Characterization of mycobacterial protein glycosyltransferase activity using synthetic peptide acceptors in a cell-free assay

Howard N. Cooper1,3, Sudagar S. Gurcha1,4, Jérôme Nigou4, Patrick J. Brennan5, John T. Belisle5, Gurdyal S. Besra4 and Douglas Young2,3

3 Centre for Molecular Microbiology and Infection, Imperial College of Science, Technology and Medicine, South Kensington, London, SW7 2AZ, England; 4 Department of Microbiology and Immunology, University of Newcastle upon Tyne, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, England; 5 Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523-1677, USA

Received on January 24, 2002; revised on March 6, 2002; accepted on March 15, 2002


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
Synthetic peptides derived from a 45-kDa glycoprotein antigen of Mycobacterium tuberculosis were shown to function as glycosyltransferase acceptors for mannose residues in a mannosyltransferase cell-free assay. The mannosyltransferase activity was localized within both isolated membranes and a P60 cell wall fraction prepared from the rapidly growing mycobacterial strain, Mycobacterium smegmatis. Incorporation of radiolabel from GDP-[14C]mannose was inhibited by the addition of amphomycin, indicating that the glycosyl donor for the peptide acceptors was a member of the mycobacterial polyprenol-P-mannose (PPM) family of activated glycosyl donors. Furthermore, a direct demonstration of transfer from the in situ generated PP[14C]Ms was also demonstrated. It was also found that the enzyme activity was sensitive to changes in overall peptide length and amino acid composition. Because glycoproteins are present on the mycobacterial cell surface and are available for interaction with host cells during infection, protein glycosyltransferases may provide novel drug targets. The development of a cell-free mannosyltransferase assay will now facilitate the cloning and biochemical characterisation of the relevant enzymes from M. tuberculosis.

Key words: mannosyltransferase/peptide acceptors/tuberculosis


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
Protein glycosylation was once considered a characteristic feature that distinguished eukaryotic from prokaryotic organisms. However, the presence of glycoproteins in bacteria was definitively demonstrated in the case of the surface layer proteins of archaebacteria (Messner and Sleytr, 1991Go), and there is now extensive evidence for their occurrence in eubacteria. In particular, proteins associated with cell surfaces or secreted into the culture medium of a range of Gram-negative and Gram-positive bacteria have been shown to be modified by covalent attachment of glycan moieties (Dobos et al., 1996Go; Erickson and Herzberg, 1993Go; Virji, 1997Go). Of the examples subjected to detailed structural analysis, the glycosylation processes most commonly involve O-linked attachment of short oligosaccharide chains resembling those found in lower eukaryotes. Several glycoproteins have been identified in pathogenic bacteria, though the precise role that glycosylation plays in the interaction with eukaryotic cell structures during infection still remains elusive (Lindenthal and Elsinghorst, 1999Go; Nassif et al., 1999Go).

In part, the initial reluctance to accept the existence of bacterial glycoproteins was due to differences in their intracellular organisation. In eukaryotic cells, proteins are modified by glycosylation within the Golgi apparatus, a structure that is absent from prokaryotes. Although structural evidence confirms the existence of bacterial glycoproteins, considerable uncertainty remains as to the mechanism and subcellular location of glycosylation events. Mutagenesis studies have identified a gene involved in glycosylation of pili in Neisseria meningitides (Jennings et al., 1998Go), and related genes have been found in several other bacteria. Mutations affecting the N. meningitis gene (PglA) have no effect on lipopolysaccharide structure in these bacteria, suggesting a specific role in protein glycosylation, although genetic studies of a related gene in Campylobacter jejuni indicated a more general role in glycosylation of both proteins and lipopolysaccharide (Szymanski et al., 1999Go). Genetic analysis of phage resistant mutants recently identified a gene encoding a putative protein glycosyl transferase in Streptomyes coelicolor (Cowlishaw and Smith, 2001Go).

The present study develops a complementary biochemical approach to the analysis of bacterial protein glycosylation and, in particular, protein glycosylation in mycobacteria. The mycobacterial genus includes relatively rapid-growing nonpathogenic soil organisms, such as Mycobacterium smegmatis, as well as slow-growing pathogenic species responsible for tuberculosis (M. tuberculosis) and leprosy (M. leprae). There is growing literature evidence of glycosylation of several M. tuberculosis proteins (Dobos et al., 1996Go; Fifis et al., 1991Go; Garbe et al., 1993Go). Detailed structural analyses were performed on a 45-kDa secreted antigen (Dobos et al., 1996Go). The antigen was found to be glycosylated at four threonine residues with short mannose oligomers (Dobos et al., 1996Go). The occurrence of other putative mycobacterial glycoproteins is based on lectin-binding assays. Analysis of the 19-kDa antigen and MPT83 of M. tuberculosis suggests a similar level of threonine-linked glycosylation (Hewinson et al., 1996Go; Herrmann et al., 1996Go). In this present communication we utilized the growing body of structural information in relation to the 45-kDa antigen of M. tuberculosis to develop a mycobacterial protein glycosylation assay.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
Development of a cell-free system for examining mannosyltransferase activity in vitro
Glycosyltransferase activities involved in the assembly of cell wall polysaccharides have been demonstrated in mycobacterial subcellular fractions incubated with appropriate substrates and radiolabelled precursors (Besra et al., 1997Go; Lee et al., 1997Go). In this article, we have examined similar preparations for protein glycosyltransferase activity. Briefly, M. smegmatis cells were broken by ultrasonication and three enzymatic extracts prepared. First, a membrane and cytosolic fraction prepared by differential high-speed centrifugation. Second, a particulate cell wall fraction prepared by Percoll density gradient centrifugation (P60 fraction). The substrate for the subsequent glycosylation reaction was a 16-residue synthetic N-biotinylated peptide corresponding to a glycosylated region of the M. tuberculosis 45-kDa antigen (peptide A3, Table I) (Dobos et al., 1996Go) and GDP-[14C]mannose.


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Table I. Synthetic peptides as acceptors in the mannosyltransferase assay
 
The peptide A3 was initially incubated with membranes together with GDP-[14C]mannose, ATP, and MgCl2. Following incubation the reaction mixture was partitioned between chloroform-methanol-water with the resulting aqueous layer dried, resuspended in water, and fractionated by Bio-Gel P-2 gel filtration chromatography. The distribution of radioactivity in column fractions during a representative experiment with and without peptide A3 is shown in Figure 1. In the absence of the peptide A3 acceptor, a radiolabeled product eluted as a broad peak, which was retained in the Bio-Gel P-2 gel filtration column. This product was later confirmed to be unused GDP-[14C]mannose in a series of control experiments performed in the absence of enzyme. It is also interesting to note that in the absence of the peptide A3 acceptor (Figure 1), the chloroform-methanol-water partition was very efficient in removing chloroform-methanol soluble C35/C50-polyprenol-P-[14C]mannose (PPM) and phosphatidylinositol [14C]mannosides (PIMs) and linear-lipomannan (LM), which are found in the insoluble residue at the interface of the partition mixture, away from the aqueous-methanol fraction. If cross-contamination had occurred, these lipid-linked [14C]labeled mannose products would have appeared near the void volume (approximately fractions 19–23) of the Bio-Gel P-2 gel filtration column.



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Fig. 1. Mannosylation of peptide A3. The distribution of radiolabel was monitored by Bio-Gel P-2 gel filtration of the assay mixture after a 1-h incubation period with GDP-[14C]mannose and work-up as described in Materials and methods. In control samples incubated in the absence of peptide A3 acceptor (broken line), a broad peak of radioactivity was observed between fractions 38 and 50 corresponding to unreacted GDP-[14C]mannose. In the presence of peptide A3 acceptor (solid line) an additional radioactive peak was observed near the void volume of the Bio-Gel P-2 gel filtration column (fractions 20–24).

 
In the presence of peptide, an additional sharp peak of radioactivity eluted near the void volume of the Bio-Gel P-2 gel filtration column (approximately fractions 20–24). Residual radioactivity in the GDP-mannose fractions was the same in the presence or absence of peptide acceptor, indicating that radiolabel incorporated into excluded peak is derived from the pool of lipid intermediates removed in chloroform-methanol-water extraction. The synthesis of the product was completely lost when the mycobacterial membrane preparations were heat-inactivated and significantly reduced (>80%) through the inclusion of ethylenediamine tetra-acetic acid, pointing to the need of divalent cations for these particular mannosyltransferases. The absence of labeling in control experiments without peptide A3 provides preliminary evidence of peptide mannosyltransferase activity within membrane subcellular fractions. The localization of the mannosyltransferase activity was further investigated by comparing membrane, cytosol, and P60 enzymatic extracts using the peptide A3 acceptor. It was clear that the mannosylation of the peptide A3 acceptor occurred with both membrane and P60 fractions, whereas cytosolic preparations were completely inactive (Figure 2). The subcelluar localization is consistent with recent studies of mannosyltransferases from mycobacteria (Besra et al., 1997Go).



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Fig. 2. Subcelluar localization of peptide A3 mannosyltransferase activity. The mannosylation of the peptide A3 acceptor was examined using membrane, cytosol, and P60 enzymatic preparations. The distribution of radiolabel with each preparation was subsequently monitored by Bio-Gel P-2 gel filtration of the crude assay mixture as described in Materials and methods, and the activity is presented as total counts recovered within the radioactive peak observed near the void volume of the Bio-Gel P-2 gel filtration column (see Figure 1).

 
Mannosylation of peptide A3 acceptor is GDP-Man- or PPM-dependent?
The mentioned studies have assumed that GDP-mannose was the immediate donor of mannose for the mannosylation of the peptide A3 acceptor. However, Takayama et al. (1973)Go and Schultz and Elbein (1974)Go described two alkali-stable mannophospholipids from M. smegmatis, a mannosyl-1-phosphoryl-decaprenol (C50-PPM) and a mannosyl-1-phosphoryl-heptaprenol (C35-PPM). Considering their role in cell wall biosynthesis from other organisms (Bugg and Brandish, 1994Go) and dolichol-P-mannose-dependent mannosyltransferase activity in mammalian glycoprotein synthesis (Colussi et al., 1997Go; Kruszewska et al., 2000Go; Strahl-Bolsinger et al., 1999Go) we investigated the identity of the direct donor for the mannosylation of peptide A3 acceptor, that is, GDP-mannose or PPM, through two independent lines of experimentation.

Firstly through the use of the lipopeptide antibiotic amphomycin, which specifically disrupts the action of a variety of translocase enzymes by chelating polyprenol monophosphates in the presence of Ca2+ ions, thus inhibiting the transfer of a range of monomeric units to polyprenol phosphate carriers (Banerjee et al., 1981Go; Besra et al., 1997Go). The second line of investigation was based on the use of a novel assay system that generates in situ labeled C35/C50 PP[14C]M within mycobacterial membranes (Besra et al., 1997Go).

It has recently been shown that amphomycin specifically inhibits the incorporation of [14C]mannose from GDP-[14C]mannose into C35/C50-PPM, and the subsequent transfer of [14C]mannose into linear-LM from these mannophospholipids, using membranes prepared from M. smegmatis (Besra et al., 1997Go). Preincubation of M. smegmatis membranes with amophomycin prior to the addition of GDP-[14C]mannose had a profound effect on the synthesis of C35/C50-PPM (data not shown) and (more important) on the synthesis of the mannosylated peptide A3 acceptor (Figure 3). Inhibition of GDP-[14C]mannose incorporation into lipid-linked intermediates in the presence of amphomycin is evident from the increased radioactivity eluting in the GDP-mannose fractions from the P2 column (Figure 3).



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Fig. 3. Inhibition of peptide mannosyltransferase activity by amphomycin. Membranes were pretreated with amphomycin as described by Besra et al. (1997)Go, and the peptide mannosyltransferase assays were performed using the peptide A3 acceptor. Initially the effect of pretreatment of mycobacterial membranes with amphomycin was assessed for inhibition of C35/C50-PP[14C]M synthesis (Besra et al., 1997Go). The incorporation of label into the peptide A3 acceptor product (fractions 22–28) in the absence (solid line) and presence (broken line) of amphomycin were analysed using Bio-Gel P-2 gel filtration chromatography of the reaction products.

 
Besra et al. (1997)Go previously developed a novel assay whereby membranes were pulsed with GDP-[14C]mannose for a 10-min incubation period. The resulting [14C]mannose-labeled membranes were reharvested by centrifugation at 100,000 x g, washed several times by resuspending in 4-morpholine propane sulfonic acid (MOPS) buffer, and reharvested following centrifugation. Analysis of the [14C]mannose-labeled membranes following extraction using CHCl3/CH3OH by thin-layer chromatography (TLC)–autoradiography demonstrated that greater than 90% of the [14C]mannose incorporation from GDP-[14C]mannose was within C35-PP[14C]M- and C50-PP[14C]M-based lipids, with the remainder associated with PI[14C]Ms (data not shown). On further incubation of the [14C]mannose-labeled membranes in the absence and presence of the peptide A3 acceptor, the results were striking (Figure 4). In the presence of peptide, the additional sharp peak of radioactivity, which eluted near the void volume of the P-2 gel filtration column as observed in previous experiments, was clearly present (see Figure 4). Interestingly, a notable feature, a consequence of extensive washing of the in situ generated membranes is apparent from the control experiments in the absence of the peptide A3 acceptor; GDP-[14C]mannose is almost completely absent (fractions 38–50). Again, the efficiency of the chloroform-methanol-water partition is clearly apparent through the removal of unused C35/C50-PP[14C]M. The absence of labeling in control experiments, amphomycin inhibition, and labeling with in situ generated [14C]mannose-labeled membranes (i.e., C35/C50-PP[14C]M), provides evidence that mannosylation of the peptide A3 acceptor occurs directly from PPM.



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Fig. 4. Bio-Gel P-2 gel filtration analysis of reaction products generated in the peptide mannosyltransferase assay using peptide A3 acceptor and in situ labeled C35/C50-PP[14C]M. Exact conditions for in situ labeling of membranous C35/C50-PP[14C]M, and subsequent use in the peptide mannosylation assay are described in Materials and methods.

 
Analysis of glycosylated peptide A3
The [14C]mannose-labeled peptide A3 product from the mannosyltransferase assay was hydrolysed using trifluoroacetic acid and TLC analysis showed the presence of mannose, in comparison with authentic sugar standards (data not shown) confirming that peptide A3 was indeed mannosylated. The [14C]mannose-labeled peptide A3 was also sensitive to jack bean {alpha}-mannosidase and resulted in quantitative removal of [14C]mannose residues from the [14C]mannose-labeled peptide A3 product (Figure 5A and B). In addition, in comparison to a standard mixture of NaB[3H]4 reduced mannose oligomers (Man-ol [M1], Man-Man-ol [M2], and Man-Man-Man-ol [M3]), reductive cleavage of the O-glycosidic linkage of the [14C]mannose-labeled peptide A3 product with NaBH4 afforded [14C]Man-ol (Figure 5C and D). The results suggest that peptide A3 is mannosylated by a single {alpha}-mannosyl residue, which is supported by the observation that a single but diffuse product is detected by TLC autoradiography and distinct from the unlabeled starting peptide A3 as revealed by charring of the TLC plates with ethanol/H2SO4 (Figure 6).Using alternative peptide acceptors (Table I), the [14C]mannose-labeled peptide A4 product also gave rise to a single product, whereas the [14C]mannose-labeled peptide A5 product when fractionated by TLC autoradiography gave rise to multiple products (Figure 6). Structural analyses have identified trimannose substitution in this region of the 45-kDa protein (Dobos et al., 1996Go), and the observation of multiple bands may represent peptides differing in their extent of glycosylation, although this has not been shown directly in this present study.



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Fig. 5. Degradative analysis of mannosylated peptide A3. The [14C]peptide products from the GDP-[14C]mannose-peptide:mannosyltransferase reaction were purified by Bio-Gel P-2 gel filtration chromatography and then analysed by Sephadex G-25 gel filtration chromatography following (A) no degradative treatment, (B) {alpha}-mannosidase treatment, and (C) reductive cleavage using NaBH4. (D) Standard mixture of NaB[3H]4 reduced mannose oligomers (Man-ol [M1], Man-Man-ol [M2], and Man-Man-Man-ol [M2]).

 


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Fig. 6. TLC-autoradiogram of [14C]peptide products from the peptide mannosyltransferase assay. The [14C]peptide products from the GDP-[14C]mannose-peptide:mannosyltransferase reaction were purified by Bio-Gel P-2 gel filtration chromatography and analyzed by autoradiography following TLC. The arrows indicate the position of the unmodified peptides. Peptides A3 and A4 gave rise to a single band, whereas peptide A5 gave rise to three bands.

 
Characteristic features of the peptide acceptor assay
The effect of variation in assay conditions was investigated initially using the peptide A3 acceptor. Incorporation of [14C] label in a linear fashion was observed over a 60-min period (Figure 7) and was dependent on protein concentration (see Figure 2). The concentration of the peptide acceptor was found to limit the extent of incorporation (Figure 8). Calculation of kinetic constants revealed that the peptide A3 acceptor possessed a Km value of 0.56 mM and Vmax value of 4.95 pmol/mg/min. Different peptides were then examined for their ability to substitute for peptide A3 in the peptide mannosyltransferase assay (see Table I). These included a second peptide from the N-terminal glycosylated region of the 45-kDa antigen (peptide A4), a peptide covering the C-terminal glycosylation site of the same protein (peptide A5) (Dobos et al., 1996Go), and a peptide corresponding to the threonine-rich region implicated in glycosylation of the 19-kDa antigen (peptide A2) (Herrmann et al., 1996Go).



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Fig. 7. Time course incorporation of [14C]label within the peptide mannosyltransferase assay. The peptide mannosyltransferase assay was performed using the peptide acceptor A3, and samples were assayed by Bio-Gel P-2 gel filtration chromatography at different time points for incorporation of [14C]mannose from GDP-[14C]mannose. The activity is presented as the total counts recovered within the radioactive peak observed near the void volume of the Bio-Gel P-2 gel filtration column (see Figure 1).

 


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Fig. 8. Effect of peptide concentration on mannosyltransferase activity. The peptide mannosyltransferase assay was performed using peptide acceptor A3, and samples were assayed by Bio-Gel P-2 gel filtration chromatography at different peptide A3 concentrations for incorporation of [14C]mannose from GDP-[14C]mannose. The activity is presented as the total counts recovered within the radioactive peak observed near the void volume of the Bio-Gel P-2 gel filtration column (see Figure 1).

 
Peptides A4 and A5 generated profiles similar to that observed with peptide A3; in contrast, no significant incorporation was seen using peptide A2. Results with the different peptides are summarized in Table II. The effect of peptide length was also investigated. Reducing the length of peptide A3 from a 16-mer to an 8-mer and inclusion in the assay at equimolar amounts resulted in a progressive decrease in labeling (Figure 9). Alteration of individual residues also influenced mannosylation. Substitution of alanine by methionine at position 13 of peptide A3 drastically reduced incorporation of label to just 15% of the incorporation observed in peptide A3 (data not shown).


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Table II. Activity of peptide acceptors in mannosyltransferase assay
 


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Fig. 9. Effect of peptide length on mannosyltransferase activity. Synthetic peptides of different lengths were prepared from the peptide A3 sequence (see Table I) and examined as peptide acceptors in the mannosyltransferase assay. The activity is presented as the total counts recovered within the radioactive peak observed near the void volume of the Bio-Gel P-2 gel filtration column (see Figure 1).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
Our results demonstrate the presence of mannosyltransferase activity in mycobacterial extracts that are able to catalyze the incorporation of radiolabeled mannose from GDP-[14C]mannose into peptide acceptors corresponding to known glycosylated sequences from the M. tuberculosis 45-kDa antigen. This provides a relatively simple biochemical assay for further characterization of glycoprotein biosynthesis in mycobacteria. Inhibition of the synthesis of the reaction products by amphomycin indicated that, as in the case of mannosyltransferases involved in mycobacterial LM and lipoarabinomannan biosynthesis, protein mannosylation proceeds via lipid-linked C35/C50-PPM intermediates. The peptide-based assay provides a useful tool for exploration of the sequence specificity of the mannosyltransferase activity. In accordance with structural data (Dobos et al., 1996Go), peptides derived from both the N-terminal and C-terminal glycosylated regions of the 45-kDa antigen were found to act as mannose acceptors. Both of these regions contain threonine-rich sequences similar to those found in eukaryotic O-linked glycoproteins, and both are positively predicted by a neural network algorithm trained on the eukaryotic glycoprotein database (Net-O-Glyc; Hansen et al., 1998Go). However, a similar threonine-rich peptide from the 19-kDa lipoprotein was negative in the mannosyltransferase assay. This observation could be explained if glycosylation of the 19-kDa lipoprotein is initiated from a sugar other than mannose; we are currently investigating this possibility by trying to identify the precise structure of the 19-kDa lipoprotein.

Alternatively, features associated with the conformation or solubility of the 19-kDa peptide may make it unsuitable as an acceptor within the format of the current assay. In contrast, to results with the 19-kDa lipoprotein, initial experiments based on the M. bovis MPB83 antigen do demonstrate mannosylation of the corresponding threonine-rich N-terminal region of this lipoprotein (Cooper and Michell, unpublished data). In the mannosyltransferase assay with the peptide A3 acceptor, substitution of a neighboring alanine residue in peptide A3 was found to block glycosylation. These results are consistent with site-directed mutants, which were used to investigate glycoprotein biosynthesis in intact mycobacteria, which illustrated that amino acids flanking the modified threonine residues have an important influence on glycosylation (Herrmann et al., 1996Go).

In summary, combining the peptide assay with structural analysis of glycosylated products will allow comparisons of glycosylation patterns in different mycobacterial species. If protein glycosylation has evolved to play a role in host cell interactions, it might be anticipated that comparison of extracts from M. smegmatis and M. tuberculosis will reveal differences in the specificity of the peptide acceptor sequences or in the extent and manner of glycosylation. Drugs that specifically target the process of mycobacterial protein glycosylation may provide a novel means of interfering with the pathogenesis of tuberculosis and would be complementary to current antimicrobial therapy strategies.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
General
M. smegmatis was grown with shaking at 37°C in a glycerol/alanine salts medium as described previously (Besra et al., 1997Go). Large-scale cultures of M. smegmatis were grown to mid-log phase (OD600 = 0.6–0.7), harvested, washed with phosphate buffered saline, and stored at –20°C until further use. Buffer A contained 50 mM MOPS, pH 7.9, 5 mM ß-mercaptoethanol, and 10 mM MgCl2. The majority of chemicals used were obtained from the Aldrich Chemical Company (Milwaukee, WI) and used without further purification. All fine chemicals were purchased from Sigma (St. Louis, MO) and Percoll was obtained from Pharmacia (Sweden). Synthetic peptides (Table I) were purchased from Akademish Ziekenhuis 2300 RC Leiden, The Netherlands. The peptides were purified by several ether precipitations and their purity checked by high-performance liquid chromatography analysis. Stock solutions were prepared by dissolving the peptides at 50 mg/ml in distilled water. The concentration of peptide used was based on dry weight. Amphomycin (a lipopeptide antibiotic that specifically inhibits polyprenyl-P-requiring translocases and the synthesis of PPM) was dissolved in 500 µl 0.1 N acetic acid, and the solution was adjusted to 0.05 M sodium acetate (pH 7.0) with 0.1 N NaOH to afford a final concentration of 2 mg/ml (Banerjee et al., 1981Go).

A [3H]mannooligosaccharide standard was generated from Saccharomyces cerevisiae mannan (Sigma). Briefly, 10 mg of S. cerevisiae mannan was subjected to acetoylsis by resuspending the sample in 1 ml of acetic anhydride/acetic acid/sulfuric acid (10:10:1, v/v/v). The reaction mixture was then incubated for 3 h at 40°C. The reaction mixture was cooled, and 1 ml of pyridine was added. The mixture was vortexed and dried under a stream of nitrogen. The acetolysis products were then deacetylated using methanol/20% ammonium solution (1:1, v/v) at 37°C for 18 h (Nigou et al., 2000Go). The reagents were then removed under a stream of nitrogen. The released oligomers of mannose (Man, M1; Man-Man, M2; and Man-Man-Man, M3) were tagged for radiometric detection using NaB[3H]4 reduction in 1 M NH4OH/ethanol (1:1, v/v) for 1 h at room temperature (Besra and Brennan, 1994Go). Samples were neutralized with acetic acid, dried, and repeatedly dried in methanol to remove borate salts (Besra and Brennan, 1994Go).

Preparation of enzymatic extracts from M. smegmatis
M. smegmatis cells (25 g wet weight) were washed with phosphate buffered saline and resuspended at 4°C in buffer A (50 mM MOPS, adjusted to pH 7.9 with KOH, 5 mM ß-mercaptoethanol, 10 mM MgCl2) supplemented with DNAse I (Type IV, Sigma) and RNAse (Microsomal Nuclease, Sigma). The mixture was then subjected to probe sonication (Soniprep 150; MSE, Crawley, Sussex, UK; 1 cm probe and 10 amplitude microns) on ice, for a total time of 10 min in 10 x 60 s cycles with 90 s cooling intervals between pulses. The resulting preparations were centrifuged initially at 27,000 x g for 20 min at 4°C to obtain the cell-wall fraction. The resulting cell wall pellet was resuspended in buffer A to a volume of 20 ml, and Percoll was added to produce a 60% (v/v) suspension. This mixture was centrifuged at 25,000 x g for 1 h at 4°C. The cell wall–containing fraction produced an upper band of diffuse particulate material that was washed three times in buffer A before being resuspended in buffer A to yield a final protein concentration of 10 mg/ml. This material is referred to as the P60 fraction. To prepare the membrane fraction and cytosol, the supernatant from the initial centrifugation step was subjected to ultracentrifugation at 100,000 x g for 1 h at 4°C. The resulting thin yellow-brown gelatinous pellet was resuspended in buffer A to yield a final protein concentration of 10 mg/ml. This is referred to as the membrane fraction and the clear supernatant as the cystosol. Protein concentrations were determined using the Micro-BCA protein assay kit (Pierce).

Conditions for generation of in situ labeled C35/C50-PP[14C]M
Enzymatically active membranes containing in situ labeled C35/C50-PP[14C]M were generated as described previously (Besra et al., 1997Go). Briefly, assays involved incubation of membranes (100 µl; 1 mg of protein) in buffer A with 0.5 µCi of GDP-[14C]mannose (Amersham Pharmacia Biotech, 303 mCi/mmol) and 1 mM ATP in a total volume of 160 µl. Assays were incubated for 10 min at 37°C and membranes recovered by centrifugation of combined multiple reactions (usually 20) at 100,000 x g for 1 h at 4°C. The supernatants from these centrifugations were removed, and the [14C]mannose labeled membranes were washed several times with cold buffer A to remove residual GDP-[14C]mannose. The [14C]mannose-labeled membranes were then carefully resuspended in buffer A to yield a final protein concentration of 10 mg/ml and 500,000 cpm/mg of protein. A 50-µl aliquot was combined with 4 ml of CHCl3/CH3OH/H2O (10:10:3, v/v/v) and incubated at room temperature for 30 min followed by the addition of 1.75 ml CHCl3 and 0.75 ml H2O. The lower organic layer of the biphasic mixture was washed three times with 2 ml CHCl3/CH3OH/H2O (3:47:48, v/v/v), dried under a stream of nitrogen, and resuspended in 200 µl CHCl3/CH2OH (2:1, v/v). The transfer of [14C]mannose from GDP-[14C]mannose to C35/C50-polyprenol phosphates generating C35/C50-PP[14C]M and phosphatidyl-myo-inositolmannosides was quantified by scintillation counting and TLC using CHCl3:CH3OH:NH4OH:H2O (65:25:0.5:3.6, v/v/v/v). Autoradiography was performed by exposing the chromatogram to X-ray film (Kodak X-Omat) for 24 h. As a result, greater than 90% of [14C]mannose incorporation from GDP-[14C]mannose into the membrane fraction was observed within C35/C50-PP[14C]M, with the remainder associated with PI[14C]Ms (Besra et al., 1997Go).

Protein mannosyltransferase assay
Membrane, cytosol and P60 fractions prepared from M. smegmatis were assayed for mannosyltransferase activity using a method adapted from Besra et al. (1997)Go. Reaction mixtures consisted of the enzymatic extracts in buffer A containing, 1 mM ATP, 0.1 mM dithiothretiol, 10 mM CaCl2, 0.5 µCi GDP-[14C]mannose (Amersham Pharmacia Biotech, 303 mCi/mmol) (omitted in the case of in situ labeled membranes) and 0.5 mg synthetic peptide (2 mM final concentration) in a total volume of 160 µl. Reactions were incubated for 1 h at 37°C and stopped by the addition of 1.6 ml of CH3OH. The reaction mixture was initially centrifuged for 5 min at full speed in a microfuge and the supernatant carefully removed. The methanol extract was mixed with 3.2 ml of CHCl3 and 0.64 ml of H2O. The upper aqueous phase was removed following centrifugation for 5 min at 6000 x g and dried under a stream of nitrogen, resuspended in 200 µl water, and applied onto a 30 ml Bio-Gel P-2 gel filtration column (20 cm x 1.5 cm; Bio-Rad). Elution from the column was performed using water at a flow rate of 0.1 ml/min, and 0.5-ml fractions collected, which were subsequently mixed with 10 ml scintillant cocktail (Econoscint, National Diagnostics), and radioactivity was measured using a BetaRack scintillation counter (Wallac). Radiolabeled fractions purified by gel filtration chromatography were, in some instances, dried under nitrogen and resuspended in methanol before being applied to aluminum-backed silica TLC plates (Merck). TLC plates were then developed using CHCl3/CH3OH/1M NH4COOCH3/NH4OH/H20 (180:140:9:9:23, v/v/v/v), dried, and exposed overnight to X-ray film. For the determination of Mg2+-dependence, protein concentration, time course, peptide length, and substrate specificity (see Table I), substrate Km, thermal stability, the corresponding factors were varied in the basic assay mixture.

Analysis of reaction products
Pretreatment of membranes with amphomycin.
Membranes were preincubated with amphomycin (16 µg/160 µl reaction mixture) at 37°C for 10 min prior to the addition of GDP-[14C]mannose.

{alpha}-Mannosidase digestion.
Purified [14C]mannose-labeled enzymatic products isolated from the Bio-Gel P-2 gel filtration column were dried and redissolved in 0.1 M sodium acetate buffer, pH 5, containing 1 mM ZnSO4 and 0.1% (w/v) sodium taurodeoxycholate, incubated at 37°C for 16 h with and without 1 U jack bean {alpha}-mannosidase (30 µl final volume) (Oxford Glycosystems). The resulting reaction products were then mixed with a NaB[3H]4 reduced mannan standard (10,000 cpm) and applied onto a 165-ml Sephadex G-25 gel filtration column (120 cm x 1.5 cm: Bio-Rad). Elution from the column was performed using water at a flow rate of 0.1 ml/min, and 0.5-ml fractions collected, which were subsequently mixed with 10 ml scintillant cocktail and radioactivity measured using a BetaRack scintillation counter.

Reductive cleavage of O-glycosidic linkage.
The purified [14C]mannose-labeled enzymatic product isolated from the Bio-Gel P-2 gel filtration column was dissolved in a freshly prepared solution of 0.1 M NaOH in 2 M NaBH4 adjusted to pH 10 and incubated at 45°C for 16 h. The reaction mixture was neutralized using 50% acetic acid, dried, resuspended in methanol, and redried to remove borate salts (Besra and Brennan, 1994Go). The reaction mixture was combined with a NaB[3H]4 reduced mannan standard (10,000 cpm), resuspended in water, and applied onto a 165-ml Sephadex G-25 gel filtration column (120 cm x 1.5 cm). Elution from the column was performed using water at a flow rate of 0.1 ml/min, and 0.5-ml fractions collected, which were subsequently mixed with 10 ml of scintillant cocktail; radioactivity measured using a BetaRack scintillation counter.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
C35/C50-P-Man, polyprenol-monophosphoryl-mannose; FAB-MS, fast atom bombardment mass-spectrometry; LM, lipomannan; MOPS, 4-morpholine propane sulfonic acid; PI, phosphatidylinositol; PIMs, phosphatidylinositol mannosides; PPM, C35/C50-polyprenol-P-mannose; TLC, thin-layer chromatography.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
This work was supported by grants from Glaxo Wellcome PLC under the auspices of the "Action TB" program (to H.N.C. and D.B.Y.) and in part by a program project from the National Cooperative Drug Discovery Group for Opportunistic Infections (NIDDG-OI), NIAID, NIH (AI-38087 to P.J.B., Program Primary Investigator) and Grant AI-35220 (to G.S.B., Lister Institute, Jenner Research Fellow) and AI-18357 (P.J.B.).


    Footnotes
 
1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed; E-mail: d.young{at}ic.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
Banerjee, D.K., Scher, M.G., and Waechter, C.J. (1981) Amphomycin: effect of the lipopeptide antibiotic on the glycosylation and extraction of dolichyl monophosphate in calf brain membranes Biochemistry, 26, 1561–1568.

Besra, G.S. and Brennan, P.J. (1994) The glycolipids of mycobacteria. In Fenselau, C. (Ed), Mass spectrometry for the characterization of microorganisms. ACS Symposium Series 541, American Chemical Society, Washington, DC, pp. 203–232.

Besra, G.S., Morehouse, C.B., Rittner, C.M., Waechter, C.J., and Brennan, P.J. (1997) Biosynthesis of mycobacterial lipoarabinomannan. J. Biol. Chem., 272, 18460–184606.[Abstract/Free Full Text]

Bugg, T.D. and Brandish, P.E. (1994) From peptidoglycan to glycoproteins: common features of lipid-linked oligosaccharide biosynthesis. FEMS Microbiol. Lett., 119, 225–262.

Colussi, P.A., Taron, C.H., Mack, J.C., and Orlean, P. (1997) Human and Saccharomyces cerevisiae dolichol phosphate mannose synthases represent two classes of the enzyme but, both function in Schizosaccharomyces pombe. Proc. Natl Acad. Sci. USA, 94, 7873–7878.[Abstract/Free Full Text]

Cowlishaw, D.A. and Smith, M.C. (2001) Glycosylation of a Streptomyces coelicolor A3(2) cell envelope protein is required for infection by bacteriophage phi C31. Mol. Microbiol., 41, 601–610.[CrossRef][ISI][Medline]

Dobos, K.M., Khoo, K.H., Swiderek, K.M., Brennan, P.J., and Belisle, J.T. (1996) Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis. J. Bacteriol., 178, 2498–2506.[Abstract]

Erickson, P.R. and Herzberg, M.C. (1993) Evidence for the covalent linkage of carbohydrate polymers to a glycoprotein from Streptococcus sanguis. J. Biol. Chem., 269, 23780–23783.

Fifis, T., Costopoulos, C., Radford, A.J., Bacic, A., and Wood, P.R. (1991) Purification and characterization of major antigens from a Mycobacterium bovis culture filtrate. Infect. Immun., 59, 800–807.[ISI][Medline]

Garbe, T., Harris, D., Vodermeier, M., Lathigra, R., Ivanyi, J., and Young, D. (1993). Expression of the Mycobacterium tuberculosis 19-kilodalton antigen in Mycobacterium smegmatis: immunological analysis and evidence of glycosylation. Infect. Immun., 61, 260–267.[Abstract]

Hansen, J.E., Lund, O., Tolstrup, N., Gooley, A.A., Williams, K.L., and Brunak, S. (1998) NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility. Glycoconj. J., 15, 115–130.[CrossRef][ISI][Medline]

Herrmann, J.L., O’Gaora, P., Gallagher, A., Thole, J.E., and Young, D.B. (1996) Bacterial glycoproteins: a link between glycosylation and proteolytic cleavage of a 19kDa antigen from Mycobacterium tuberculosis. EMBO J., 15, 3547–3554.[Abstract]

Hewinson, R.G., Michell, S.L., Russell, W.P., McAdam, R.A., and Jacobs, W.J. (1996). Molecular characterization of MPT83: a seroreactive antigen of Mycobacterium tuberculosis with homology to MPT70. Scand. J. Immunol., 43, 490–499.[ISI][Medline]

Jennings, M.P., Virji, M., Evans, D., Foster, V., Srikhanta, Y.N., Steeghs, L., van der Ley, P., and Moxon, E.R. (1998) Identification of a novel gene involved in pilin glycosylation in Neisseria meningitidis. Mol. Microbiol., 29, 975–984.[CrossRef][ISI][Medline]

Kruszewska, J. S., Saloheimo, M., Migdalski, A., Orlean, P., Penttila, M., and Palamarczyk, G. (2000) Dolichol phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccharomyces pombe class of the enzyme. Glycobiology, 10, 983–991[Abstract/Free Full Text]

Lee, R.E., Brennan, P.J., and Besra, G.S. (1997) Mycobacterial arabinan biosynthesis: the use of synthetic arabinoside acceptors in the development of an arabinosyl transfer assay. Glycobiology, 7, 1121–1128.[Abstract]

Lindenthal, C. and Elsinghorst, E. (1999) Identification of a glycoprotein produced by enterotoxigenic Escherichia coli. Infect. Immun., 67, 4084–4091.[Abstract/Free Full Text]

Messner, P. and Sleytr, U.B. (1991). Bacterial surface layer glycoproteins. Glycobiology, 1, 545–551.[Abstract]

Nassif, X., Pujol, C., Morand, P., and Eugene, E. (1999) Interactions of pathogenic Neisseria with host cells. Is it possible to assemble the puzzle? Mol. Microbiol., 32, 1124–1132.[CrossRef][ISI][Medline]

Nigou, J., Vercellone, A., and Puzo, G. (2000) New structural insights into the molecular deciphering of mycobacterial lipoglycan binding to C-type lectins: lipoarabinomannan glycoform characterization and quantification by capillary electrophoresis at the subnanomole level. J. Mol. Biol., 299, 1353–1363.[CrossRef][ISI][Medline]

Schultz, J. and Elbein, A.D. (1974) Biosynthesis of mannosyl- and glucosyl-phosphoryl polyprenols in Mycobacterium smegmatis. Evidence for oligosaccharide-phosphoryl polyprenols. Arch. Biochem. Biophys., 16, 311–322.

Strahl-Bolsinger, S., Gentzsch, M., and Tanner, W. (1999) Protein O-mannosylation. Biochim. Biophys. Acta, 1426, 297–307.[ISI][Medline]

Szymanski, C.M., Yao, R., Ewing, C.P., Trust, T.J., and Guerry, P. (1999) Evidence for a system of general protein glycosylation in Campylobacter jejuni. Mol. Microbiol., 32, 1022–1030.[CrossRef][ISI][Medline]

Takayama, K., Schnoes, H.K., and Semmler, E.J. (1973) Characterization of the alkali-stable mannophospholipids of Mycobacterium smegmatis. Biochim. Biophys. Acta, 316, 212–21.[ISI][Medline]

Virji, M. (1997) Post-translational modifications of meningococcal pili. Identification of common substituents: glycans and alpha-glycerophosphate—a review. Gene, 192, 141–147.[CrossRef][ISI][Medline]