Prokaryotic Glycoproteins Research Articles

ProGlycProt V3.0: updated insights into prokaryotic glycoproteins and their glycosyltransferases.

ProGlycProt is a comprehensive database of experimentally validated information about protein glycosylation in prokaryotes, including the glycoproteins, glycosyltransferases, and their accessory enzymes. The first release of ProGlycProt featured experimentally validated information on glycoproteins only. For the second release in 2019, the size and scope of the database were expanded twofold, and experimental data on cognate glycosyltransferases and their accessory proteins was incorporated. The growing research and technology interest in microbial glycoproteins and their enzymes is evident from the steady rise in academic publications and patents in this area. Accordingly, the third update comprises a new section on patents related to glycosylation methods, novel glycosyltransferases, and technologies developed therefrom. The structure gallery is reorganized, wherein the number and quality of the models are upgraded with the help of AlphaFold2. Over the years, the influx of experimental proteomics data into public repositories like PRIDE has surged. Harnessing this legacy data for in-silico glycoprotein identification is a smart approach. Version 3.0 adds 45N-glycoprotein entries annotated from MS datasets available on PRIDE and reviewed by independent research groups. With a 67% rise in entries corresponding to 119 genera of prokaryotes, the ProGlycProt continues to be the exclusive database of experimentally validated comprehensive information about protein glycosylation in prokaryotes.

Two RmlC homologs catalyze dTDP-4-keto-6-deoxy-d-glucose epimerization in Pseudomonas putida KT2440

l-Rhamnose is an important monosaccharide both as nutrient source and as building block in prokaryotic glycoproteins and glycolipids. Generation of those composite molecules requires activated precursors being provided e. g. in form of nucleotide sugars such as dTDP-β-l-rhamnose (dTDP-l-Rha). dTDP-l-Rha is synthesized in a conserved 4-step reaction which is canonically catalyzed by the enzymes RmlABCD. An intact pathway is especially important for the fitness of pseudomonads, as dTDP-l-Rha is essential for the activation of the polyproline specific translation elongation factor EF-P in these bacteria. Within the scope of this study, we investigated the dTDP-l-Rha-biosynthesis route of Pseudomonas putida KT2440 with a focus on the last two steps. Bioinformatic analysis in combination with a screening approach revealed that epimerization of dTDP-4-keto-6-deoxy-d-glucose to dTDP-4-keto-6-deoxy-l-mannose is catalyzed by the two paralogous proteins PP_1782 (RmlC1) and PP_0265 (RmlC2), whereas the reduction to the final product is solely mediated by PP_1784 (RmlD). Thus, we also exclude the distinct RmlD homolog PP_0500 and the genetically linked nucleoside diphosphate-sugar epimerase PP_0501 to be involved in dTDP-l-Rha formation, other than suggested by certain databases. Together our analysis contributes to the molecular understanding how this important nucleotide-sugar is synthesized in pseudomonads.

Improving designer glycan production in Escherichia coli through model-guided metabolic engineering

Asparagine-linked (N-linked) glycosylation is the most common protein modification in eukaryotes, affecting over two-thirds of the proteome. Glycosylation is also critical to the pharmacokinetic activity and immunogenicity of many therapeutic proteins currently produced in complex eukaryotic hosts. The discovery of a protein glycosylation pathway in the pathogen Campylobacter jejuni and its subsequent transfer into laboratory strains of Escherichia coli has spurred great interest in glycoprotein production in prokaryotes. However, prokaryotic glycoprotein production has several drawbacks, including insufficient availability of non-native glycan precursors. To address this limitation, we used a constraint-based model of E. coli metabolism in combination with heuristic optimization to design gene knockout strains that overproduced glycan precursors. First, we incorporated reactions associated with C. jejuni glycan assembly into a genome-scale model of E. coli metabolism. We then identified gene knockout strains that coupled optimal growth to glycan synthesis. Simulations suggested that these growth-coupled glycan overproducing strains had metabolic imbalances that rerouted flux toward glycan precursor synthesis. We then validated the model-identified knockout strains experimentally by measuring glycan expression using a flow cytometric-based assay involving fluorescent labeling of cell surface-displayed glycans. Overall, this study demonstrates the promising role that metabolic modeling can play in optimizing the performance of a next-generation microbial glycosylation platform.

Emerging facets of prokaryotic glycosylation.

Glycosylation of proteins is one of the most prevalent post-translational modifications occurring in nature, with a wide repertoire of biological implications. Pathways for the main types of this modification, the N- and O-glycosylation, can be found in all three domains of life-the Eukarya, Bacteria and Archaea-thereby following common principles, which are valid also for lipopolysaccharides, lipooligosaccharides and glycopolymers. Thus, studies on any glycoconjugate can unravel novel facets of the still incompletely understood fundamentals of protein N- and O-glycosylation. While it is estimated that more than two-thirds of all eukaryotic proteins would be glycosylated, no such estimate is available for prokaryotic glycoproteins, whose understanding is lagging behind, mainly due to the enormous variability of their glycan structures and variations in the underlying glycosylation processes. Combining glycan structural information with bioinformatic, genetic, biochemical and enzymatic data has opened up an avenue for in-depth analyses of glycosylation processes as a basis for glycoengineering endeavours. Here, the common themes of glycosylation are conceptualised for the major classes of prokaryotic (i.e. bacterial and archaeal) glycoconjugates, with a special focus on glycosylated cell-surface proteins. We describe the current knowledge of biosynthesis and importance of these glycoconjugates in selected pathogenic and beneficial microbes.

Glycobiology of the oral pathogen Porphyromonas gingivalis and related species

Until recently, glycoproteins had only been described in eukaryotes. However, advances in detection methods and genome analyses have allowed the discovery of N-linked or O-linked glycoproteins, similar to those found in eukaryotes, in some bacterial species. These prokaryotic glycoproteins play roles in adhesion, solubility, formation of protein complexes, protection from protein degradation, and changes in antigenicity. Periodontal pathogen Porphyromonas gingivalis secretes virulence proteins via the type IX secretion system, some of which localize on the cell surface by binding to lipopolysaccharide (LPS). These virulence proteins have a conserved C-terminal domain (CTD) region, which is used as a secretion signal. However, it is still uncertain how the secreted proteins on the cell surface bind to LPS. In this review, we discuss the synthesis of P. gingivalis O polysaccharide, which plays a role in anchoring the CTD protein on the cell surface, and recent discoveries of glycoproteins in P. gingivalis as well as other species in the phylum Bacteroidetes.

Surface-exposed Glycoproteins of Hyperthermophilic Sulfolobus solfataricus P2 Show a Common N-Glycosylation Profile

Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the N-glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of S. solfataricus, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex4(GlcNAc)2 plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of S. acidocaldarius, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of S. solfataricus are N-glycosylated by the same sulfated oligosaccharide and we identified 56 N-glycosylation sites in this subproteome.

GlycoPP: A Webserver for Prediction of N- and O-Glycosites in Prokaryotic Protein Sequences

Glycosylation is one of the most abundant post-translational modifications (PTMs) required for various structure/function modulations of proteins in a living cell. Although elucidated recently in prokaryotes, this type of PTM is present across all three domains of life. In prokaryotes, two types of protein glycan linkages are more widespread namely, N- linked, where a glycan moiety is attached to the amide group of Asn, and O- linked, where a glycan moiety is attached to the hydroxyl group of Ser/Thr/Tyr. For their biologically ubiquitous nature, significance, and technology applications, the study of prokaryotic glycoproteins is a fast emerging area of research. Here we describe new Support Vector Machine (SVM) based algorithms (models) developed for predicting glycosylated-residues (glycosites) with high accuracy in prokaryotic protein sequences. The models are based on binary profile of patterns, composition profile of patterns, and position-specific scoring matrix profile of patterns as training features. The study employ an extensive dataset of 107 N-linked and 116 O-linked glycosites extracted from 59 experimentally characterized glycoproteins of prokaryotes. This dataset includes validated N-glycosites from phyla Crenarchaeota, Euryarchaeota (domain Archaea), Proteobacteria (domain Bacteria) and validated O-glycosites from phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria (domain Bacteria). In view of the current understanding that glycosylation occurs on folded proteins in bacteria, hybrid models have been developed using information on predicted secondary structures and accessible surface area in various combinations with training features. Using these models, N-glycosites and O-glycosites could be predicted with an accuracy of 82.71% (MCC 0.65) and 73.71% (MCC 0.48), respectively. An evaluation of the best performing models with 28 independent prokaryotic glycoproteins confirms the suitability of these models in predicting N- and O-glycosites in potential glycoproteins from aforementioned organisms, with reasonably high confidence. A web server GlycoPP, implementing these models is available freely at http:/www.imtech.res.in/raghava/glycopp/.

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.

ProGlycProt: a repository of experimentally characterized prokaryotic glycoproteins

ProGlycProt (http://www.proglycprot.org/) is an open access, manually curated, comprehensive repository of bacterial and archaeal glycoproteins with at least one experimentally validated glycosite (glycosylated residue). To facilitate maximum information at one point, the database is arranged under two sections: (i) ProCGP—the main data section consisting of 95 entries with experimentally characterized glycosites and (ii) ProUGP—a supplementary data section containing 245 entries with experimentally identified glycosylation but uncharacterized glycosites. Every entry in the database is fully cross-referenced and enriched with available published information about source organism, coding gene, protein, glycosites, glycosylation type, attached glycan, associated oligosaccharyl/glycosyl transferases (OSTs/GTs), supporting references, and applicable additional information. Interestingly, ProGlycProt contains as many as 174 entries for which information is unavailable or the characterized glycosites are unannotated in Swiss-Prot release 2011_07. The website supports a dedicated structure gallery of homology models and crystal structures of characterized glycoproteins in addition to two new tools developed in view of emerging information about prokaryotic sequons (conserved sequences of amino acids around glycosites) that are never or rarely seen in eukaryotic glycoproteins. ProGlycProt provides an extensive compilation of experimentally identified glycosites (334) and glycoproteins (340) of prokaryotes that could serve as an information resource for research and technology applications in glycobiology.

Bioanalytical tools for the discovery of eukaryotic glycoproteins applied to the analysis of bacterial glycoproteins

The commonly accepted theory that prokaryotes lack the ability to glycosylate their proteins has been disproved recently. The field of bacterial glycoprotein research is no longer considered novel owing to the rapid progress in analytical technologies and genome sequencing that has been made in the last few years. Enhanced interest in glycoprotein discovery in bacteria can be explained by a proven correlation between the presence of glycosylation and bacterial pathogenicity. Eukaryotic and prokaryotic organisms’ features share certain similarities. However, with respect to inherent differences between these two distinct domains of life, the use of bioanalytical tools for glycoprotein analysis in eukaryotic systems often needs modification to be applied successfully to bacteria. In this article, we draw attention to the differences between eukaryotic and prokaryotic glycoproteins. We also focus on the main bottlenecks that may be encountered in the search for glycosylation in concrete bacterium and outline a possible work-flow for the exploration of glycoproteins in bacteria.

Mass spectrometric profiling ofO-linked glycans released directly from glycoproteins in gels using in-gel reductive β-elimination

Glycosylation is a widespread PTM of proteins; the carbohydrate moieties provide various functional, immunological and structural aspects of both eukaryotic and prokaryotic glycoproteins. Traditional strategies used to analyse glycoprotein O-glycans involve glycoprotein isolation, followed by glycan release using solution-phase base-catalysed beta-elimination. However, in a proteomics context, mixtures of proteins and glycoproteins are routinely separated using SDS-PAGE. We have therefore developed a method to enable the profiling of O-linked glycans directly from glycoproteins on gels. This is achieved using in-gel reductive beta-elimination followed by mass spectrometric analysis of the released glycans. Here we describe our demonstration of the feasibility of this approach, our development and optimisation of the procedure using bovine submaxillary gland glycoproteins as a standard, and then show its usefulness by applying the developed procedure to the analysis of the O-glycans from a glycoprotein band from a Coomassie-stained SDS-PAGE separation of a mixture of Mycobacterium avium capsular proteins and glycoproteins. The procedure has been shown to be applicable to both CBB- and silver-stained gels. The method offers a quick and easy way to identify the O-glycans from gel-separated glycoproteins within gel-based proteomics workflows.

Bacterial glycoproteins: functions, biosynthesis and applications.

Although widely distributed in eukaryotic cells glycoproteins appear to be rare in prokaryotic organisms. The prevalence of the misconception that bacteria do not glycosylate their proteins has been a subject matter of discussion for a long time. Glycoconjugates that are linked to proteins or peptides, generated by the ribosomal translational mechanism have been reported only in the last two to three decades in a few prokaryotic organisms. Most studied prokaryotic glycoproteins are the S-layer glycoproteins of Archeabacteria. Apart from these, membrane-associated, surface-associated, secreted glycoproteins and exoenzymes glycoproteins are also well documented in both, Archea and Eubacteria. From the recent literature, it is now clear that prokaryotes are capable of glycosylating proteins. In general, prokaryotes are deprived of the cellular organelles required for glycosylation. In prokaryotes many different glycoprotein structures have been observed that display much more variation than that observed in eukaryotes. Besides following similar mechanisms in the process of glycosylation, prokaryotes have also been shown to use mechanisms that are different from those found in eukaryotes. The knowledge pertaining to the functional aspects of prokaryotic glycoproteins is rather scarce. This review summarizes developments and understanding relating to characteristics, synthesis, and functions of prokaryotic glycoproteins. An extensive summary of glycosylation that has been reported to occur in bacteria has also been tabulated. Various possible applications of these diverse biomolecules in biotechnology, vaccine development, pharmaceutics and diagnostics are also touched upon.

Prokaryotic glycosylation.

With the advances of molecular biology and with improved analytical techniques a significant change of perception has taken place regarding prokaryotic glycoproteins. Glycosylation of proteins from prokaryotes is no longer considered a specific feature of certain organisms but has been demonstrated for many archaea and bacteria. Besides the occurrence of glycosylated enzymes, antigens and other cell envelope components, surface layer (S-layer) glycoproteins represent the best-studied examples of glycosylated prokaryotic proteins. They are widely distributed among archaeal wild-type strains, but among bacteria they have been mainly observed with Gram-positive organisms. There is, in general, an enormous increase of reports on the presence of glycosylated proteins among prokaryotes. For their isolation and characterization a great number of methods are available, aiming at the identification of the covalent linkage between the carbohydrate and the polypeptide portion. So far, several differences in structure and biosynthesis have been observed in comparison to eukaryotic glycoproteins. In this review we introduce a protocol which has been successfully applied to the investigation of the complex structures, linkage units, and polypeptide consensus sequences of glycosylated bacterial S-layer proteins.

Prokaryotic glycosylation

With the advances of molecular biology and with improved analytical techniques a significant change of perception has taken place regarding prokaryotic glycoproteins. Glycosylation of proteins from prokaryotes is no longer considered a specific feature of certain organisms but has been demonstrated for many archaea and bacteria. Besides the occurrence of glycosylated enzymes, antigens and other cell envelope components, surface layer (S-layer) glycoproteins represent the best-studied examples of glycosylated prokaryotic proteins. They are widely distributed among archaeal wild-type strains, but among bacteria they have been mainly observed with Gram-positive organisms. There is, in general, an enormous increase of reports on the presence of glycosylated proteins among prokaryotes. For their isolation and characterization a great number of methods are available, aiming at the identification of the covalent linkage between the carbohydrate and the polypeptide portion. So far, several differences in structure and biosynthesis have been observed in comparison to eukaryotic glycoproteins. In this review we introduce a protocol which has been successfully applied to the investigation of the complex structures, linkage units, and polypeptide consensus sequences of glycosylated bacterial S-layer proteins.

Glycoproteins in prokaryotes.

Rather recently it has become clear that prokaryotes (Archaea and Bacteria) are able to glycosylate proteins. A literature survey revealed the different types of glycoproteins. They include mainly surface layer (S-layer) proteins, flagellins, and polysaccharide-degrading enzymes. Only in a few cases is structural information available. Many different structures have been observed that display much more variation than that observed in eukaryotes. A few studies have given evidence for the function of the prokaryotic glycoprotein glycans. Also from the biosynthetic point of view, information is rather scarce. Due to their different cell structure, prokaryotes have to use mechanisms different from those found in eukaryotes to glycosylate proteins. However, from the fragmented data available for the prokaryotic glycoproteins, similarities with the eukaryotic system can be noticed.

Biochemistry of S-layers.

During evolution prokaryotes have developed different envelope structures exterior to the cell wall proper. Among these surface components are regularly arranged S-layers and capsules. The structural characterization and the detailed chemical analysis of these surface molecules is a prerequisite to understand their biosynthesis and functional role(s) at the molecular level. Of particular interest are the glycosylated S-layer proteins which belong to the first prokaryotic glycoproteins ever described. Their characterization was performed on strains belonging to the thermophilic Bacillaceae and included structural studies and experiments to learn about the pathways for the glycan biosynthesis of S-layer glycoproteins. As an example for non-glycosylated S-layer proteins those of Lactobacillus helveticus strains are described in detail. Recently, a novel type of bacterial glycoconjugate was observed in the cell envelope of the extremely halophilic archaeon Natronococcus occultus which consists of a glycosylated polyglutamyl polymer. Beside the conventional biochemical techniques for the analysis new sophisticated instrumental methods such as X-ray photoelectron spectroscopy and matrix-assisted laser desorption ionization or electrospray ionization mass spectrometry have been introduced for the analysis of the protein and glycan portions of these cell surface macromolecules.

Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis.

Chemical evidence for the true glycosylation of mycobacterial proteins was recently provided in the context of the 45-kDa MPT 32 secreted protein of Mycobacterium tuberculosis (K. Dobos, K. Swiderek, K.-H. Khoo, P. J. Brennan, and J. T. Belisle, Infect. Immun. 63:2846-2853, 1995). However, the full extent and nature of glycosylation as well as the location of glycosylated amino acids remained undefined. First, to examine the nature of the covalently attached sugars, the 45-kDa protein was obtained from cells metabolically labeled with D-[U-14C] glucose and subjected to compositional analysis, which revealed mannose as the only covalently bound sugar. Digestion of the protein with the endoproteinase subtilisin and analysis of products by liquid chromatography-electrospray-mass spectrometry on the basis of fragments demonstrating neutral losses of hexose (m/z 162) or pentose (m/z 132) revealed five glycopeptides, S7, S18, S22, S29, and S41 among a total of 50 peptides, all of which produced only m/z 162 fragmentation ion deletions. Fast atom bombardment-mass spectrometry, N-terminal amino acid sequencing, and alpha-mannosidase digestion demonstrated universal O glycosylation of Thr residues with a single alpha-D-Man, mannobiose, or mannotriose unit. Linkages within the mannobiose and mannotriose were all alpha 1-2, as proven by gas chromatography-mass spectrometry of oligosaccharides released by beta-elimination. Total sequences of many of the glycosylated and nonglycosylated peptides combined with published information on the deduced amino acid sequence of the entire 45-kDa protein demonstrated that the sites of glycosylation were located in Pro-rich domains near the N terminus and C terminus of the polypeptide backbone. Specifically, the Thr residues at positions 10 and 18 were substituted with alpha-D-Manp(1-->2)alpha-D-Manp, the Thr residue at position 27 was substituted with a single alpha-D-Manp, and Thr-277 was substituted with either alpha-D-Manp, alpha-D-Manp(1-->2)alpha-D-Manp, or alpha-D-Manp(1--> 2)alpha-D-Manp(1-->2)alpha-D-Manp. This report further corroborates the existence of true prokaryotic glycoproteins, defines the complete structure of a mycobacterial mannoprotein and the first complete structure of a mannosylated mycobacterial protein, and establishes the principles for the study of other mycobacterial glycoproteins.

Structure and biosynthesis of prokaryotic glycoproteins.

Abstract Glycoproteins as components of cell surfaces are not restricted to eukaryotes. The prokaryotic glycoprotein studied in greatest detail so far is the cell surface glycoprotein of the archaebacterium Halobacterium halobium . This bacterial glycoprotein contains 3 different types of glycoconjugates, and each type of glycoconjugate involves a different carbohydrate-protein linkage unit: 1. 1) One glycosaminoglycan chain, constructed from a repeating sulfated pentasaccharide block, is linked to one protein molecule via the novel N -glycosyl linkage unit asparaginyl- N -acetylgalactosamine. 2. 2) Ten sulfated oligosaccharides that contain glucose, glucuronic acid and iduronic acid are bound to the protein via the hitherto unknown N -glycosyl linkage unit asparaginylglucose. 3. 3) About 15 disaccharides, glucosylgalactose, are O -glycosyl-linked to a cluster of threonine residues close to the C-terminus of the core protein. The overall structure of the cell surface glycoprotein of halobacteria is thus reminiscent of animal proteoglycans and a functional role of the glycosaminoglycan chain in maintaining the rod shape of halobacteria is discussed. Biosynthesis of the two N -glycosyl linkage units involves dolichol monophosphate and dolicholdiphosphate-linked saccharide precursors. Sulfation and epimerization of the glycoconjugates occur at the lipid-linked level and the mature saccharides are transferred to the protein core on the cell surface. The sulfated oligosaccharides that finally become bound to asparagine via glucose are transiently methylated at their lipid-linked stage and this transient chemical modification seems to be required for the biosynthesis of the corresponding N -glycosyl bond.

Structure and biosynthesis of prokaryotic glycoproteins

Glycoproteins as components of cell surfaces are not restricted to eukaryotes. The prokaryotic glycoprotein studied in greatest detail so far is the cell surface glycoprotein of the archaebacterium Halobacterium halobium. This bacterial glycoprotein contains 3 different types of glycoconjugates, and each type of glycoconjugate involves a different carbohydrate-protein linkage unit: 1. 1) One glycosaminoglycan chain, constructed from a repeating sulfated pentasaccharide block, is linked to one protein molecule via the novel N-glycosyl linkage unit asparaginyl- N-acetylgalactosamine. 2. 2) Ten sulfated oligosaccharides that contain glucose, glucuronic acid and iduronic acid are bound to the protein via the hitherto unknown N-glycosyl linkage unit asparaginylglucose. 3. 3) About 15 disaccharides, glucosylgalactose, are O-glycosyl-linked to a cluster of threonine residues close to the C-terminus of the core protein. The overall structure of the cell surface glycoprotein of halobacteria is thus reminiscent of animal proteoglycans and a functional role of the glycosaminoglycan chain in maintaining the rod shape of halobacteria is discussed. Biosynthesis of the two N-glycosyl linkage units involves dolichol monophosphate and dolicholdiphosphate-linked saccharide precursors. Sulfation and epimerization of the glycoconjugates occur at the lipid-linked level and the mature saccharides are transferred to the protein core on the cell surface. The sulfated oligosaccharides that finally become bound to asparagine via glucose are transiently methylated at their lipid-linked stage and this transient chemical modification seems to be required for the biosynthesis of the corresponding N-glycosyl bond.

Purification and characterization of a prokaryotic glycoprotein from the cell envelope of Halobacterium salinarium.

The glycoprotein which accounts for approximately 50% of the protein and all of the nonlipid carbohydrate of the cell envelope of Halobacterium salinarium (Mescher, M. F., Strominger, J. L., and Watson S. W. (1974) J. Bacteriol. 120, 945-954) has been purified and partially characterized. The glycoprotein has an apparent molecular weight of 200,000, is extremely acidic, and has a carbohydrate content of approximately 10 to 12%. The carbohydrate included neutral hexoses, amino sugar, and uronic acid. Information regarding the number, composition, and mode of attachment of the carbohydrate chains was obtained by isolation and examination of the glycopeptides derived from degradation of cell envelope protein with trypsin and pronase. Trypsin digestion resulted in two glycopeptides. One of these was large (approximately 55,000 daltons) and had most of the neutral hexose linked to it. The carbohydrate moieties consisted of di- and trisaccharides of glucosylgalactose and (uronic acid, glucose)-galactose attached via O-glycosidic linkages between galactose and threonine. The other tryptic glycopeptide had a relatively large heterosaccharide attached to it via an alkaline-stable linkage. The heterosaccharide contained 1 glucose, 8 to 9 galactose, 1 mannose, and 10 to 11 glucosamine residues, and approximately 6 residues of an unidentified amino augar. The alkaline stability of the linkage and the amino acid composition of glycopeptides resulting from Pronase digestion of the tryptic glycopeptide showed that the heterosaccharide was attached to an asparagine residue, presumably via an N-glycosylamine bond to the amide group. The intact glycoprotein has a single N-linked heterosaccharide, 22 to 24 O-linked disaccharides, and 12 to 14 O-linked trisaccharides per molecule. N- and O-glycosidic linkages are the most common carbohydrate-protein linkages in mammalian glycoproteins but, to our knowledge, this is the first report of either type of linkage in a prokaryotic cell envelope protein.