Identification and characterization of the unique N-linked glycan common to the flagellins and S-layer glycoprotein of Methanococcus voltae

The flagellum of Methanococcus voltae is composed of four structural flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3. These proteins possess a total of 15 potential N-linked sequons (NX(S/T)) and show a mass shift on an SDS-polyacrylamide gel indicating significant post-translational modification. We describe here the structural characterization of the flagellin glycan from M. voltae using mass spectrometry to examine the proteolytic digests of the flagellin proteins in combination with NMR analysis of the purified glycan using a sensitive, cryogenically cooled probe. Nano-liquid chromatography-tandem mass spectrometry analysis of the proteolytic digests of the flagellin proteins revealed that they are post-translationally modified with a novel N-linked trisaccharide of mass 779 Da that is composed of three sugar residues with masses of 318, 258, and 203 Da, respectively. In every instance the glycan is attached to the peptide through the asparagine residue of a typical N-linked sequon. The glycan modification has been observed on 14 of the 15 sequon sites present on the four flagellin structural proteins. The novel glycan structure elucidated by NMR analysis was shown to be a trisaccharide composed of beta-ManpNAcA6Thr-(1-4)-beta-Glc-pNAc3NAcA-(1-3)-beta-GlcpNAc linked to Asn. In addition, the same trisaccharide was identified on a tryptic peptide of the S-layer protein from this organism implicating a common N-linked glycosylation pathway.     

AglC and AglK are involved in biosynthesis and attachment of diacetylated glucuronic acid to the N-glycan in Methanococcus voltae

Recent advances in the field of prokaryotic N-glycosylation have established a foundation for the pathways and proteins involved in this important posttranslational protein modification process. To continue the study of the Methanococcus voltae N-glycosylation pathway, characteristics of known eukaryotic, bacterial, and archaeal proteins involved in the N-glycosylation process were examined and used to select candidate M. voltae genes for investigation as potential glycosyl transferase and flippase components. The targeted genes were knocked out via linear gene replacement, and the resulting effects on N-glycan assembly were identified through flagellin and surface (S) layer protein glycosylation defects. This study reports the finding that deletion of two putative M. voltae glycosyl transferase genes, designated aglC (for archaeal glycosylation) and aglK, interfered with proper N-glycosylation. This resulted in flagellin and S-layer proteins with significantly reduced apparent molecular masses, loss of flagellar assembly, and absence of glycan attachment. Given previous knowledge of both the N-glycosylation pathway in M. voltae and the general characteristics of N-glycosylation components, it appears that AglC and AglK are involved in the biosynthesis or transfer of diacetylated glucuronic acid within the glycan structure. In addition, a knockout of the putative flippase candidate gene (Mv891) had no effect on N-glycosylation but did result in the production of giant cells with diameters three to four times that of wild-type cells.     

Identification of a putative acetyltransferase gene, MMP0350, which affects proper assembly of both flagella and pili in the archaeon Methanococcus maripaludis

Glycosylation is a posttranslational modification utilized in all three domains of life. Compared to eukaryotic and bacterial systems, knowledge of the archaeal processes involved in glycosylation is limited. Recently, Methanococcus voltae flagellin proteins were found to have an N-linked trisaccharide necessary for proper flagellum assembly. Current analysis by mass spectrometry of Methanococcus maripaludis flagellin proteins also indicated the attachment of an N-glycan containing acetylated sugars. To identify genes involved in sugar biosynthesis in M. maripaludis, a putative acetyltransferase was targeted for in-frame deletion. Deletion of this gene (MMP0350) resulted in a flagellin molecular mass shift to a size comparable to that expected for underglycosylated or completely nonglycoslyated flagellins, as determined by immunoblotting. Assembled flagellar filaments were not observed by electron microscopy. Interestingly, the deletion also resulted in defective pilus anchoring. Mutant cells with a deletion of MMP0350 had very few, if any, pili attached to the cell surface compared to a nonflagellated but piliated strain. However, pili were obtained from culture supernatants of this strain, indicating that the defect was not in pilus assembly but in stable attachment to the cell surface. Complementation of MMP0350 on a plasmid restored pilus attachment, but it was unable to restore flagellation, likely because the mutant ceased to make detectable flagellin. These findings represent the first report of a biosynthetic gene involved in flagellin glycosylation in archaea. Also, it is the first gene to be associated with pili, linking flagellum and pilus structure and assembly through posttranslational modifications.     

Identification of genes involved in the acetamidino group modification of the flagellin N-linked glycan of Methanococcus maripaludis

N-linked glycosylation of protein is a posttranslational modification found in all three domains of life. The flagellin proteins of the archaeon Methanococcus maripaludis are known to be modified with an N-linked tetrasaccharide consisting of N-acetylgalactosamine (GalNAc), a diacetylated glucuronic acid (GlcNAc3NAc), an acetylated and acetamidino-modified mannuronic acid with a substituted threonine group (ManNAc3NAmA6Thr), and a novel terminal sugar residue [(5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose]. To identify genes involved in biosynthesis of the component sugars of this glycan, three genes, mmp1081, mmp1082, and mmp1083, were targeted for in-frame deletion, based on their annotation and proximity to glycosyltransferase genes known to be involved in assembly of the glycan. Mutants carrying a deletion in any of these three genes remained flagellated and motile. A strain with a deletion of mmp1081 had lower-molecular-mass flagellins in Western blots. Mass spectrometry of purified flagella revealed a truncated glycan with the terminal sugar absent and the threonine residue and the acetamidino group missing from the third sugar. No glycan modification was seen in either the Δmmp1082 or Δmmp1083 mutant grown in complex Balch III medium. However, a glycan identical to the Δmmp1081 glycan was observed when the Δmmp1082 or Δmmp1083 mutant was grown under ammonia-limited conditions. We hypothesize that MMP1082 generates ammonia and tunnels it through MMP1083 to MMP1081, which acts as the amidotransferase, modifying the third sugar residue of the M. maripaludis glycan with the acetamidino group.     

Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway

We describe in this report the characterization of the recently discovered N-linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N-linked heptasaccharide. Furthermore, we show that C. jejuni- and E. coli-derived pathways can interact in the biosynthesis of N-linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP-GlcNAc: undecaprenylphosphate GlcNAc-1-phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N-linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni-derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N-linked glycan occurs on a lipid-linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N-linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.     

Identification of genes involved in the assembly and attachment of a novel flagellin N-linked tetrasaccharide important for motility in the archaeon Methanococcus maripaludis

Recently, the flagellin proteins of Methanococcus maripaludis were found to harbour an N -linked tetrasaccharide composed of N -acetylgalactosamine, di-acetylated glucuronic acid, an acetylated and acetamidino-modified mannuronic acid linked to threonine, and a novel terminal sugar [( 5S )-2-acetamido-2,4-dideoxy-5-O-methyl-α-L- erythro -hexos-5-ulo-1,5-pyranose]. To identify genes involved in the assembly and attachment of this glycan, in-frame deletions were constructed in putative glycan assembly genes. Successful deletion of genes encoding three glycosyltransferases and an oligosaccharyltransferase (Stt3p homologue) resulted in flagellins of decreased molecular masses as evidenced by immunoblotting, indicating partial or completely absent glycan structures. Deletion of the oligosaccharyltransferase or the glycosyltransferase responsible for the transfer of the second sugar in the chain resulted in flagellins that were not assembled into flagella filaments, as evidenced by electron microscopy. Deletions of the glycosyltransferases responsible for the addition of the third and terminal sugars in the glycan were confirmed by mass spectrometry analysis of purified flagellins from these mutants. Although flagellated, these mutants had decreased motility as evidenced by semi-swarm plate analysis with the presence of each additional sugar improving movement capabilities.     

Distinct glycan-charged phosphodolichol carriers are required for the assembly of the pentasaccharide N-linked to the Haloferax volcanii S-layer glycoprotein

In Archaea, dolichol phosphates have been implicated as glycan carriers in the N-glycosylation pathway, much like their eukaryal counterparts. To clarify this relation, highly sensitive liquid chromatography/mass spectrometry was employed to detect and characterize glycan-charged phosphodolichols in the haloarchaeon Haloferax volcanii. It is reported that Hfx. volcanii contains a series of C(55) and C(60) dolichol phosphates presenting saturated isoprene subunits at the α and ω positions and sequentially modified with the first, second, third and methylated fourth sugar subunits comprising the first four subunits of the pentasaccharide N-linked to the S-layer glycoprotein, a reporter of N-glycosylation. Moreover, when this glycan-charged phosphodolichol pool was examined in cells deleted of agl genes encoding glycosyltransferases participating in N-glycosylation and previously assigned roles in adding pentasaccharide residues one to four, the composition of the lipid-linked glycans was perturbed in the identical manner as was S-layer glycoprotein N-glycosylation in these mutants. In contrast, the fifth sugar of the pentasaccharide, identified as mannose in this study, is added to a distinct dolichol phosphate carrier. This represents the first evidence that in Archaea, as in Eukarya, the oligosaccharides N-linked to glycoproteins are sequentially assembled from glycans originating from distinct phosphodolichol carriers.     

N-linked glycosylation is required for nicotinic receptor assembly but not for subunit associations with calnexin

We investigated how asparagine (N)-linked glycosylation affects assembly of acetylcholine receptors (AChRs) in the endoplasmic reticulum (ER). Block of N-linked glycosylation inhibited AChR assembly whereas block of glucose trimming partially blocked assembly at the late stages. Removal of each of seven glycans had a distinct effect on AChR assembly, ranging from no effect to total loss of assembly. Because the chaperone calnexin (CN) associates with N-linked glycans, we examined CN interactions with AChR subunits. CN rapidly associates with 50% or more of newly synthesized AChR subunits, but not with subunits after maturation. Block of N-linked glycosylation or trimming did not alter CN-AChR subunit associations nor did subunit mutations prevent N-linked glycosylation. Additionally, CN associations with subunits lacking N-linked glycans occurred without subunit aggregation or misfolding. Our data indicate that CN associates with AChR subunits without N-linked glycan interactions. Furthermore, CN-subunit associations only occur early in AChR assembly and have no role in events later that require N-linked glycosylation.     

Characterization of the structurally diverse N-linked glycans of Campylobacter species

The Gram-negative bacterium Campylobacter jejuni encodes an extensively characterized N-linked protein glycosylation system that modifies many surface proteins with a heptasaccharide glycan. In C. jejuni, the genes that encode the enzymes required for glycan biosynthesis and transfer to protein are located at a single pgl gene locus. Similar loci are also present in the genome sequences of all other Campylobacter species, although variations in gene content and organization are evident. In this study, we have demonstrated that only Campylobacter species closely related to C. jejuni produce glycoproteins that interact with both a C. jejuni N-linked-glycan-specific antiserum and a lectin known to bind to the C. jejuni N-linked glycan. In order to further investigate the structure of Campylobacter N-linked glycans, we employed an in vitro peptide glycosylation assay combined with mass spectrometry to demonstrate that Campylobacter species produce a range of structurally distinct N-linked glycans with variations in the number of sugar residues (penta-, hexa-, and heptasaccharides), the presence of branching sugars, and monosaccharide content. These data considerably expand our knowledge of bacterial N-linked glycan structure and provide a framework for investigating the role of glycosyltransferases and sugar biosynthesis enzymes in glycoprotein biosynthesis with practical implications for synthetic biology and glycoengineering.     

Glycobiology of surface layer proteins

Over the last two decades, a significant change of perception has taken place regarding prokaryotic glycoproteins. For many years, protein glycosylation was assumed to be limited to eukaryotes; but now, a wealth of information on structure, function, biosynthesis and molecular biology of prokaryotic glycoproteins has accumulated, with surface layer (S-layer) glycoproteins being one of the best studied examples. With the designation of Archaea as a second prokaryotic domain of life, the occurrence of glycosylated S-layer proteins had been considered a taxonomic criterion for differentiation between Bacteria and Archaea. Extensive structural investigations, however, have demonstrated that S-layer glycoproteins are present in both domains. Among Gram-positive bacteria, S-layer glycoproteins have been identified only in bacilli. In Gram-negative organisms, their presence is still not fully investigated; presently, there is no indication for their existence in this class of bacteria. Extensive biochemical studies of the S-layer glycoprotein from Halobacterium halobium have, at least in part, unravelled the glycosylation pathway in Archaea; molecular biological analyses of these pathways have not been performed, so far. Significant observations concern the occurrence of unusual linkage regions both in archaeal and bacterial S-layer glycoproteins. Regarding S-layer glycoproteins of bacteria, first genetic data have shed some light into the molecular organization of the glycosylation machinery in this domain. In addition to basic S-layer glycoprotein research, the biotechnological application potential of these molecules has been explored. With the development of straightforward molecular biological methods, fascinating possibilities for the expression of prokaryotic glycoproteins will become available. S-layer glycoprotein research has opened up opportunities for the production of recombinant glycosylation enzymes and tailor-made S-layer glycoproteins in large quantities, which are commercially not yet available. These bacterial systems may provide economic technologies for the production of biotechnologically and medically important glycan structures in the future.     

Genetic organization of chromosomal S-layer glycan biosynthesis loci of Bacillaceae

S-layer glycoproteins are cell surface glycoconjugates that have been identified in archaea and in bacteria. Usually, S-layer glycoproteins assemble into regular, crystalline arrays covering the entire bacterium. Our research focuses on thermophilic Bacillaceae, which are considered a suitable model system for studying bacterial glycosylation. During the past decade, investigations of S-layer glycoproteins dealt with the elucidation of the highly variable glycan structures by a combination of chemical degradation methods and nuclear magnetic resonance spectroscopy. It was only recently that the molecular characterization of the genes governing the formation of the S-layer glycoprotein glycan chains has been initiated. The S-layer glycosylation (slg) gene clusters of four of the 11 known S-layer glycan structures from members of the Bacillaceae have now been studied. The clusters are approximately 16 to approximately 25 kb in size and transcribed as polycistronic units. They include nucleotide sugar pathway genes that are arranged as operons, sugar transferase genes, glycan processing genes, and transporter genes. So far, the biochemical functions only of the genes required for nucleotide sugar biosynthesis have been demonstrated experimentally. The presence of insertion sequences and the decrease of the G + C content at the slg locus suggest that the investigated organisms have acquired their specific S-layer glycosylation potential by lateral gene transfer. In addition, S-layer protein glycosylation requires the participation of housekeeping genes that map outside the cluster. The gene encoding the respective S-layer target protein is transcribed monocistronically and independently of the slg cluster genes. Its chromosomal location is not necessarily in close vicinity to the slg gene cluster.     

Biosynthesis of the N-linked glycan in Campylobacter jejuni and addition onto protein through block transfer

In eukaryotes, N-linked protein glycosylation is a universal modification involving addition of preformed oligosaccharides to select Asn-Xaa-Ser/Thr motifs and influencing multiple biological events. We recently demonstrated that Campylobacter jejuni is the first member of the Bacteria to possess an N-linked glycan pathway. In this study, high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to probe and quantitate C. jejuni N-glycan biosynthesis in vivo. To confirm HR-MAS NMR findings, glycosylation mutants were screened for chicken colonization potential, and glycoproteins were examined by mass spectrometry and lectin blotting. Consistent with the mechanism in eukaryotes, the combined data indicate that bacterial glycans are assembled en bloc, emphasizing the evolutionary conservation of protein N glycosylation. We also show that under the conditions examined, PglG plays no role in glycan biosynthesis, PglI is the glucosyltransferase and the putative ABC transporter, and WlaB (renamed PglK) is required for glycan assembly. These studies underpin the mechanism of N-linked protein glycosylation in Bacteria and provide a simple model system for investigating protein glycosylation and for exploitation in glycoengineering.     

Genetic organization of chromosomal S-layer glycan biosynthesis loci of Bacillaceae

S-layer glycoproteins are cell surface glycoconjugates that have been identified in archaea and in bacteria. Usually, S-layer glycoproteins assemble into regular, crystalline arrays covering the entire bacterium. Our research focuses on thermophilic Bacillaceae, which are considered a suitable model system for studying bacterial glycosylation. During the past decade, investigations of S-layer glycoproteins dealt with the elucidation of the highly variable glycan structures by a combination of chemical degradation methods and nuclear magnetic resonance spectroscopy. It was only recently that the molecular characterization of the genes governing the formation of the S-layer glycoprotein glycan chains has been initiated. The S-layer glycosylation (slg) gene clusters of four of the 11 known S-layer glycan structures from members of the Bacillaceae have now been studied. The clusters are ～16 to ～25 kb in size and transcribed as polycistronic units. They include nucleotide sugar pathway genes that are arranged as operons, sugar transferase genes, glycan processing genes, and transporter genes. So far, the biochemical functions only of the genes required for nucleotide sugar biosynthesis have been demonstrated experimentally. The presence of insertion sequences and the decrease of the G+C content at the slg locus suggest that the investigated organisms have acquired their specific S-layer glycosylation potential by lateral gene transfer. In addition, S-layer protein glycosylation requires the participation of housekeeping genes that map outside the cluster. The gene encoding the respective S-layer target protein is transcribed monocistronically and independently of the slg cluster genes. Its chromosomal location is not necessarily in close vicinity to the slg gene cluster. Published in 2004.     

Stimulation of N-linked glycosylation and lipid-linked oligosaccharide synthesis by stress responses in metazoan cells

Endoplasmic reticulum (ER) stress responses comprising the unfolded protein response (UPR) are activated by conditions that disrupt folding and assembly of proteins inside the ER lumenal compartment. Conditions known to be proximal triggers of the UPR include saturation of chaperones with misfolded protein, redox imbalance, disruption of Ca2+ levels, interference with N-linked glycosylation, and failure to dispose of terminally misfolded proteins. Potentially, ER stress responses can reprogram cells to correct all of these problems and thereby restore ER function to normal. This article will review literature on stimulation of N-linked glycosylation by ER stress responses, focusing on metazoan systems. The mechanisms involved will be contrasted with those mediating stimulation of N-linked glycosylation by cytoplasmic stress responses. This information will interest readers who study the biological roles of stress responses, the functions of N-linked glycans, and potential strategies for treatment of genetic disorders of N-linked glycosylation.     

Variable site-occupancy classification of N-linked glycosylation using artificial neural networks

A novel neural-network-based model has been developed for the prediction of N-linked glycosylation characteristics related to glycosylation site-occupancy. Intracellular oligosaccharide transfer to a polypeptide is known to be either robust or dependent upon culture conditions during pharmaceutical production. This glycan attachment is classified by the model as robust or variable and is based on an input of the polypeptide primary sequence around the site of glycosylation. The glycosylation model utilizes multiple recurrent neural networks followed by a perceptron classifier. The input length of the polypeptide chain around the site of glycosylation (glycosylation window) was optimized through multiple independent training sessions. Incorporation of five residues prior (n - 5) to the site of glycosylation (n) and four residues beyond (n + 4) the glycan attachment site led to optimal network performance. The size of the glycosylation window for site-occupancy determination is much larger than has been previously reported. This model was developed to evaluate the effects of theoretical polypeptide mutations on glycosylation site-occupancy characteristics. Following correct prediction of the model testing data set, 20 independent networks were used to predict site-occupancy characteristics of wild-type and mutants of the rabies virus glycoprotein (rgp). Simulation results strongly correlated with previously published experimental results (Kasturi, L.; Hegang, C.; Shakin-Eshleman, S. H. Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosacchride acceptors. Biochem. J. 1997, 323, 415-419. Mellquist, J. L.; Kasturi, L.; Spitalnik, S. L.; Shakin-Eshleman, S. H. The amino acid following an Asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency. Biochemistry 1998, 37, 6833-6837). Further simulations on purely theoretical sequences suggested that influences of charged residues were a subset of multiple mechanisms in the determination of glycosylation site-occupancy.     

Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel Flagellum Accessory Proteins

Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii has been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes, flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes of M. voltae were successfully overexpressed as His-tagged proteins in Escherichia coli. These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionated M. voltae cells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly, flaD was found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes of M. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.     

The expanding horizons of asparagine-linked glycosylation

Asparagine-linked glycosylation involves the sequential assembly of an oligosaccharide onto a polyisoprenyl donor, followed by the en bloc transfer of the glycan to particular asparagine residues within acceptor proteins. These N-linked glycans play a critical role in a wide variety of biological processes, such as protein folding, cellular targeting and motility, and the immune response. In the past decade, research in the field of N-linked glycosylation has achieved major advances, including the discovery of new carbohydrate modifications, the biochemical characterization of the enzymes involved in glycan assembly, and the determination of the biological impact of these glycans on target proteins. It is now firmly established that this enzyme-catalyzed modification occurs in all three domains of life. However, despite similarities in the overall logic of N-linked glycoprotein biosynthesis among the three kingdoms, the structures of the appended glycans are markedly different and thus influence the functions of elaborated proteins in various ways. Though nearly all eukaryotes produce the same nascent tetradecasaccharide (Glc(3)Man(9)GlcNAc(2)), heterogeneity is introduced into this glycan structure after it is transferred to the protein through a complex series of glycosyl trimming and addition steps. In contrast, bacteria and archaea display diversity within their N-linked glycan structures through the use of unique monosaccharide building blocks during the assembly process. In this review, recent progress toward gaining a deeper biochemical understanding of this modification across all three kingdoms will be summarized. In addition, a brief overview of the role of N-linked glycosylation in viruses will also be presented.     

N-Glycosylation of Haloferax volcanii Flagellins Requires Known Agl Proteins and Is Essential for Biosynthesis of Stable Flagella

N-glycosylation, a posttranslational modification required for the accurate folding and stability of many proteins, has been observed in organisms of all domains of life. Although the haloarchaeal S-layer glycoprotein was the first prokaryotic glycoprotein identified, little is known about the glycosylation of other haloarchaeal proteins. We demonstrate here that the glycosylation of Haloferax volcanii flagellins requires archaeal glycosylation (Agl) components involved in S-layer glycosylation and that the deletion of any Hfx. volcanii agl gene impairs its swimming motility to various extents. A comparison of proteins in CsCl density gradient centrifugation fractions from supernatants of wild-type Hfx. volcanii and deletion mutants lacking the oligosaccharyltransferase AglB suggests that when the Agl glycosylation pathway is disrupted, cells lack stable flagella, which purification studies indicate consist of a major flagellin, FlgA1, and a minor flagellin, FlgA2. Mass spectrometric analyses of FlgA1 confirm that its three predicted N-glycosylation sites are modified with covalently linked pentasaccharides having the same mass as that modifying its S-layer glycoprotein. Finally, the replacement of any of three predicted N-glycosylated asparagines of FlgA1 renders cells nonmotile, providing direct evidence for the first time that the N-glycosylation of archaeal flagellins is critical for motility. These results provide insight into the role that glycosylation plays in the assembly and function of Hfx. volcanii flagella and demonstrate that Hfx. volcanii flagellins are excellent reporter proteins for the study of haloarchaeal glycosylation processes.     

Overexpression of Methanococcus voltae Flagellin Subunits in Escherichia coli and Pseudomonas aeruginosa: a Source of Archaeal Preflagellin

Methanococcus voltae is a flagellated member of the Archaea. Four highly similar flagellin genes have previously been cloned and sequenced, and the presence of leader peptides has been demonstrated. While the flagellins of M. voltae are predicted from their gene sequences to be approximately 22 to 25 kDa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified flagella revealed flagellin subunits with apparent molecular masses of 31 and 33 kDa. Here we describe the expression of a M. voltae flagellin in the bacteria Escherichia coli and Pseudomonas aeruginosa. Both of these systems successfully generated a specific expression product with an apparently uncleaved leader peptide migrating at approximately 26.5 kDa. This source of preflagellin was used to detect the presence of preflagellin peptidase activity in the membranes of M. voltae. In addition to the native flagellin, a hybrid flagellin gene containing the sequence encoding the M. voltae FlaB2 mature protein fused to the P. aeruginosa pilin (PilA) leader peptide was constructed and transformed into both wild-type P. aeruginosa and a prepilin peptidase (pilD) mutant of P. aeruginosa. Based on migration in SDS-PAGE, the leader peptide appeared to be cleaved in the wild-type cells. However, the archaeal flagellin could not be detected by immunoblotting when expressed in the pilD mutant, indicating a role of the peptidase in the ultimate stability of the fusion product. When the +5 position of the mature flagellin portion of the pilin-flagellin fusion was changed from glycine to glutamic acid (as in the P. aeruginosa pilin) and expressed in both wild-type and pilD mutant P. aeruginosa, the product detected by immunoblotting migrated slightly more slowly in the pilD mutant, indicating that the fusion was likely processed by the prepilin peptidase present in the wild type. Potential assembly of the cleaved fusion product by the type IV pilin assembly system in a P. aeruginosa PilA-deficient strain was tested, but no filaments were noted on the cell surface by electron microscopy.     

From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni

The gram-negative bacterium Campylobacter jejuni was recently discovered to contain a general N-linked protein glycosylation pathway. Central to this pathway is PglB, a homologue of the Stt3p subunit of the eukaryotic oligosaccharyl transferase (OT), which is involved in the transfer of an oligosaccharide from a polyisoprenyl pyrophosphate carrier to the asparagine side chain of proteins within the conserved glycosylation sites D/E-X1-N-X2-S/T, where X1 and X2 can be any amino acids except proline. Using a library of peptide substrates and a quantitative radioactivity-based in vitro assay, we assessed the amino acids at each position of the consensus glycosylation sequence for their impact on glycosylation efficiency, whereby the sequence DQNAT was found to be the optimal acceptor substrate. In the context of a full-length folded protein, the differences between variations of the glycosylation sequences were found to be consistent with the trends observed from their peptidyl counterparts, though less dramatic because of additional influences. In addition to characterizing the acceptor preferences of PglB, we also assessed the selectivity toward the glycan donor. Interestingly, despite recent reports of relaxed selectivity toward the glycan donor, PglB was not found to be capable of utilizing glycosyl donors such as dolichyl-pyrophosphate-chitobiose, which is the minimum substrate for the eukaryotic OT process.