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1.
Biosynthesis of the N-linked glycan in Campylobacter jejuni and addition onto protein through block transfer
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Kelly J Jarrell H Millar L Tessier L Fiori LM Lau PC Allan B Szymanski CM 《Journal of bacteriology》2006,188(7):2427-2434
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. 相似文献
2.
Linton D Dorrell N Hitchen PG Amber S Karlyshev AV Morris HR Dell A Valvano MA Aebi M Wren BW 《Molecular microbiology》2005,55(6):1695-1703
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. 相似文献
3.
Post-translational glycosylation is a universal modification of proteins in eukarya, archaea and bacteria. Two recent publications describe the first confirmed report of a bacterial N-linked glycosylation pathway in the human gastrointestinal pathogen Campylobacter jejuni. In addition, an O-linked glycosylation pathway has been identified and characterized in C. jejuni and the related species Campylobacter coli. Both pathways have similarity to the respective N- and O-linked glycosylation processes in eukaryotes. In bacteria, homologues of the genes in both pathways are found in other organisms, the complex glycans linked to the glycoproteins share common biosynthetic precursors and these modifications could play similar biological roles. Thus, Campylobacter provides a unique model system for the elucidation and exploitation of glycoprotein biosynthesis. 相似文献
4.
The O-linked protein glycosylation pathway in Neisseria gonorrhoeae is responsible for the synthesis of a complex oligosaccharide on undecaprenyl diphosphate and subsequent en bloc transfer of the glycan to serine residues of select periplasmic proteins. Protein glycosylation (pgl) genes have been annotated on the basis of bioinformatics and top-down mass spectrometry analysis of protein modifications in pgl-null strains [Aas, F. E., et al. (2007) Mol. Microbiol. 65, 607-624; Vik, A., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 4447-4452], but relatively little biochemical analysis has been performed to date. In this report, we present the expression, purification, and functional characterization of seven Pgl enzymes. Specifically, the enzymes studied are responsible for synthesis of an uncommon uridine diphosphate (UDP)-sugar (PglD, PglC, and PglB-acetyltransferase domain), glycan assembly (PglB-phospho-glycosyltransferase domain, PglA, PglE, and PglH), and final oligosaccharide transfer (PglO). UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-hexose (DATDH), which is the first sugar in glycan biosynthesis, was produced enzymatically, and the stereochemistry was assigned as uridine diphosphate N'-diacetylbacillosamine (UDP-diNAcBac) by nuclear magnetic resonance characterization. In addition, the substrate specificities of the phospho-glycosyltransferase, glycosyltransferases, and oligosaccharyltransferase (OTase) were analyzed in vitro, and in most cases, these enzymes exhibited strong preferences for the native substrates relative to closely related glycans. In particular, PglO, the O-linked OTase, and PglB(Cj), the N-linked OTase from Campylobacter jejuni, preferred the native N. gonorrhoeae and C. jejuni substrates, respectively. This study represents the first comprehensive biochemical characterization of this important O-linked glycosylation pathway and provides the basis for further investigations of these enzymes as antibacterial targets. 相似文献
5.
Microbial cell surface glycans in the form of glycolipids and glycoproteins frequently play important roles in cell-cell interaction and host immune responses. Given the likely importance of these surface structures in the survival and pathogenesis of Campylobacter jejuni, a concerted effort has been made to characterise these determinants genetically and structurally since the genome was sequenced in 2000. We review the considerable progress made in characterising the Campylobacter glycome including the lipooligosaccharide (LOS), the capsule and O- and N-linked protein glycosylation systems, and speculate on the roles played by glycan surface structures in the life-cycle of C. jejuni. 相似文献
6.
N-linked protein glycosylation is required for full competence in Campylobacter jejuni 81-176
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The recent sequencing of the virulence plasmid of Campylobacter jejuni 81-176 revealed the presence of genes homologous to type IV secretion systems (TFSS) that have subsequently been found in Helicobacter pylori and Wolinella succinogenes. Mutational analyses of some of these genes have implicated their involvement in intestinal epithelial cell invasion and natural competence. In this report, we demonstrate that one of these type IV secretion homologs, Cjp3/VirB10, is a glycoprotein. Treatment with various glycosidases and binding to soybean agglutinin indicated that the structure of the glycan present on VirB10 contains a terminal GalNAc, consistent with previous reports of N-linked glycans in C. jejuni. Site-directed mutagenesis of five putative N-linked glycosylation sites indicated that VirB10 is glycosylated at two sites, N32 and N97. Mutants in the N-linked general protein glycosylation (pgl) system of C. jejuni are significantly reduced in natural transformation, which is likely due, in part, to lack of glycosylation of VirB10. The natural transformation defect in a virB10 mutant can be complemented in trans by using a plasmid expressing wild-type VirB10 or an N32A substitution but not by using a mutant expressing VirB10 with an N97A substitution. Taken together, these results suggest that glycosylation of VirB10 specifically at N97 is required for the function of the TFSS and for full competence in C. jejuni 81-176. 相似文献
7.
Szymanski CM Michael FS Jarrell HC Li J Gilbert M Larocque S Vinogradov E Brisson JR 《The Journal of biological chemistry》2003,278(27):24509-24520
Glycomics, the study of microbial polysaccharides and genes responsible for their formation, requires the continuous development of rapid and sensitive methods for the identification of glycan structures. In this study, methods for the direct analysis of sugars from 108 to 1010 cells are outlined using the human gastrointestinal pathogen, Campylobacter jejuni. Using capillary-electrophoresis coupled with sensitive electrospray mass spectrometry, we demonstrate variability in the lipid A component of C. jejuni lipooligosaccharides (LOSs). In addition, these sensitive methods have permitted the detection of phase-variable LOS core structures that were not observed previously. High resolution magic angle spinning (HR-MAS) NMR was used to examine capsular polysaccharides directly from campylobacter cells and showed profiles similar to those observed for purified polysaccharides analyzed by solution NMR. This method also exhibited the feasibility of campylobacter serotyping, mutant verification, and preliminary sugar analysis. HR-MAS NMR examination of growth from individual colonies of C. jejuni NCTC11168 indicated that the capsular glycan modifications are also phase-variable. These variants show different staining patterns on deoxycholate-PAGE and reactivity with immune sera. One of the identified modifications was a novel -OP=O(NH2)OMe phosphoramide, not observed previously in nature. In addition, HR-MAS NMR detected the N-linked glycan, GalNAc-alpha1,4-GalNAc-alpha1,4-[Glc-beta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac, where Bac is 2,4-diacetamido-2,4,6-trideoxy-d-glucopyranose, in C. jejuni and Campylobacter coli. The presence of this common heptasaccharide in multiple campylobacter isolates demonstrates the conservation of the N-linked protein glycosylation pathway in this organism and describes the first report of HR-MAS NMR detection of N-linked glycans on glycoproteins from intact bacterial cells. 相似文献
8.
Michael Welti 《Glycoconjugate journal》2013,30(1):51-56
In the majority of congenital disorders of glycosylation, the assembly of the glycan precursor GlcNAc2Man9Glc3 on the polyprenol carrier dolichyl-pyrophosphate is compromised. Because N-linked glycosylation is essential to life, most types of congenital disorders of glycosylation represent partial losses of enzymatic activity. Consequently, increased availability of substrates along the glycosylation pathway can be beneficial to increase product formation by the compromised enzymes. Recently, we showed that increased dolichol availability and improved N-linked glycosylation can be achieved by inhibition of squalene biosynthesis. This review summarizes the current knowledge on the biosynthesis of dolichol-linked glycans with respect to deficiencies in N-linked glycosylation. Additionally, perspectives on therapeutic treatments targeting dolichol and dolichol-linked glycan biosynthesis are examined. 相似文献
9.
Glycosylation of proteins in prokaryotes has been known for the last few decades. Glycan structures and/or the glycosylation pathways have been experimentally characterized in only a small number of prokaryotes. Even this has become possible only during the last decade or so, primarily due to technological and methodological developments. Glycosylated proteins are diverse in their function and localization. Glycosylation has been shown to be associated with a wide range of biological phenomena. Characterization of the various types of glycans and the glycosylation machinery is critical to understand such processes. Such studies can help in the identification of novel targets for designing drugs, diagnostics, and engineering of therapeutic proteins. In view of this, the experimentally characterized pgl system of Campylobacter jejuni, responsible for N-linked glycosylation, has been used in this study to identify glycosylation loci in 865 prokaryotes whose genomes have been completely sequenced. Results from the present study show that only a small number of organisms have homologs for all the pgl enzymes and a few others have homologs for none of the pgl enzymes. Most of the organisms have homologs for only a subset of the pgl enzymes. There is no specific pattern for the presence or absence of pgl homologs vis-à-vis the 16S rRNA sequence-based phylogenetic tree. This may be due to differences in the glycan structures, high sequence divergence, horizontal gene transfer or non-orthologous gene displacement. Overall, the presence of homologs for pgl enzymes in a large number of organisms irrespective of their habitat, pathogenicity, energy generation mechanism, etc., hints towards the ubiquity of N-linked glycosylation in prokaryotes. 相似文献
10.
Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure 总被引:1,自引:0,他引:1
Neisseria gonorrhoeae expresses an O-linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a 'top-down' mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O-acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N-linked glycosylation system that adds N-acetylgalactosamine onto undecaprenylpyrophosphate-linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O-linked di- and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid-linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O- and N-linked pathways can be combined in novel glycoengineering strategies. 相似文献
11.
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. 相似文献
12.
Cell surface layers (S-layers) are common structures of the bacterial cell envelope with a lattice-like appearance that are formed by a self-assembly process. Frequently, the constituting S-layer proteins are modified with covalently linked glycan chains facing the extracellular environment. S-layer glycoproteins from organisms of the Bacillaceae family possess long, O-glycosidically linked glycans that are composed of a great variety of sugar constituents. The observed variations already exceed the display found in eukaryotic glycoproteins. Recent investigations of the S-layer protein glycosylation process at the molecular level, which has lagged behind the structural studies due to the lack of suitable molecular tools, indicated that the S-layer glycoprotein glycan biosynthesis pathway utilizes different modules of the well-known biosynthesis routes of lipopolysaccharide O-antigens. The genetic information for S-layer glycan biosynthesis is usually present in S-layer glycosylation (slg) gene clusters acting in concert with housekeeping genes. To account for the nanometer-scale cell surface display feature of bacterial S-layer glycosylation, we have coined the neologism 'nanoglycobiology'. It includes structural and biochemical aspects of S-layer glycans as well as molecular data on the machinery underlying the glycosylation event. A key aspect for the full potency of S-layer nanoglycobiology is the unique self-assembly feature of the S-layer protein matrix. Being aware that in many cases the glycan structures associated with a protein are the key to protein function, S-layer protein glycosylation will add a new and valuable component to an 'S-layer based molecular construction kit'. In our long-term research strategy, S-layer nanoglycobiology shall converge with other functional glycosylation systems to produce 'functional' S-layer neoglycoproteins for diverse applications in the fields of nanobiotechnology and vaccine technology. Recent advances in the field of S-layer nanoglycobiology have made our overall strategy a tangible aim of the near future. 相似文献
13.
Nina M. van Sorge Nancy M. C. Bleumink Sandra J. van Vliet Eirikur Saeland W. -Ludo van der Pol Yvette van Kooyk Jos P. M. van Putten 《Cellular microbiology》2009,11(12):1768-1781
An increasing number of bacterial pathogens produce an array of glycoproteins of unknown function. Here we report that Campylobacter jejuni proteins that are modified by the N -linked glycosylation machinery encoded by the pgl locus bind the human Macrophage Galactose-type lectin (MGL). MGL receptor binding was abrogated by EDTA and N -acetylgalactosamine (GalNAc) and was successfully transferred to Escherichia coli by introducing the C. jejuni pgl locus together with a glycan acceptor protein. In addition to glycoproteins, C. jejuni lipooligosaccharide with a terminal GalNAc residue was recognized by MGL. Recombinant E. coli expressing the C. jejuni pgl locus in the absence of a suitable glycan acceptor protein produced altered lipopolysaccharide glycoforms that gained MGL reactivity. Infection assays demonstrated high levels of GalNAc-dependent interaction of the recombinant E. coli with MGL-transfected mammalian cells. In addition, interleukin-6 production by human dendritic cells was enhanced by C. jejuni lacking N -linked glycans compared with wild-type bacteria. Collectively, our results provide evidence that both N -linked glycoproteins and distinct lipooligosaccharide glycoforms of C. jejuni are ligands for the human C-type lectin MGL and that the C. jejuni N -glycosylation machinery can be exploited to target recombinant bacteria to MGL-expressing eukaryotic cells. 相似文献
14.
Lowell J Brady Jyoti Velayudhan Devi B Visone Ken C Daugherty Jeff L Bartron Michael Coon Cabot Cornwall Peter J Hinckley Lisa Connell-Crowley 《MABS-AUSTIN》2015,7(3):562-570
Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays. This work highlights the challenges of developing high-quality biosimilar candidates and the need for modern biotechnology capabilities. These results show that high-quality analytics, combined with sensitive cell-based assays to study in vivo mechanisms of action, is an essential part of biosimilar development. 相似文献
15.
《MABS-AUSTIN》2013,5(3):562-570
Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays. This work highlights the challenges of developing high-quality biosimilar candidates and the need for modern biotechnology capabilities. These results show that high-quality analytics, combined with sensitive cell-based assays to study in vivo mechanisms of action, is an essential part of biosimilar development. 相似文献
16.
Creuzenet C 《FEBS letters》2004,559(1-3):136-140
Campylobacter jejuni encodes numerous sugar-nucleotide-modifying enzymes potentially involved in the biosynthesis of surface carbohydrates. One of them, CJ1293, is involved in flagellin glycosylation but its biochemical activity remains unknown. Using over-expressed and purified protein, we demonstrate that CJ1293 has UDP-GlcNAc-specific C(6) dehydratase activity. Catalysis occurs without addition of cofactor, suggesting internal recycling of NAD(P)(+). The K(m) for UDP-GlcNAc of 50 microM indicates that CJ1293 has higher affinity for its substrate than previously characterized homologues. Based on enzymatic data, we propose that CJ1293 catalyzes the first step in the biosynthesis of bacillosamine, a sugar found in C. jejuni's protein glycosylation motifs. 相似文献
17.
Campylobacter jejuni has a general N-linked glycosylation pathway, encoded by the pgl gene cluster. In C. jejuni, a heptasaccharide is transferred from an undecaprenyl pyrophosphate donor [GalNAc-alpha1,4-GalNAc-alpha1,4-(Glcbeta1,3)-GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-alpha1-PP-undecaprenyl, where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] to the asparagine side chain of target proteins at the Asn-X-Ser/Thr motif. In this study, we have cloned, overexpressed in Escherichia coli, and purified PglC, the glycosyl-1-phosphate transferase responsible for the first step in the biosynthesis of the undecaprenyl-linked heptasaccharide donor. In addition, we report the first synthetic route to uridine 5'-diphosphobacillosamine. Using the uridine 5'-diphosphobacillosamine and undecaprenyl phosphate, we demonstrate the ability of PglC to produce undecaprenyl pyrophosphate bacillosamine using radiolabeled HPLC and mass spectral analysis. In addition, we revealed that PglC does not accept uridine 5'-diphospho-N-acetylglucosamine or uridine 5'-diphospho-N-acetylgalactosamine as substrates but will accept uridine 5'-diphospho-6-hydroxybacillosamine, an analogue of bacillosamine that retains the C-6 hydroxyl functionality from the biosynthetic precursor. The in vitro characterization of PglC as a bacillosamine 1-phosphoryl transferase provides direct evidence for the early steps in the C. jejuni N-linked glycosylation pathway, and the coupling of PglC with the latter glycosyltransferases (PglA, PglJ, PglH, and PglI) allows for the "one-pot" chemoenzymatic synthesis of the undecaprenyl pyrophosphate heptasaccharide donor. 相似文献
18.
The Campylobacter coli 72Dz/92 cjaA gene (orthologue of cj0982c of C. jejuni NCTC 11168) product is a highly immunogenic, amino acid-binding protein. CjaA was palmitic acid-modified when processed in E. coli. In addition, site-directed mutagenesis of the Cys residue of the LAAC motif of its signal sequence confirmed that CjaA is a lipoprotein when processed in Campylobacter. Localization of the protein appeared to be host dependent. In Campylobacter, CjaA was recovered mainly as an inner-membrane protein, whereas in E. coli most of the protein was present in the periplasmic space. Interestingly, antiserum raised against Campylobacter glycine-extracted material also recognized CjaA produced by Campylobacter and Escherichia coli, indicating that at least part of the protein may be surface exposed. Site-directed mutagenesis of the Asn residues of two putative N-linked glycosylation sites (NIS and NFT) showed that CjaA is glycosylated and that only the first N-X-S/T sequeon serves as a glycan acceptor. 相似文献
19.
Sparrow LG Lawrence MC Gorman JJ Strike PM Robinson CP McKern NM Ward CW 《Proteins》2008,71(1):426-439
The human insulin receptor (IR) homodimer is heavily glycosylated and contains a total of 19 predicted N-linked glycosylation sites in each monomer. The recent crystal structure of the IR ectodomain shows electron density consistent with N-linked glycosylation at the majority of sites present in the construct. Here, we describe a refined structure of the IR ectodomain that incorporates all of the N-linked glycans and reveals the extent to which the attached glycans mask the surface of the IR dimer from interaction with antibodies or other potential therapeutic binding proteins. The usefulness of Fab complexation in the crystallization of heavily glycosylated proteins is also discussed. The compositions of the glycans on IR expressed in CHO-K1 cells and the glycosylation deficient Lec8 cell line were determined by protease digestion, glycopeptide purification, amino acid sequence analysis, and mass spectrometry. Collectively the data reveal: multiple species of complex glycan at residues 25, 255, 295, 418, 606, 624, 742, 755, and 893 (IR-B numbering); multiple species of high-mannose glycan at residues 111 and 514; a single species of complex glycan at residue 671; and a single species of high-mannose glycan at residue 215. Residue 16 exhibited a mixture of complex, hybrid, and high-mannose glycan species. Of the remaining five predicted N-linked sites, those at residues 397 and 906 were confirmed by amino acid sequencing to be glycosylated, while that at residue 78 and the atypical (NKC) site at residue 282 were not glycosylated. The peptide containing the final site at residue 337 was not recovered but is seen to be glycosylated in the electron density maps of the IR ectodomain. The model of the fully glycosylated IR reveals that the sites carrying high-mannose glycans lie at positions of relatively low steric accessibility. 相似文献
20.
The human pathogen Campylobacter jejuni possesses a general N-linked glycosylation system that is known to play a role in pathogenicity; however, a detailed understanding of this role remains elusive. A considerable hindrance to studying bacterial N-glycosylation in vivo is the absence of small molecule inhibitors to reversibly control the process. This report describes a pathway-screening assay that targets the early enzymes of C. jejuni N-glycan biosynthesis that would enable identification of inhibitors to the first four steps in the pathway. The assay includes PglF, PglE, PglD, PglC, and PglA; the enzymes involved in the biosynthesis of an undecaprenyl diphosphate-linked disaccharide and monitors the transfer of [3H]GalNAc from the hydrophilic UDP-linked carrier to the lipophilic UndPP-diNAcBac (2,4-diacetamido-2,4,6-trideoxyglucose). The optimized assay has a Z'-factor calculated to be 0.77, indicating a robust assay suitable for screening. The diacylglycerol kinase from Streptococcus mutans, which provides a convenient method for phosphorylating undecaprenol, has been included in a modified version of the assay thereby allowing the screen to be conducted with entirely commercially available substrates. 相似文献