. Despite its phylogenetic proximity to M. voltae, Methanococcus maripaludis produces glycoproteins that carry a distinct NATURE REVIEWS | MICROBIOLOGY N‑linked glycan (FIGS 1,2c). Recent studies have revealed that in M. maripaludis, modified proteins are decorated with a tetrasaccharide that can be distinguished from its M. voltae counterpart through the use of N‑acetylgalactosamine instead of GlcNAc as the linking first sugar, by the presence of a 3‑acetamidino moiety on the third sugar subunit (which is otherwise common to the N‑linked glycans in both species) and by the novel sugar ((5S)-2‑acetamido2,4‑dideoxy-5-O-α‑l‑erythro-hexos‑5‑ulo1,5‑pyranose) found at the fourth and final position of the M. maripaludis glycan31. This novel sugar is the first example of a naturally occurring diglycoside of an aldulose. It has also been shown that MMP1685, the major structural subunit of M. maripaludis pilin, carries the same tetrasaccharide modified by a hexose branch attached to the N‑acetylgalactosamine subunit 32 (FIG. 1). M. maripaludis relies on a distinct set of Agl enzymes for N‑glycosylation compared with M. voltae, with the exception of AglA, which adds a similar third sugar to the di‑N‑acetylglucuronic acid at position two in both organisms, and AglB, which serves as the OST in both organisms27,33(FIG. 2b,c). To date, only a limited number of the species-specific M. maripaludis pathway components have been identified — namely, AglO, which is involved in adding the second sugar subunit, and AglL, which has been assigned a role in adding the final sugar subunit and/or the Thr attached to the third sugar of the glycan33 (the former role is more likely, given the homology of AglL to other glycosyltransferases). Moreover, the absence of the Thr moiety from the third N‑glycan subunit in cells lacking AglL could mean that the Thr modification occurs only after addition of the final sugar. Finally, it has been reported that AglY generates ammonia, which is subsequently tunnelled through AglZ to AglX, the amidotransferase that modifies the third sugar residue for the M. maripaludis N‑linked glycan with an acetamidino group before addition of the modified sugar by AglA34 (FIG. 2c). In both M. voltae27 and M. maripaludis33 (and also in Hfx. volcanii 15), N‑glycosylation of the archaeal flagellum, or archaellum35, is important for its assembly and therefore for cellular motility, underscoring the importance of this post-translational modification in archaea. Branching out in a thermoacidophile At present, the majority of insights into archaeal N‑glycosylation have come VOLUME 11 | MARCH 2013 | 153 © 2013 Macmillan Publishers Limited. All rights reserved PROGRESS a Haloferax volcanii OCH3 OCH3 AglS N AglB OCH3 N AglR AglJ Mannose AglD Hexuronic acid AglG UDP AglM UDP AglE UDP Hexose OCH3 AglI N Asn AglP SAM S-adenosylmethionine SAM AglM UDP AglF P b Methanococcus voltae T T N AglB AglH N-acetylmannuronic acid AglC AglK Di-N-acetylglucuronic acid T AglA N-acetylglucosamine T T Thr N Asn c Methanococcus maripaludis T T AglB AglO T AglA AglY N-acetylgalactosamine T AglZ Q + H2O (5S)-2-acetamido-2,4-dideoxy-5-O-methylα-L-erythro-hexos-5-ulo-1,5-pyranose 3-acetamidino-N-acetylmannuronic acid Di-N-acetylglucuronic acid AglL AglX N NH3 E N-acetylmannuronic acid T Thr Q Gln E Glu N Asn Figure 2 | N‑glycosylation pathways in archaea. Our current understanding of the N‑glycosylation Nature Reviews | Microbiology pathways of three archaeal species, on the basis of both in vitro and in vivo findings. In each case, various Agl (archaeal glycosylation) proteins catalyse the assembly and attachment of the N‑linked glycan, as well as the biosynthesis of the glycan sugar subunits. In each pathway, the glycans are assembled on a dolichol phosphate molecule. a | Haloferax volcanii. b | Methanococcus voltae. The N‑linked glycan can include an additional 220 kDa or 262 kDa sugar moiety as a fourth subunit. c | Methanococcus maripaludis. In this species, pili are glycosylated with a variation on the shown glycan, in which an additional hexose subunit branches from the N-acetylgalactosamine. 154 | MARCH 2013 | VOLUME 11 from studies of members of the phylum Euryarchaeota, a major archaeal phylum that includes the halophiles and the methanogens. Recently, however, studies on Sulfolobus acidocaldarius, a member of a second major archaeal phylum, Crenarchaeota, have also enhanced our understanding of this post-translational modification. In S. acidocaldarius, an organism that grows optimally at pH 2–3 and at 75 °C –80 °C36, the S‑layer protein SlaA is highly N‑glycosylated, particularly in the carboxy‑ terminal region, where a tri-branched hexasaccharide is detected every 30–40 residues37. Previously, the same N‑linked glycan had been shown to decorate cytochrome b558/566 in this species38. This N‑linked hexasaccharide has attracted attention for several reasons. First, the glycan contains 6‑sulphoquinovose (6‑deoxy‑6‑sulphoglucose) (FIG. 1), an unusual sugar that is commonly found only in the photosynthetic membranes of plants and phototrophic bacteria39. Second, like N‑linked oligosaccharides in higher eukaryotes1,2, the S. acidocaldarius glycan is N‑linked via a chitobiose core, namely a pair of GlcNAc residues. Finally, the multibranched structure of this archaeal hexa saccharide is a property that is shared by the N‑linked glycans that decorate eukaryotic glycoproteins1–3. With a well-stocked genetic toolbox now available for manipulating S. acidocaldarius — including tools such as markers for selection, expression vectors containing inducible promoters and the ability to create deletion strains40 — we have begun to define the pathway responsible for the biogenesis of the N‑linked tri-branched glycan41. Such efforts have, however, been complicated by the limited homology of S. acidocaldarius glycosyltransferases to their counterparts in other species. Moreover, although glycosyltransferase-encoding genes are clustered in the vicinity of aglB in many euryarchaeotes, this is not the case in S. acidocaldarius and most other crenarchaeotes42. Nonetheless, gene deletion and mass spectrometry have been successfully used to identify at least one gene involved in the S. acidocaldarius N‑glycosylation pathway, namely agl3 (Saci_0423), which was shown to encode a UDP-sulphoquinovose synthase41. The same study predicted that the neighbouring genes, Saci_0421, Saci_0422 and Saci_0424, encode additional N‑glycosylation pathway components, namely Agl1 (annotated as a membrane-bound glycosyltransferase), Agl2 (annotated as a dTDP-glucose pyro phosphorylase) and Agl4 (annotated as www.nature.com/reviews/micro © 2013 Macmillan Publishers Limited. All rights reserved PROGRESS a glucokinase), respectively. In addition, another study described a hexose-charged dolichol phosphate that is possibly involved in S. acidocaldarius N‑glycosylation; this dolichol phosphate is saturated at the α-, ω- and internal-position isoprenes and is shorter than the dolichol phosphates seen in other species43. Structural insights into OST function In archaea (as in bacteria and eukaryotes), N‑glycosylation occurs at Asn residues within an Asn‑Xaa‑Ser/Thr motif, known as the sequon (in which Xaa is any residue except Pro)44,45, although N‑glycosylation of Asn‑Xaa‑Asn/Leu/Val has been reported in Hbt. salinarum46. In some bacteria, such as C. jejuni, the sequon can be extended to Asp/Glu‑Xaa‑Asn‑Xaa‑Ser/Thr 47 (TABLE 1). In all three domains, N‑linked glycans are transferred from polyprenol carriers to target Asn residues by the actions of OSTs. In S. cerevisiae and higher eukaryotes, the OST is a multisubunit complex based on the catalytic subunit Stt348. In bacteria and archaea, OSTs consist of a single subunit, the Stt3 homologues protein glycosylation B (PglB) and AglB, respectively 6,27,49–51; Stt3‑based single-subunit OSTs can also be found in lower eukaryotes52 (TABLE 1). Archaeal OSTs possess traits that are seemingly unique to this domain of organisms. For instance, although Hbt. salinarum encodes a single AglB, this enzyme is apparently able to catalyse the transfer of two distinct glycans to the S‑layer glycoprotein, one from a dolichol phosphate carrier and another from a dolichol pyrophosphate carrier 53. A comparable situation might occur in Hfx. volcanii, in which the single AglB identified would have to attach the two distinct N‑linked glycans that decorate the S‑layer glycoprotein, despite the fact that different linking sugars are used in each case (see below)54. However, many archaeal species encode multiple AglBs, although the physiological significance of this observation remains unclear 42. Future studies on Methanosarcina spp., which encode multiple AglBs42 and for which advanced genetic tools are available55, could help elucidate this point. Recently, the crystal structures were solved for the catalytic carboxy‑terminal domains of AglB proteins from the hyperthermophiles Pyrococcus furiosus and Archaeoglobus fulgidus, and these structures have shed new light on the workings of these OSTs50,51, identifying novel sequence motifs involved in OST catalytic activity other than the evolutionarily conserved Trp-Trp-Asp- Tyr-Gly consensus motif 42,56–58. Despite the low sequence homology between Stt3, PglB and AglB proteins, an alignment based on the P. furiosus AglB and C. jejuni PglB structures revealed that a so‑called DK motif (DXXKXX(M/I), in which X is any amino acid) is restricted to eukaryotic Stt3 and most archaeal AglB proteins, whereas bacterial PglB and the remaining AglB proteins contain a distinct MI motif (MXXIXXX(I/V/W), in which X is any amino acid) at this position56 (TABLE 1). Further analysis of the crystal structure of a bacterial PglB in complex with an acceptor substrate revealed that the DK and MI motifs contribute to the recognition pocket surrounding the Ser/Thr residue at the +2 position upstream of the glycosylated Asn in the target protein, and that this pocket also contains the invariable Trp-Trp-Asp portion of the Trp-Trp-AspTyr-Gly consensus motif of Stt3–PglB–AglB proteins58. The structural information obtained about the carboxy‑terminal catalytic domain of one of the three A. fulgidus AglB proteins, AF_0329 (which is the shortest Stt3–PglB–AglB protein known), served to further classify these proteins by identifying AglB proteins presenting a variant of the DK motif 51 (TABLE 1). Finally, by analysing the in vitro activity of P. furiosus AglB, the transfer of a lipid-linked heptasaccharide to an acceptor peptide was demonstrated. This glycan, comprising two N‑acetylhexosamines, two hexoses, a hexuronic acid and two pentose branches50, represents yet another distinct archaeal N‑linked glycan structure (FIG. 1). Coping with extremes Archaea are found in a broad range of environments that are often extreme in nature59. It is thus possible that the enormous diversity seen in archaeal N‑glycosylation reflects species-specific means of coping with the environmental challenges encountered. The observation that S. acidocaldarius cells lacking Agl3 grow increasingly poorly as the NaCl concentration of the growth medium increases41 suggests a physiological role for cell surface N‑glycosylation in this species. As the mutant cells do not contain sulphoquinovose, they lack the negative charges that are normally provided by this sugar, which are thought to contribute to the hydrated shell that surrounds the cell. Therefore, S‑layer stability in the mutant strain becomes compromised in an increasingly saline environment, affecting growth. Indeed, an enhanced surface charge in the face of increased salinity was also offered as the reason for the higher proportion of NATURE REVIEWS | MICROBIOLOGY sulphated sugars in the N‑linked glycans decorating the S‑layer glycoprotein of the extreme halophile Hbt. salinarum, relative to the proportion in the N-linked glycans modifying the same protein in Hfx. volcanii, a moderate halophile60. Moreover, disruption of Hfx. volcanii N‑glycosylation renders the S‑layer surrounding the cell more susceptible to proteolytic degradation18–20. However, recent studies have shown that the interplay between N‑glycosylation and environmental conditions is more complex than previously imagined54. When grown in medium containing 3.4 M NaCl, the Hfx. volcanii S‑layer glycoprotein residues Asn13 and Asn83 are modified by an N‑linked pentasaccharide. However, when the same cells are grown in medium containing 1.75 M NaCl, significant changes in the N‑linked glycan profile of the protein are observed. Although Asn13 and Asn83 are still modified by the same pentasaccharide, such modification occurs on a substantially smaller proportion of the glycoprotein. More strikingly, Asn498, a position that is not modified when the cells are grown at the higher salinity, carries a novel tetra saccharide comprising a sulphated hexose, two hexoses and a terminal rhamnose sub unit (FIG. 1). At present, it is not clear what advantage is offered to Hfx. volcanii by such differential N‑glycosylation in response to changes in environmental salinity. It is equally striking that the Hfx. volcanii S‑layer glycoprotein can be simultaneously modified by two distinct N‑linked glycans. Apart from for the Hbt. salinarum S‑layer glycoprotein53, this phenomenon has not been reported for any other species or protein4. Outlook With more archaeal genome sequences becoming readily available and as new and improved molecular tools for working with the different archaeal species start to accumulate, we are beginning to make major advances in our understanding of archaeal N‑glycosylation. Such efforts will elucidate how protein glycosylation contributes to the remarkable ability of archaea to overcome the physical challenges associated with the extreme environments that they inhabit. These efforts will also provide novel insight into the evolution of this universal posttranslational modification. In addition, we can expect to see new applied uses for extremophilic enzymes that can be tailored to meet specific needs via glyco-engineering 61. Looking forward, the future certainly looks sweet. VOLUME 11 | MARCH 2013 | 155 © 2013 Macmillan Publishers Limited. All rights reserved PROGRESS Jerry Eichler is at the Department of Life Sciences, Ben Gurion University of the Negev, P.O. 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Cell surface glycoproteins from Thermoplasma acidophilum are modified with an N‑linked glycan containing 6‑C‑sulfofucose. Glycobiology 22, 1256–1267 (2012). Acknowledgements Work in the author’s laboratory is supported by the Israel Science Foundation (grant 8/11) and the US Army Research Office (W911NF-11‑1‑520). Competing interests statement The author declares no competing financial interests. FURTHER INFORMATION Jerry Eichler’s homepage: http://lifeserv.bgu.ac.il/wb/jeichler/ ALL LINKS ARE ACTIVE IN THE ONLINE PDF www.nature.com/reviews/micro © 2013 Macmillan Publishers Limited. All rights reserved