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.     

Defining the topology of the N-glycosylation pathway in the halophilic archaeon Haloferax volcanii

In Eukarya, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For Archaea, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to target proteins on the external surface of the cell. To remedy this situation, subcellular localization and topology predictive algorithms, protease accessibility, and immunoblotting, together with cysteine modification following site-directed mutagenesis, were enlisted to define the topology of Haloferax volcanii proteins experimentally proven to participate in the N-glycosylation process. AglJ and AglD, involved in the earliest and latest stages, respectively, of assembly of the pentasaccharide decorating the H. volcanii S-layer glycoprotein, were shown to present their soluble N-terminal domain, likely containing the putative catalytic site of each enzyme, to the cytosol. The same holds true for Alg5-B, Dpm1-A, and Mpg1-D, proteins putatively involved in this posttranslational event. The results thus point to the assembly of the pentasaccharide linked to certain Asn residues of the H. volcanii S-layer glycoprotein as occurring within the cell.     

Diversity in prokaryotic glycosylation: an archaeal-derived N-linked glycan contains legionaminic acid

VP4, the major structural protein of the haloarchaeal pleomorphic virus, HRPV‐1, is glycosylated. To define the glycan structure attached to this protein, oligosaccharides released by β‐elimination were analysed by mass spectrometry and nuclear magnetic resonance spectroscopy. Such analyses showed that the major VP4‐derived glycan is a pentasaccharide comprising glucose, glucuronic acid, mannose, sulphated glucuronic acid and a terminal 5‐N‐formyl‐legionaminic acid residue. This is the first observation of legionaminic acid, a sialic acid‐like sugar, in an archaeal‐derived glycan structure. The importance of this residue for viral infection was demonstrated upon incubation with N‐acetylneuraminic acid, a similar monosaccharide. Such treatment reduced progeny virus production by half 4 h post infection. LC‐ESI/MS analysis confirmed the presence of pentasaccharide precursors on two different VP4‐derived peptides bearing the N‐glycosylation signal, NTT. The same sites modified by the native host, Halorubrum sp. strain PV6, were also recognized by the Haloferax volcanii N‐glycosylation apparatus, as determined by LC‐ESI/MS of heterologously expressed VP4. Here, however, the N‐linked pentasaccharide was the same as shown to decorate the S‐layer glycoprotein in this species. Hence, N‐glycosylation of the haloarchaeal viral protein, VP4, is host‐specific. These results thus present additional examples of archaeal N‐glycosylation diversity and show the ability of Archaea to modify heterologously expressed proteins.     

Cytosolic invertases contribute to cellulose biosynthesis and influence carbon partitioning in seedlings of Arabidopsis thaliana

In plants, UDP‐glucose is the direct precursor for cellulose biosynthesis, and can be converted into other NDP‐sugars required for the biosynthesis of wall matrix polysaccharides. UDP‐glucose is generated from sucrose by two distinct metabolic pathways. The first pathway is the direct conversion of sucrose to UDP‐glucose and fructose by sucrose synthase. The second pathway involves sucrose hydrolysis by cytosolic invertase (CINV), conversion of glucose to glucose‐6‐phosphate and glucose‐1‐phosphate, and UDP‐glucose generation by UDP‐glucose pyrophosphorylase (UGP). Previously, Barratt et al. (Proc. Natl Acad. Sci. USA, 106, 2009 and 13124) have found that an Arabidopsis double mutant lacking CINV1 and CINV2 displayed drastically reduced growth. Whether this reduced growth is due to deficient cell wall production caused by limited UDP‐glucose supply, pleiotropic effects, or both, remained unresolved. Here, we present results indicating that the CINV/UGP pathway contributes to anisotropic growth and cellulose biosynthesis in Arabidopsis. Biochemical and imaging data demonstrate that cinv1 cinv2 seedlings are deficient in UDP‐glucose production, exhibit abnormal cellulose biosynthesis and microtubule properties, and have altered cellulose organization without substantial changes to matrix polysaccharide composition, suggesting that the CINV/UGP pathway is a key metabolic route to UDP‐glucose synthesis in Arabidopsis. Furthermore, differential responses of cinv1 cinv2 seedlings to exogenous sugar supplementation support a function of CINVs in influencing carbon partitioning in Arabidopsis. From these data and those of previous studies, we conclude that CINVs serve central roles in cellulose biosynthesis and carbon allocation in Arabidopsis.     

Receptor quality control in the endoplasmic reticulum for plant innate immunity

Pattern recognition receptors in eukaryotes initiate defence responses on detection of microbe‐associated molecular patterns shared by many microbe species. The Leu‐rich repeat receptor‐like kinases FLS2 and EFR recognize the bacterial epitopes flg22 and elf18, derived from flagellin and elongation factor‐Tu, respectively. We describe Arabidopsis ‘priority in sweet life’ (psl) mutants that show de‐repressed anthocyanin accumulation in the presence of elf18. EFR accumulation and signalling, but not of FLS2, are impaired in psl1, psl2, and stt3a plants. PSL1 and PSL2, respectively, encode calreticulin3 (CRT3) and UDP‐glucose:glycoprotein glycosyltransferase that act in concert with STT3A‐containing oligosaccharyltransferase complex in an N‐glycosylation pathway in the endoplasmic reticulum. However, EFR‐signalling function is impaired in weak psl1 alleles despite its normal accumulation, thereby uncoupling EFR abundance control from quality control. Furthermore, salicylic acid‐induced, but EFR‐independent defence is weakened in psl2 and stt3a plants, indicating the existence of another client protein than EFR for this immune response. Our findings suggest a critical and selective function of N‐glycosylation for different layers of plant immunity, likely through quality control of membrane‐localized regulators.     

N-linked glycosylation in Archaea: two paths to the same glycan

N‐linked protein glycosylation occurs in all three branches of life, eukaryotes, bacteria and archaea. The simplest system is that of the bacterium, Campylobacter jejuni, in which a heptasaccharide glycan is added to multiple proteins from a single lipid carrier molecule. In the eukaryotic system a conserved tetradecasaccharide modification is first added to target proteins, but is then modified by trimming and addition of other glycans from additional carrier molecules resulting in a diverse array of glycans of distinct functionality. In the halophilic Archaea from the Dead Sea, Haloferax volcanii, the surface array or S‐layer protein is glycosylated with a pentasaccharide. This glycan is synthesized from two separate carrier molecules, one that carries a tetrasaccharide and another that carries the terminal mannose, in a process that is analogous to that of eukaryotes. In this issue of Molecular Microbiology the glycosylation of the S‐layer of another halophilic Archaea from the Dead Sea, Haloarcula marismortui is characterized ( Calo et al., 2011 ). This S‐layer is glycosylated with the same pentasaccharide as that of Hfx. volcanii, but the intact pentasaccharide is synthesized on a single carrier molecule in Har. marismortui in a process that more closely resembles that of the bacterial N‐linked system.     

Structure of the external aldimine form of PglE,an aminotransferase required for N,N'‐diacetylbacillosamine biosynthesis

N,N'‐diacetylbacillosamine is a novel sugar that plays a key role in bacterial glycosylation. Three enzymes are required for its biosynthesis in Campylobacter jejuni starting from UDP‐GlcNAc. The focus of this investigation, PglE, catalyzes the second step in the pathway. It is a PLP‐dependent aminotransferase that converts UDP‐2‐acetamido‐4‐keto‐2,4,6‐trideoxy‐d ‐glucose to UDP‐2‐acetamido‐4‐amino‐2,4,6‐trideoxy‐d ‐glucose. For this investigation, the structure of PglE in complex with an external aldimine was determined to a nominal resolution of 2.0 Å. A comparison of its structure with those of other sugar aminotransferases reveals a remarkable difference in the manner by which PglE accommodates its nucleotide‐linked sugar substrate.     

Molecular architectures of Pen and Pal: Key enzymes required for CMP‐pseudaminic acid biosynthesis in Bacillus thuringiensis

Bacillus thuringiensis is a soil‐dwelling Gram positive bacterium that has been utilized as a biopesticide for well over 60 years. It is known to contain flagella that are important for motility. One of the proteins found in flagella is flagellin, which is post‐translationally modified by O‐glycosylation with derivatives of pseudaminic acid. The biosynthetic pathway for the production of CMP‐pseudaminic acid in B. thuringiensis, starting with UDP‐N‐acetyl‐d ‐glucosamine (UDP‐GlcNAc), requires seven enzymes. Here, we report the three‐dimensional structures of Pen and Pal, which catalyze the first and second steps, respectively. Pen contains a tightly bound NADP(H) cofactor whereas Pal is isolated with bound NAD(H). For the X‐ray analysis of Pen, the site‐directed D128N/K129A mutant variant was prepared in order to trap its substrate, UDP‐GlcNAc, into the active site. Pen adopts a hexameric quaternary structure with each subunit showing the bilobal architecture observed for members of the short‐chain dehydrogenase/reductase superfamily. The hexameric quaternary structure is atypical for most members of the superfamily. The structure of Pal was determined in the presence of UDP. Pal adopts the more typical dimeric quaternary structure. Taken together, Pen and Pal catalyze the conversion of UDP‐GlcNAc to UDP‐4‐keto‐6‐deoxy‐l ‐N‐acetylaltrosamine. Strikingly, in Gram negative bacteria such as Campylobacter jejuni and Helicobacter pylori, only a single enzyme (FlaA1) is required for the production of UDP‐4‐keto‐6‐deoxy‐l ‐N‐acetylaltrosamine. A comparison of Pen and Pal with FlaA1 reveals differences that may explain why FlaA1 is a bifunctional enzyme whereas Pen and Pal catalyze the individual steps leading to the formation of the UDP‐sugar product. This investigation represents the first structural analysis of the enzymes in B. thuringiensis that are required for CMP‐pseudaminic acid formation.     

Haloferax volcanii AglB and AglD are involved in N-glycosylation of the S-layer glycoprotein and proper assembly of the surface layer

In this study, the effects of deleting two genes previously implicated in Haloferax volcanii N-glycosylation on the assembly and attachment of a novel Asn-linked pentasaccharide decorating the H. volcanii S-layer glycoprotein were considered. Mass spectrometry revealed the pentasaccharide to comprise two hexoses, two hexuronic acids and an additional 190 Da saccharide. The absence of AglD prevented addition of the final hexose to the pentasaccharide, while cells lacking AglB were unable to N-glycosylate the S-layer glycoprotein. In AglD-lacking cells, the S-layer glycoprotein-based surface layer presented both an architecture and protease susceptibility different from the background strain. By contrast, the absence of AglB resulted in enhanced release of the S-layer glycoprotein. H. volcanii cells lacking these N-glycosylation genes, moreover, grew significantly less well at elevated salt levels than did cells of the background strain. Thus, these results offer experimental evidence showing that N-glycosylation endows H. volcanii with an ability to maintain an intact and stable cell envelope in hypersaline surroundings, ensuring survival in this extreme environment.     

Different glycosyltransferases are involved in lipid glycosylation and protein N-glycosylation in the halophilic archaeon <Emphasis Type="Italic">Haloferax volcanii</Emphasis>

Both the lipid and the protein components of biological membranes can be modified by the covalent addition of polysaccharides. Whereas eukaryal and bacterial pathways of lipid and protein glycosylation are relatively well defined, considerably less is known of the parallel processes in Archaea. Recent efforts have identified glycosyltransferases involved in N-glycosylation of the surface-layer glycoprotein of the halophilic archaeon Haloferax volcanii. In the present study, the involvement of these same glycosyltransferases in the biosynthesis of Hfx. volcanii glycolipids was considered by performing nuclear magnetic resonance analysis of the glycolipid fraction of Hfx. volcanii cells deleted of genes encoding those glycosyltransferases, as well as the oligosaccharyltransferase, AglB. The results reveal that different glycosyltransferases are involved in the biosynthesis of N-linked glycoproteins and glycolipids in Archaea.     

Systematic identification of the protein substrates of UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferase‐T1/T2/T3 using a human proteome microarray

O‐GalNAc glycosylation is the initial step of the mucin‐type O‐glycosylation. In humans, it is catalyzed by a family of 20 homologous UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferases (ppGalNAc‐Ts). So far, there is very limited information on their protein substrate specificities. In this study, we developed an on‐chip ppGalNAc‐Ts assay that could rapidly and systematically identify the protein substrates of each ppGalNAc‐T. In detail, we utilized a human proteome microarray as the protein substrates and UDP‐GalNAz as the nucleotide sugar donor for click chemistry detection. From a total of 16 368 human proteins, we identified 570 potential substrates of ppGalNAc‐T1, T2, and T3. Among them, 128 substrates were overlapped, while the rest were isoform specific. Further cluster analysis of these substrates showed that the substrates of ppGalNAc‐T1 had a closer phylogenetic relationship with that of ppGalNAc‐T3 compared with ppGalNAc‐T2, which was consistent with the topology of the phylogenetic tree of these ppGalNAc‐Ts. Taken together, our microarray‐based enzymatic assay comprehensively reveals the substrate profile of the ppGalNAc‐T1, T2, and T3, which not only provides a plausible explanation for their partial functional redundancy as reported, but clearly implies some specialized roles of each enzyme in different biological processes.     

The functional vanGCd cluster of Clostridium difficile does not confer vancomycin resistance

vanG_Cd, a cryptic gene cluster highly homologous to the vanG gene cluster of Enterococcus faecalis is largely spread in Clostridium difficile. Since emergence of vancomycin resistance would have dramatic clinical consequences, we have evaluated the capacity of the vanG_Cd cluster to confer resistance. We showed that expression of vanG_Cd is inducible by vancomycin and that VanG_Cd, VanXY_Cd and VanT_Cd are functional, exhibiting D‐Ala : D‐Ser ligase, D,D‐dipeptidase and D‐Ser racemase activities respectively. In other bacteria, these enzymes are sufficient to promote vancomycin resistance. Trans‐complementation of C. difficile with the vanC resistance operon of Enterococcus gallinarum faintly impacted the MIC of vancomycin, but did not promote vancomycin resistance in C. difficile. Sublethal concentration of vancomycin led to production of UDP‐MurNAc‐pentapeptide[D‐Ser], suggesting that the vanG_Cd gene cluster is able to modify the peptidoglycan precursors. Our results indicated amidation of UDP‐MurNAc‐tetrapeptide, UDP‐MurNAc‐pentapeptide[D‐Ala] and UDP‐MurNAc‐pentapeptide[D‐Ser]. This modification is passed on the mature peptidoglycan where a muropeptide Tetra‐Tetra is amidated on the meso‐diaminopimelic acid. Taken together, our results suggest that the vanG_Cd gene cluster is functional and is prevented from promoting vancomycin resistance in C. difficile.     

Differences between rice and wheat in ribulose-1,5-bisphosphate regeneration capacity per unit of leaf-N content

The photosynthetic rates under saturating CO₂ conditions per unit of leaf‐N content were higher in wheat than in rice. This suggested that ribulose‐1,5‐bisphosphate (RuBP) regeneration capacity is greater in wheat. Therefore, the biochemical factor(s) for this difference were examined between rice and wheat. Soluble protein‐N, insoluble‐N, and trichloroacetic acid (TCA) soluble‐N contents were found not to differ between the two species. The activities of several Calvin cycle enzymes such as RuBP carboxylase, NADP‐glyceraldehyde‐3‐phosphate dehydrogenase, phosphoglycerate kinase and chloroplastic fructose‐1,6‐bisphosphatase (cpFBPase) activities per unit of leaf‐N content were all higher in wheat than in rice. Among them, cpFBPase activity was most highly correlated with CO₂‐saturated photosynthesis. The V_max activity of sucrose‐phosphate synthase (SPS) for UDP‐glucose was almost the same between the two species and its K_m value was a little lower in rice. Chlorophyll content and its a/b ratio did not differ. Cytochrome (Cyt) f content was greater in wheat, whereas coupling factor 1 content was greater in rice. Cyt f content was highly correlated with CO₂‐saturated photosynthesis, irrespective of the two species. The results thus suggested that higher RuBP regeneration capacity in wheat leaves is most closely related to a greater Cyt f content and that another candidate is cpFBPase.     

Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases

Asparagine‐linked glycosylation is catalysed by oligosaccharyltransferase (OTase). In Trypanosoma brucei OTase activity is catalysed by single‐subunit enzymes encoded by three paralogous genes of which TbSTT3B and TbSTT3C can complement a yeast Δstt3 mutant. The two enzymes have overlapping but distinct peptide acceptor specificities, with TbSTT3C displaying an enhanced ability to glycosylate sites flanked by acidic residues. TbSTT3A and TbSTT3B, but not TbSTT3C, are transcribed in the bloodstream and procyclic life cycle stages of T. brucei. Selective knockdown and analysis of parasite protein N‐glycosylation showed that TbSTT3A selectively transfers biantennary Man₅GlcNAc₂ to specific glycosylation sites whereas TbSTT3B selectively transfers triantennary Man₉GlcNAc₂ to others. Analysis of T. brucei glycosylation site occupancy showed that TbSTT3A and TbSTT3B glycosylate sites in acidic to neutral and neutral to basic regions of polypeptide, respectively. This embodiment of distinct specificities in single‐subunit OTases may have implications for recombinant glycoprotein engineering. TbSTT3A and TbSTT3B could be knocked down individually, but not collectively, in tissue culture. However, both were independently essential for parasite growth in mice, suggesting that inhibiting protein N‐glycosylation could have therapeutic potential against trypanosomiasis.     

An oligosaccharyltransferase from Leishmania major increases the N‐glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana

N‐glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central‐protein complex facilitating the N‐glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N‐glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single‐subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well‐established production platform for recombinant proteins. A fluorescent protein‐tagged LmSTT3D variant was predominately found in the ER and co‐located with plant oligosaccharyltransferase subunits. Co‐expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N‐glycosylation site occupancy on all N‐glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N‐glycosylation efficiency in plants.