Sophie Yurist-Doutsch

Sweet to the extreme: protein glycosylation in Archaea

Post‐translational modifications account for much of the biological diversity generated at the proteome level. Of these, glycosylation is the most prevalent. Long thought to be unique to Eukarya, it is now clear that both Bacteria and Archaea are also capable of N‐glycosylation, namely the covalent linkage of oligosaccharides to select target asparagine residues. However, while the eukaryal and bacterial N‐glycosylation pathways are relatively well defined, little is known of the parallel process in Archaea. Of late, however, major advances have been made in describing the process of archaeal N‐glycosylation. Such efforts have shown, as is often the case in archaeal biology, that protein N‐glycosylation in Archaea combines particular aspects of the eukaryal and bacterial pathways along with traits unique to this life form. For instance, while the oligosaccharides of archaeal glycoproteins include nucleotide‐activated sugars formed by bacterial pathways, the lipid carrier on which such oligosaccharides are assembled is the same as used in eukaryal N‐glycosylation. By contrast, transfer of assembled oligosaccharides to their protein targets shows Archaea‐specific properties. Finally, addressing N‐glycosylation from an archaeal perspective is providing new general insight into this event, as exemplified by the solution of the first crystal structure of an oligosaccharide transferase from an archaeal source.

aglF, aglG and aglI, novel members of a gene island involved in the N-glycosylation of the Haloferax volcanii S-layer glycoprotein

Proteins in all three domains of life can experience N‐glycosylation. The steps involved in the archaeal version of this post‐translational modification remain largely unknown. Hence, as the next step in ongoing efforts to identify components of the N‐glycosylation pathway of the halophilic archaeon Haloferax volcanii, the involvement of three additional gene products in the biosynthesis of the pentasaccharide decorating the S‐layer glycoprotein was demonstrated. The genes encoding AglF, AglI and AglG are found immediately upstream of the gene encoding the archaeal oligosaccharide transferase, AglB. Evidence showing that AglF and AglI are involved in the addition of the hexuronic acid found at position three of the pentasaccharide is provided, while AglG is shown to contribute to the addition of the hexuronic acid found at position two. Given their proximities in the H. volcanii genome, the transcription profiles of aglF, aglI, aglG and aglB were considered. While only aglF and aglI share a common promoter, transcription of the four genes is co‐ordinated, as revealed by determining transcript levels in H. volcanii cells raised in different growth conditions. Such changes in N‐glycosylation gene transcription levels offer additional support for the adaptive role of this post‐translational modification in H. volcanii.

AglP is a S‐adenosyl‐L‐methionine‐dependent methyltransferase that participates in the N‐glycosylation pathway of Haloferax volcanii

While pathways for N‐glycosylation in Eukarya and Bacteria have been solved, considerably less is known of this post‐translational modification in Archaea. In the halophilic archaeon Haloferax volcanii, proteins encoded by the agl genes are involved in the assembly and attachment of a pentasaccharide to select asparagine residues of the S‐layer glycoprotein. AglP, originally identified based on the proximity of its encoding gene to other agl genes whose products were shown to participate in N‐glycosylation, was proposed, based on sequence homology, to serve as a methyltransferase. In the present report, gene deletion and mass spectrometry were employed to reveal that AglP is responsible for adding a 14 Da moiety to a hexuronic acid found at position four of the pentasaccharide decorating the Hfx. volcanii S‐layer glycoprotein. Subsequent purification of a tagged version of AglP and development of an in vitro assay to test the function of the protein confirmed that AglP is a S‐adenosyl‐L‐methionine‐dependent methyltransferase.

N-glycosylation in Archaea: on the coordinated actions of Haloferax volcanii AglF and AglM

Like Eukarya and Bacteria, Archaea are also capable of performing N‐glycosylation. In the halophilic archaeon Haloferax volcanii, N‐glycosylation is mediated by the products of the agl gene cluster. In the present report, this gene cluster was expanded to include an additional sequence, aglM, shown to participate in the biosynthesis of hexuronic acids contained within a pentasaccharide decorating the S‐layer glycoprotein, a reporter H. volcanii glycoprotein. In response to different growth conditions, changes in the transcription profile of aglM mirrored changes in the transcription profiles of aglF, aglG and aglI, genes encoding confirmed participants in the H. volcanii N‐glycosylation pathway, thus offering support to the hypothesis that in H. volcanii, N‐glycosylation serves an adaptive role. Following purification, biochemical analysis revealed AglM to function as a UDP‐glucose dehydrogenase. In a scoupled reaction with AglF, a previously identified glucose‐1‐phosphate uridyltransferase, UDP‐glucuronic acid was generated from glucose‐1‐phosphate and UTP in a NAD+‐dependent manner. These experiments thus represent the first step towards in vitro reconstitution of the archaeal N‐glycosylation process.

Two Distinct N-Glycosylation Pathways Process the Haloferax volcanii S-Layer Glycoprotein upon Changes in Environmental Salinity

ABSTRACT N-glycosylation in Archaea presents aspects of this posttranslational modification not seen in either Eukarya or Bacteria. In the haloarchaeon Haloferax volcanii, the surface (S)-layer glycoprotein can be simultaneously modified by two different N-glycans. Asn-13 and Asn-83 are modified by a pentasaccharide, whereas Asn-498 is modified by a tetrasaccharide of distinct composition, with N-glycosylation at this position being related to environmental conditions. Specifically, N-glycosylation of Asn-498 is detected when cells are grown in the presence of 1.75 but not 3.4 M NaCl. While deletion of genes encoding components of the pentasaccharide assembly pathway had no effect on the biosynthesis of the tetrasaccharide bound to Asn-498, deletion of genes within the cluster spanning HVO_2046 to HVO_2061 interfered with the assembly and attachment of the Asn-498-linked tetrasaccharide. Transfer of the “low-salt” tetrasaccharide from the dolichol phosphate carrier upon which it is assembled to S-layer glycoprotein Asn-498 did not require AglB, the oligosaccharyltransferase responsible for pentasaccharide attachment to Asn-13 and Asn-83. Finally, although biogenesis of the low-salt tetrasaccharide is barely discernible upon growth at the elevated salinity, this glycan was readily detected under such conditions in strains deleted of pentasaccharide biosynthesis pathway genes, indicative of cross talk between the two N-glycosylation pathways. IMPORTANCE In the haloarchaeon Haloferax volcanii, originally from the Dead Sea, the pathway responsible for the assembly and attachment of a pentasaccharide to the S-layer glycoprotein, a well-studied glycoprotein in this species, has been described. More recently, it was shown that in response to growth in low salinity, the same glycoprotein is modified by a novel tetrasaccharide. In the present study, numerous components of the pathway used to synthesize this “low-salt” tetrasaccharide are described. As such, this represents the first report of two N-glycosylation pathways able to simultaneously modify a single protein as a function of environmental salinity. Moreover, and to the best of our knowledge, the ability to N-glycosylate the same protein with different and unrelated glycans has not been observed in either Eukarya or Bacteria or indeed beyond the halophilic archaea, for which similar dual modification of the Halobacterium salinarum S-layer glycoprotein was reported. In the haloarchaeon Haloferax volcanii, originally from the Dead Sea, the pathway responsible for the assembly and attachment of a pentasaccharide to the S-layer glycoprotein, a well-studied glycoprotein in this species, has been described. More recently, it was shown that in response to growth in low salinity, the same glycoprotein is modified by a novel tetrasaccharide. In the present study, numerous components of the pathway used to synthesize this “low-salt” tetrasaccharide are described. As such, this represents the first report of two N-glycosylation pathways able to simultaneously modify a single protein as a function of environmental salinity. Moreover, and to the best of our knowledge, the ability to N-glycosylate the same protein with different and unrelated glycans has not been observed in either Eukarya or Bacteria or indeed beyond the halophilic archaea, for which similar dual modification of the Halobacterium salinarum S-layer glycoprotein was reported.

Manual Annotation, Transcriptional Analysis, and Protein Expression Studies Reveal Novel Genes in the agl Cluster Responsible for N Glycosylation in the Halophilic Archaeon Haloferax volcanii

While Eukarya, Bacteria, and Archaea are all capable of protein N glycosylation, the archaeal version of this posttranslational modification is the least understood. To redress this imbalance, recent studies of the halophilic archaeon Haloferax volcanii have identified a gene cluster encoding the Agl proteins involved in the assembly and attachment of a pentasaccharide to select Asn residues of the surface layer glycoprotein in this species. However, because the automated tools used for rapid annotation of genome sequences, including that of H. volcanii, are not always accurate, a reannotation of the agl cluster was undertaken in order to discover genes not previously recognized. In the present report, reanalysis of the gene cluster that includes aglB, aglE, aglF, aglG, aglI, and aglJ, which are known components of the H. volcanii protein N-glycosylation machinery, was undertaken. Using computer-based tools or visual inspection, together with transcriptional analysis and protein expression approaches, genes encoding AglP, AglQ, and AglR are now described.

AglS, a Novel Component of the Haloferax volcanii N-Glycosylation Pathway, Is a Dolichol Phosphate-Mannose Mannosyltransferase

In Haloferax volcanii, a series of Agl proteins mediates protein N-glycosylation. The genes encoding all but one of the Agl proteins are sequestered into a single gene island. The same region of the genome includes sequences also suspected but not yet verified as serving N-glycosylation roles, such as HVO_1526. In the following, HVO_1526, renamed AglS, is shown to be necessary for the addition of the final mannose subunit of the pentasaccharide N-linked to the surface (S)-layer glycoprotein, a convenient reporter of N-glycosylation in Hfx. volcanii. Relying on bioinformatics, topological analysis, gene deletion, mass spectrometry, and biochemical assays, AglS was shown to act as a dolichol phosphate-mannose mannosyltransferase, mediating the transfer of mannose from dolichol phosphate to the tetrasaccharide corresponding to the first four subunits of the pentasaccharide N-linked to the S-layer glycoprotein.

AglQ Is a Novel Component of the Haloferax volcanii N-Glycosylation Pathway

N-glycosylation is a post-translational modification performed by members of all three domains of life. Studies on the halophile Haloferax volcanii have offered insight into the archaeal version of this universal protein-processing event. In the present study, AglQ was identified as a novel component of the pathway responsible for the assembly and addition of a pentasaccharide to select Asn residues of Hfx. volcanii glycoproteins, such as the S-layer glycoprotein. In cells deleted of aglQ, both dolichol phosphate, the lipid carrier used in Hfx. volcanii N-glycosylation, and modified S-layer glycoprotein Asn residues only presented the first three pentasaccharide subunits, pointing to a role for AglQ in either preparing the third sugar for attachment of the fourth pentasaccharide subunit or processing the fourth sugar prior to its addition to the lipid-linked trisaccharide. To better define the precise role of AglQ, shown to be a soluble protein, bioinformatics tools were recruited to identify sequence or structural homologs of known function. Site-directed mutagenesis experiments guided by these predictions identified residues important for AglQ function. The results obtained point to AglQ acting as an isomerase in Hfx. volcanii N-glycosylation.

The Cell Envelopes of Haloarchaea: Staying in Shape in a World of Salt

The haloarchaea possess various cell envelope types, composed of different polymers such as S-layers, heteropolysaccharides, or glutaminylglucans. These cell wall polymers are described below.

Salty and Sweet: Protein Glycosylation in Haloferax volcanii

Ever since the discovery of the first glycosylated archaeal protein, namely the Halobacterium salinarum surface-layer glycoprotein some 35 years ago, research on haloarchaea has been at the forefront of efforts to decipher the archaeal version of N-glycosylation, a universal post-translational modification. Now, with the availability of sufficient numbers of genome sequences and the development of appropriate experimental tools, the possibility for detailed molecular analysis of archaeal N-glycosylation pathways is being realized, using haloarchaeal species as model systems. In this chapter, current understanding of N-glycosylation in Archaea and the contribution of studies on Haloferax volcanii to such endeavors are described.