National Research Council of Canada, Institute for Biological Sciences, 100 Sussex Drive, Ottawa, Ontario, K1A 0R6 Canada
Received on January 8, 2004; revised on February 19, 2004; accepted on March 4, 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: glycosyltransferase / lipooligosaccharide / Neisseria
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The biosynthesis of bacterial lipopolysaccharides (LPSs) has been extensively investigated over the past 10 years by genetic approaches (Raetz and Whitfield, 2002, and references within). Genetic analysis has identified a large number of genes, and various mutations or strain comparisons have been made to clarify the functions of these genes (Gilbert et al., 2000
; Kahler et al., 1996
; Wakarchuk et al., 1996
). The enzymology of the biosynthesis of the lipid A portion has been investigated for Escherichia coli LPS (Raetz and Whitfield, 2002
, and references within). The enzymes involved in the biosynthesis of the inner core structures have been examined only in a selection of organisms (Noah et al., 2001
). The outer core enzymes from a variety of species have been examined as recombinant enzymes (Gilbert et al., 1997
, 2000
; Wakarchuk et al., 1998
).
The study of the enzymology of glycosyltransferases involved in the biosynthesis of the LOS in N. meningitidis and N. gonorrhoeae so far has been limited to the outer core enzymes, which make the sialyl-lacto-N-neotetraose mimic (Blixt et al., 1999; Gilbert et al., 1997
; Wakarchuk et al., 1998
), or the Pk epitope mimic (Persson et al., 2001
), but the enzymes that add residues directly onto the inner core heptose residues have not been characterized to date. The studies of one of the enzymes involved (LgtK), which adds the
-1,2-linked GlcNAc to heptose (II) (Hep II) (Figure 1) has so far been limited to genetic approaches in N. meningitidis in which the gene has been inactivated by insertional mutagenesis (Kahler et al., 1996
). Although the genetic studies have suggested a function for this gene, no characterization of the enzyme's biochemical properties has been reported.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To evaluate the enzyme activity, we were only able to partially purify the protein using a partitioning strategy. Because the enzyme is found associated with the membrane fraction, we used this initially to enrich the enzyme. For further characterization of the enzyme we used the MalE-fusion version of the protein, which could be purified as a soluble enzyme to near homogeneity in a single step on an amylose affinity column (data not shown). We recovered 33% of enzyme activity loaded on the amylose column, and this was stable in 20% glycerol for up to 1 week.
Enzyme activity
To investigate enzyme activity, we initially used a core oligosaccharide derived from the truncated LPS of the lgtK knockout mutant as an acceptor, as there had been suggestions in the literature that the PEtn at the 3 position of Hep II might be required for transfer of the -GlcNAc residue (Rahman et al., 2001
). We did not use the core oligosaccharide derived from the N. gonorrhoeae knockout, however, because it contained two PEtn residues on Hep II, which we felt might not reflect the true biosynthetic intermediate. For this reason we used the core oligosaccharide from the corresponding N. meningitidis immunotype L3 lgtK mutant, which had only one PEtn, at the 3 position of Hep II (van der Ley et al., 1997
). This core oligosaccharide would be a trisaccharide similar to the structure shown in Figure 1, with the addition of phosphoethanolamine on Hep II. The enzyme activity was analyzed by examining the reaction mixtures with CE-MS, an example of this being in Figure 3. Because MS data is difficult to quantitate, the yield of product could only be estimated as around 1% or 2%. This analysis does show that only a trace of product could be detected indicating the enzyme is only very weakly active on this oligosaccharide. This result suggests the core oligosaccharide is a poor acceptor for this enzyme. In the product ion scan shown in Figure 3, the two peaks around 3 min are due to some positively charged impurities in the sample, because they were ahead of electroendoosmotic flow (
5 min). The peaks at 9 and 10 min were artifact spikes, due to the fluctuation of electrospray instrument at this high sensitivity setting.
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The biochemical characterization of the inner core LPS biosynthetic enzymes has not been extensive. The current literature has reports of work on the heptosyltransferase I from E. coli (Kadrmas and Raetz, 1998) and the KDO-transferases from Chlamydia (Lobau et al., 1995
). The enzymology of these proteins would be facilitated if suitable soluble synthetic acceptors could be used. These previous reports have not included attempts to characterize the enzyme using synthetic acceptors and have used radiolabeled LPS precursors as acceptors. Our initial attempts to assay the LgtK enzyme were performed with core oligosaccharides (no lipid A) derived from the lgtK mutant strain of N. meningitidis L3, which contains a phosphoethanolamine residue at O3 of Hep II. Reports in the literature suggested that the presence of the phosphoethanolamine group on the 3-position of Hep II would be required for enzyme activity (Rahman et al., 2001
).
Our approach was to use CE-MS analysis to determine if the enzyme reactions were producing any product. Despite repeated attempts to optimize the reaction using the core oligosaccharide, we were unable to obtain more than 1% or 2% of the product in these reactions. In previous work we have been able to see other enzymes adding sugars to core oligosaccharides using this approach (Thibault et al., 2003). We interpreted this to mean that the core oligosaccharide was in fact a poor acceptor for this enzyme. It is also possible that the lipid A portion of the molecule may be required for this enzyme to perform its reaction; however, it was impossible to do assays with the lipid Acontaining material because it forms high-molecular-weight aggregates. When we employed the synthetic acceptor FEX-
-Man or FEX-
-Hep, we were able to obtain quantitative conversion to product on the milligram scale with enzyme derived from either N. meningitidis or N. gonorrhoeae and to verify that the product being formed was indeed an
-1,2-linked GlcNAc residue. Our data suggests that the 3-linked phosphoethanolamine on Hep II could go on after the
-1,2-linked GlcNAc residue, and that the presence of the 3- and 6-linked phosphoethanolamine on Hep II in the lgtK mutant might simply reflect the low specificity of the phosphoethanolamine transferase with the LPS generated in the mutant strain. A direct test of this hypothesis could be to examine LgtK reactions on a synthetic acceptor with phosphoethanolamine at O3. The synthesis of such an acceptor, however, is not trivial, and to date we have not attempted to make such an acceptor.
We also noticed that the LgtK enzyme from N. meningitidis was less active toward the synthetic acceptor than the enzyme from N. gonorrhoeae. The explanation for the differential activity is not simple, although there is a short sequence difference from aa 243253 in LgtK-Ng, which may be responsible, but we have not verified this experimentally.
We investigated the expression of the LgtK enzyme in E. coli and found that it could be overexpressed and enriched as an active enzyme when assayed with synthetic acceptors. Attempts to further purify the protein using conventional ion-exchange chromatography were unsuccessful because the enzyme rapidly lost activity (data not shown). We were able to express the enzyme as a fusion with the MalE protein from E. coli. A comparison of the native enzyme and MalE fusion showed that making the fusion protein resulted in only a slight loss of activity and that the purified MalE-LgtK fusion protein also lost activity (data not shown), but it was stable enough to use to collect data with the FE labeled acceptors. We were able to show that the enzyme was specific for UDP-GlcNAc, but our synthetic acceptors, though useful for measuring enzyme activity, were not suitable for collecting kinetic parameters.
We previously developed many synthetic acceptors to examine outer core enzymes from Neisseria and other bacterial pathogens (Gilbert et al. 1997, 2000
; Wakarchuk and Cunningham, 2003
) and have used this approach to start a study of a key control point enzyme, LgtK from N. gonorrhoeae. So far the analysis has been performed with only monosaccharide acceptors with a variety of aglycones. As we saw with other bacterial glycosyltransferases, acceptors with the larger aglycone FEX performed better in the assays (four- to sevenfold higher specific activity). A comparison of the acceptors FEX-
-Man and FEX-
-Hep showed the enzyme did not discriminate much between these two synthetic acceptors. We did notice that the
-Man acceptor was better than the
-Hep. This is somewhat surprising, but perhaps it reflects that the accessible part of Hep II is limited to the axial hydroxyl at C2 and that the C7 position of Hep is not a critical contact point for this enzyme. The preliminary kinetic data also suggests that these synthetic acceptors are not optimalwe were unable to obtain meaningful kinetic data because of very strong acceptor inhibition at higher concentrations of FEX-
-Man/Hep. The reason we observed substrate inhibition with the FEX-aminophenyl-
-Man and Hep acceptors is not known, but it is likely the large planar FEX aglycone may have a negative effect on the enzyme activity of LgtK at the relatively high acceptor concentrations we used. Attempts to use the smaller acceptor p-nitrophenyl-
-Man for kinetic assays were also unsuccessful, as we could not see the apparent acceptor saturation of the enzyme up to the limit of solubility of the acceptor (>15 mM). These data also clearly indicate that the nature of the aglycone is important for this enzyme and that size and shape play a role in how the acceptor is recognized. This remains one of the most important considerations when characterizing glycosyltransferases from many sources.
We were able to measure an apparent Km for UDP-GlcNAc and showed that this is the only UDP-linked sugar that the enzyme would use as a donor. Because we were unable to determine other kinetic parameters with these acceptor substrates, we have avoided over interpretation of this data. The data we did collect shows the Km(app) values would be in the mM range, which is similar to what has been seen with many other synthetic acceptors used with many other glycosyltransferases. It may be the aglycone that contributes to the substrate inhibition, but this will need to be tested by synthesis of several analogs where the aglycones have different properties to FEX. We have attempted to use other LPS-derived molecules to measure the enzyme activity, but molecules containing the lipid A portion are poorly soluble in water and tend to aggregate, making these poor acceptors. We have also invested some effort in using the oligosaccharide derived from the LPS by mild acid hydrolysis, but obtaining large amounts of this material with and without the phosphoethanolamine is not practical at present.
In conclusion, we have shown that the LgtK protein from N. gonorrhoeae and N. meningitidis has -1,2-N-acetyl- glucosaminyltransferase activity on synthetic acceptors, which is the biochemical demonstration of an activity that has been previously only been inferred by structural analysis of knockout mutants of the bacterium. We have also shown that the enzyme activity does not depend on having a 3-substitued D-manno configured acceptor. What is not known is if the presence of the 3-substitution enhances activity, although our data suggest the 3-substituted Hep II was not a good acceptor for this enzyme. Clearly other synthetic acceptors are required to provide definitive data on the role of the phosphoethanolamine substitution in the activity of LgtK to the detailed examination of the acceptor specificity of LgtK. We are continuing to characterize this important enzyme to obtain further structurefunction data.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of lgtK mutants of N. meningitidis and N. gonorrhoeae
Knockout strains in which the lgtK gene had been inactivated were constructed essentially as was previously described by (Kahler et al., 1996; van der Ley et al., 1997
). The strains used were N. meningitidis MC58 and N. gonorrhoeae FA1090. Correct single insertions of the antibiotic resistance cassette were detected by polymerase chain reaction and Southern blot analysis (data not shown).
Bacterial growth and generation of LOS, core oligosaccharide, and O-deacylated LPS
N. gonorrhoeae lgtK::kanR was grown with shaking overnight at 37°C in Columbia broth supplemented with 30 µg/ml kanamycin, and biomass was then harvested by centrifugation. LOSs were isolated by the hot waterphenol extraction of bacterial cells (Westphal and Jann, 1965) as a gel-like pellet on ultracentrifugation of the aqueous phase. The LOS pellet was lyophilized and then purified on a column of Bio-Gel P-2 (1 cm x 100 cm) with water as eluent. Some of the LOS preparation was then treated with 1% acetic acid at 100°C for 1 h with subsequent removal of the insoluble lipid A by centrifugation (5,000 x g) to yield the core oligosaccharide, which was further purified by chromatography on a column of Bio-Gel P-2 with water as eluent. LOS was O-deacylated with anhydrous hydrazine under mild conditions as described previously (Holst et al., 1991
). Briefly, 15 mg was treated with 1 ml anhydrous hydrazine for 1 h at 37°C. The sample was then cooled to 0°C, and the excess hydrazine was destroyed with the addition of 5 ml cold acetone. The precipitate was centrifuged at 5,000 x g for 15 min and then washed with acetone (3 x 2 ml) and then acetone:water (4:1 x 3 ml) and lyophilized.
Synthesis of synthetic acceptors FEX--Man and FEX-
-LD-Heptose
Aminophenyl-glycosides were made from the corresponding nitrophenyl-glycosides by catalytic hydrogenation (Hudlicky, 1984). These were then labeled with FEX and purified as previously described for FCHASE-aminophenylglycosides (Gilbert et al., 1997
). Synthesis of the
-L,D-heptose compounds was performed partly as described in the literature (Garegg et al., 1992
) and as will be described. The numbering of the compound structures is explained in Figure 6. Compound 1 was commercially available (Sigma-Aldrich, St. Louis, MO).
|
p-Nitrophenyl 2,3,4,6,7-Penta-O-acetyl-L-glycero--D-mannoheptopyranoside
Trichloroacetimidate (4, 70 mg, 0.124 mmole) was dissolved in dry methylene chloride (8 ml) under an argon atmosphere and p-nitrophenol (35 mg, 0.248 mmole) was added followed by 4A molecular sieves (150 mg). The mixture was stirred for 1 h at room temperature and then chilled in an icecalcium chloride bath before triethylsilyl trifluoromethane sulfonate (40 µl, 0.173 mmole) was added. After 1.5 h, TLC showed disappearance of trichloroacetimidate, and the reaction was quenched with triethylamine (0.4 ml). The reaction mixture was diluted with methylene chloride and then absorbed on a small amount of silica gel. This silica gel was placed on a small silica gel column and eluted using 35:65 ethyl acetate: hexanes to yield (5, 58 mg; 86%). []D + 83.9° (c 0.13 chloroform); 1H NMR (CDCl3):
8.22 (d, 2H, Ph), 7.15 (d, 2H, Ph), 5.67 (d, 1H, J1,2 2.8 Hz, H-1), 5.51 (dd, 1H, J2,3 3.7 and J3,410.3 Hz, H-3), 5.46 (m, 1H, H-2), 5.42 (brt, 1H, J 10.1 Hz, H-4), 5.21 (dt, 1H, J5,6 2.0 and J6,7 6.8 Hz, H-6), 4.08 (dd, 1H, J5,6 2.0 and J4,5 10.0 Hz, H-5), 4.054.01 (m, 2H, H-7 and H-7'), 2.22, 2.21, 2.02, 2.01, 1.67 (5x s, 3H, OAc). FAB-MS (positive-ion): analytical calculated for C23H27NO14Na: 564.4. Found: m/z 564.4.
p-Nitrophenyl L-glycero-D-mannoheptopyranoside
Peracetate (5, 50 mg, 0.092 mmole) was dissolved in dry methanol (10 ml) and 1 M sodium methoxide solution (0.5 ml) was added. The reaction was complete in 2 h at room temperature. The mixture was neutralized using 101 Rexyn (H+) resin, and after filtration the solvent was evaporated. The product was purified by trituration overnight in diethyl ether. The resulting yellow solid was filtered off and rinsed with more diethyl ether and dried to yield (6, 23 mg; 75%). []D +134.8° (c 0.21 methanol); 1H NMR (CDCl3):
8.25 (d, 2H, Ph), 7.28 (d, 2H, Ph), 5.70 (s, 1H, H-1), 4.05 (m, 1H, H-2), 3.99 (t, 1H, J 9.5 Hz, H-4), 3.963.89 (m, 2H, H-6 and H-3), 3.573.50 (m, 2H, H-5 and H-7), 3.313.28 (m, 1H, H-7'); 13C NMR (CDCl3):
100.04 (C-1), 74.50 (C-5), 72.64 (C-3), 71.68 (C-2), 70.72 (C-6), 67.51 (C-4), 64.80 (C-7); FAB-MS (positive-ion): analytical calculated for C13H17NO9: 331.27. Found: m/z 331.37.
Sugar linkage analysis by methylation
Methylation analysis was performed with the NaOH/DMSO/CH3I procedure (Ciucanu and Kerek, 1984) on the product of the LgtK enzyme reaction that had been performed with the synthetic acceptor FEX-
-Man. The hydrolysis was performed in 4 M trifluoroacetic acid for 4 h at 100°C followed by reduction with NaBD4 in H2O overnight, then acetylated with acetic anhydride at 100°C for 2 h using residual sodium acetate as catalyst. The methyl ether derivatives were characterized by gas-liquid chromatography MS using a Hewlett-Packard chromatograph equipped with a 30 M DB-17 capillary column (180°C to 230°C at 2.5°C/min). MS was done in the electron impact mode and recorded on a Varian Saturn II mass spectrometer.
NMR
NMR experiments were acquired on a Varian Inova 500 MHz spectrometer using a 5 mm triple resonance probe with the 1H coil nearest to the sample and with a Z gradient coil. The measurements were performed on the TLC-purified enzyme reaction product starting from the synthetic acceptor FEX--Man. All measurements were made at 25°C, with the monodeuterated water resonance at 4.78 ppm. The previously lyophilized sample (2 mg) was dissolved in 0.6 ml D2O. All NMR experiments were performed as previously described (Gilbert et al., 2000
)
CE-MS and CE-MS/MS
A crystal Model 310 CE instrument (AYI Unicam, Boston, MA) was coupled to an API 3000 mass spectrometer (Perkin-Elmer/Sciex, Concord, Canada) via a micro-Ionspray interface. A sheath solution (isopropanol-methanol, 2:1) was delivered at a flow rate of 1 µl/min to a low dead volume tee (250 µm ID, Chromatographic Specialties, Brockville, Canada). All aqueous solutions were filtered through a 0.45-µm filter (Millipore, Bedford, MA) before use. An electrospray stainless steel needle (27-gauge) was butted against the low dead volume tee and enabled the delivery of the sheath solution to the end of the capillary column. The separations were obtained on 90-cm length bare fused-silica capillary using 10 mM ammonium acetate/ammonium hydroxide in deionized water, pH 9.0, containing 5% methanol. A voltage of 20 kV was typically applied at the injection. The outlet of the capillary was tapered to
15 µm ID using a laser puller (Sutter Instruments, Novato, CA). Mass spectra were acquired with dwell times of 3.0 ms per step of 1 m/z unit in full-mass scan mode. The MS/MS data were acquired with dwell times of 1.0 ms per step of 1 m/z unit. Fragment ions formed by collision activation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell were mass-analyzed by scanning the third quadrupole.
Production of the enzyme
Cultures of pCW::LgtK(and the MalE version) were grown first overnight at 30°C in 2YT supplemented with 150 µg/ml ampicillin. Fresh cultures (200 ml) were started with an inoculum of 0.5 A600, this culture was grown at 20°C until A600 = 0.5, at which point ispropylthiogalactoside was added to 0.5 mM. Growth was continued for 3648 h, and then the cells were harvested by centrifugation and frozen at 20°C. Extracts were made by resuspending the cells in cold 50 mM HEPES, pH 7.5, with 10% glycerol (with 0.1% Polyoxyethylene octyl ether (POE) for MalE-fusion protein workup) and then sonication (for less than 0.5 g cells) or disruption with nitrogen pressure in an Emulsiflex C5 (Avestin). Protease inhibitors were added as suggested by the manufacturer (Complete Protease Inhibitor Tablets, Roche). A clarified supernatant was made by centrifugation at 4°C at 15,00020,000 x g. The supernatant was decanted, and the pellet was resuspended pellet in half the volume of cold buffer. Polyethylene glycol precipitations were performed on 20,000 x g supernatants by adding NaCl and PEG8000 to a final concentrations of 4% PEG, 0.2 M NaCl. These mixtures were incubated at 4°C for 1 h with occasional mixing. The precipitate was collected by centrifugation at 15,00020,000 x g for 30 min. The supernatant was removed and the pellet resuspended with cold 50 mM HEPES, pH 7.5, 10% glycerol. Detergent solubilization was performed on the 4% PEG precipitate, by adding 1% POE followed by incubation at 4°C for 20 min. Soluble enzyme was recovered after centrifugation at 20,000 x g for 30 min. To remove POE, the 1% POE supernatant was dialyzed against 500 volumes of 20 mM HEPES, pH 7.4, 20% glycerol at 4°C for 16 h. Some improvement of recovery of enzyme activity could be observed with the addition of 5 mM dithiothreitol to the buffers.
The MalE-fusion protein was purified by chromatography of the clarified 20,000 x g supernatant on an amylose affinity column as suggested by the manufacturer (New England Biolabs). To stabilize the protein, glycerol (15% v/v) and 0.1% POE were added to the column buffers. Briefly from 3.6 g cells (500 ml culture) we obtained 10.3 U of activity. The specific activity of the purified fusion protein was 57 mU/mg using FCHASE--Mannoside, which was a 5.8-fold increase in specific activity.
Glycosyltransferase assays
Enzyme activity was quantitated using the CE assay we previously described (Gilbert et al., 1997). The acceptors used in these assays were FEX-
-Mannoside, FEX-
-L,D-Heptoside, p-nitrophenyl-
-mannoside, and p-nitrophenyl-
-L,D-heptoside. The reactions contained, 0.25 mM FEX containing acceptor, or 0.515 mM p-nitrophenyl-glycoside acceptor, 50 mM HEPES, pH 8.5, 10 mM MnCl2, 5 mM UDP-GlcNAc, and 5 µl extract in a 10 µl reaction. Reactions were incubated at 37°C for 560 min. Reactions were stopped with an equal volume of 50% acetonitrile, 1% sodium dodecyl sulfate, 10 mM ethylenediamine tetra-acetic acid, then were diluted with water prior to analysis by CE. A unit of enzyme activity is defined as the amount of enzyme that converts 1 µmol of acceptor to product per minute of the reaction. The data were analysed by fitting the data using the Michaelis-Menten equation using GraphPad Prism version 3.03 for Windows (GraphPad Software, San Diego, CA).
Preparative reactions with FEX--Man were performed with
1 mg of acceptor in a reaction volume of 1 ml (
1.2 mM). The progress of the reaction was monitored by CE, the reaction mixture was collected on a SepPak C18 cartridge after dilution to 10 ml with water, desalted with 10 ml of water, and then eluted with 70% acetonitrile. After removal of the solvent, the reaction mixture was separated on 0.5 mm TLC plates developed in ethyl acetate:methanol:water: acetic acid (7:2:1:0.1). The product had an Rf of 0.4 in this solvent. The product was scraped off the plate and eluted in warm water. This material was again desalted with a SepPak C18 cartridge and eluted in 70% acetonitrile. The dry product was used for NMR and methylation analysis as described.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
Abbreviations |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blixt, O., Van, I.D., Norberg, T., and van den Eijnden, D.H. (1999) High-level expression of the Neisseria meningitidis lgtA gene in Escherichia coli and characterization of the encoded N-acetylglucosaminyltransferase as a useful catalyst in the synthesis of GlcNAc beta 13Gal and GalNAc beta 1
3Gal linkages. Glycobiology, 9, 10611071.
Ciucanu, I., and Kerek, F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res., 131, 209217.[CrossRef][ISI]
Coutinho, P.M. and Henrissat, B. (1999) Carbohydrate-active enzymes server. Online database available at http://afmb.cnrs-mrs.fr/~cazy/CAZY/index.html.
Garegg, P.J., Oscarson, S., Ritzen, H., and Szonyi, M. (1992) Synthesis of 2-(4-trifluoroacetamidophenyl)ethyl O-(L-glycero-alpha-D-manno- hepto pyranosyl)-(1-7)-O-(L-glycero-alpha-D-manno- heptopyranosyl)-(1-3)- L-glycero-alpha-D-manno-heptopyranoside, corresponding to the heptose region of the Salmonella ra core structure. Carbohydr. Res., 228, 121128.[CrossRef][ISI][Medline]
Gilbert, M., Cunningham, A.M., Watson, D.C., Martin, A., Richards, J.C., and Wakarchuk, W.W. (1997) Characterization of a recombinant Neisseria meningitidis alpha-2,3-sialyltransferase and its acceptor specificity. Eur. J. Biochem., 249, 187194.[Abstract]
Gilbert, M., Brisson, J.R., Karwaski, M.F., Michniewicz, J., Cunningham, A.M., Wu, Y., Young, N.M., and Wakarchuk, W.W. (2000) Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)H and (13)C NMR analysis. J. Biol. Chem., 275, 38963906.
Holst, O., Brade, L., Kosma, P., and Brade, H. (1991) Structure, serological specificity, and synthesis of artificial glycoconjugates representing the genus-specific lipopolysaccharide epitope of Chlamydia spp. J. Bacteriol., 173, 18621866.[ISI][Medline]
Hudlicky, M. (1984) Reductions in organic chemistry. ACS monograph series, Ellis Horwod, Chichester, U.K.
Kadrmas, J.L. and Raetz, C.R. (1998) Enzymatic synthesis of lipopolysaccharide in Escherichia coli. Purification and properties of heptosyltransferase I. J. Biol. Chem., 273, 27992807.
Kahler, C.M., Carlson, R.W., Rahman, M.M., Martin, L.E., and Stephens, D.S. (1996) Two glycosyltransferase genes, lgtF and rfaK, constitute the lipooligosaccharide ice (inner core extension) biosynthesis operon of Neisseria meningitidis. J. Bacteriol., 178, 66776684.[Abstract]
Lobau, S., Mamat, U., Brabetz, W., and Brade, H. (1995) Molecular cloning, sequence analysis, and functional characterization of the lipopolysaccharide biosynthetic gene kdtA encoding 3-deoxy-alpha-D-manno-octulosonic acid transferase of Chlamydia pneumoniae strain TW-183. Mol. Microbiol., 18, 391399.[CrossRef][ISI][Medline]
Mandrell, R.E. and Apicella, M.A. (1993) Lipo-oligosaccharides (LOS) of mucosal pathogens: molecular mimicry and host-modification of LOS. Immunobiology, 187, 382402.[ISI][Medline]
Noah, C., Brabetz, W., Gronow, S., and Brade, H. (2001) Cloning, sequencing, and functional analysis of three glycosyltransferases involved in the biosynthesis of the inner core region of Klebsiella pneumoniae lipopolysaccharide. J. Endotoxin Res., 7, 2533.[ISI][Medline]
Persson, K., Ly, H.D., Dieckelmann, M., Wakarchuk, W.W., Withers, S.G., and Strynadka, N.C. (2001) Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. Nat. Struct. Biol., 8, 166175.[CrossRef][ISI][Medline]
Raetz, C.R. and Whitfield, C. (2002) Lipopolysaccharide endotoxins. Annu. Rev. Biochem., 71, 635700.[CrossRef][ISI][Medline]
Rahman, M.M., Kahler, C.M., Stephens, D.S., and Carlson, R.W. (2001) The structure of the lipooligosaccharide (LOS) from the alpha-1,2-N-acetyl glucosamine transferase (rfaK(NMB)) mutant strain CMK1 of Neisseria meningitidis: implications for LOS inner core assembly and LOS-based vaccines. Glycobiology, 11, 703709.
Romanowska, E., Gamian, A., Lugowski, C., Romanowska, A., Dabrowski, J., Hauck, M., Opferkuch, H.J., and der Lieth, C.W. (1988) Structure elucidation of the core regions from Citrobacter O4 and O36 lipopolysaccharides by chemical and enzymatic methods, gas chromatography/mass spectrometry, and NMR spectroscopy at 500 MHz. Biochemistry, 27, 41534161.[ISI][Medline]
Thibault, P., Martin, A., Gilbert, M., Wakarchuk, W., and Richards, J.C. (2003) Analysis of bacterial glycolipids by capillary electrophoresis-electrospray mass spectrometry. Methods Mol. Biol., 213, 241259.[Medline]
Unkmeir, A., Kammerer, U., Stade, A., Hubner, C., Haller, S., Kolb-Maurer, A., Frosch, M., and Dietrich, G. (2002) Lipooligosaccharide and polysaccharide capsule: virulence factors of Neisseria meningitidis that determine meningococcal interaction with human dendritic cells. Infect. Immun., 70, 24542462.
van der Ley P., Kramer, M., Martin, A., Richards, J.C., and Poolman, J.T. (1997) Analysis of the icsBA locus required for biosynthesis of the inner core region from Neisseria meningitidis lipopolysaccharide. FEMS Microbiol. Lett., 146, 247253.[CrossRef][ISI][Medline]
Wakarchuk, W.W. and Cunningham, A.M. (2003) Capillary electrophoresis as an assay method for monitoring glycosyltransferase activity. Methods Mol. Biol., 213, 263274.[Medline]
Wakarchuk, W.W., Campbell, R.L., Sung, W.L., Davoodi, J., and Yaguchi, M. (1994) Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase. Protein. Sci., 3, 467475.
Wakarchuk, W., Martin, A., Jennings, M.P., Moxon, E.R., and Richards, J.C. (1996) Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis. J. Biol. Chem., 271, 1916619173.
Wakarchuk, W.W., Cunningham, A., Watson, D.C., and Young, N.M. (1998) Role of paired basic residues in the expression of active recombinant galactosyltransferases from the bacterial pathogen Neisseria meningitidis. Protein Eng., 11, 295302.[CrossRef][ISI][Medline]
Westphal, O. and Jann, K (1965) Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem., 5, 8391.