Department of Microbiology, Colorado State University, Fort Collins, CO 80523-1677, USA1
Department of Chemistry, Ohio State University, Columbus, OH 43210-1185, USA2
Author for correspondence: Patrick J. Brennan. Tel: +1 970 491 6700. Fax: +1 970 491 1815. e-mail: Patrick.Brennan{at}ColoState.edu
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ABSTRACT |
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Keywords: M. tuberculosis, LAM, monoclonal antibodies, epitope definition, hexa-Araf motif
Abbreviations: AG, arabinogalactan; AraLAM, LAM with naked Araf termini; LAM, lipoarabinomannan; LM, lipomannan; ManLAM, mannose-capped LAM; mAGP, mycolylarabinogalactanpeptidoglycan covalent complex
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INTRODUCTION |
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mAb CS-35 was raised against M. leprae LAM in this laboratory. This antibody has been used as a reference antibody for the characterization of mAbs against LAM and to study a variety of cellular functions such as the role of LAM in the interaction of mycobacteria with macrophages (Hamasur et al., 1999 ; Means et al., 1999
; Schlesinger et al., 1994
, 1996
). However, the epitope structures recognized by this antibody have not been identified. The internal segments of arabinans, as they appear in both LAM and AG, consist of linear 5-linked
-D-Araf residues and some branched 3,5-linked
-D-Araf units substituted with 5-linked
-D-Araf residues at both branched positions (Fig. 1
). The non-reducing terminal regions of the arabinans also contain 3,5-linked
-D-Araf residues substituted at both branched positions with the disaccharide ß-D-Araf-(1
2)-
-D-Araf (Daffe et al., 1990
). In the present study, several lines of evidence indicate that structural features within the terminal branched hexa-Araf arrangement constitute the epitope of mAb CS-35 and possibly many like antibodies.
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METHODS |
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Electrophoresis and Western blotting.
Purified LAM and LM preparations (5 µg) were applied to a Tricine 1020% gel (Invitrogen) and electrophoresis was carried out at a constant 125 V for approximately 90 min. A sample of prestained molecular mass standards was also run on the same gel. After electrophoresis, gels were transblotted to nitrocellulose in Tris/glycine/methanol buffer under 56 V constant voltage. The nitrocellulose membranes were blocked in PBS containing 0·025% Tween-80 (PBST) and 1% BSA. CS-35 (diluted 1:1000) in PBST was added and incubated overnight at room temperature. The nitrocellulose membranes were then washed three times with PBST and incubated with anti-mouse IgG alkaline phosphatase conjugated antibodies (Sigma) for 1 h at room temperature. Bound antibodies were detected with 5-bromo-4-choloro-3-indolyl phosphate (BCIP)/p-nitro blue tetrazolium chloride (NBT) substrate (Sigma).
ELISA.
Indirect ELISA, which measures binding of antibody to the immobilized polysaccharide-containing antigens, and competitive ELISA, which measures the ability of synthetic oligosaccharides to inhibit binding of antibody to immobilized AG, were carried out as previously described (Britton et al., 1985 ) with some modifications. Polysaccharide solutions (50 µl) were prepared in PBS pH 7·8 and applied to the wells of a flat bottom microtitre plate. The polysaccharides were immobilized on the untreated microtitre plates by incubating at 4 °C overnight. Non-specific antibody-binding sites were blocked by incubation for 1 h with 1% (w/v) ovalbumin and 0·05% Tween-80 in PBS (blocking buffer). mAb CS-35 (100 µl) diluted 1:1000 was added to the wells and allowed to incubate for 1 h. The wells were then washed with PBS containing 0·05% Tween-80 (washing buffer) (3x200 µl). Plates were incubated for 1 h with 100 µl antimouse IgGalkaline phosphatase conjugate (Sigma) diluted 1:2000 in washing buffer and washed with PBS (3x200 µl). The alkaline phosphatase activity was measured using p-nitrophenyl phosphate as substrate (Kirkegard and Perry Laboratories). The liberated p-nitrophenol was quantified by measuring the absorbance at 405 nm.
Competitive ELISA.
The synthesis of a range of oligosaccharides reflective of the termini of LAM and AG has been described (DSouza & Lowary, 2000 ; Yin et al., 2002
). These were prepared as the methyl glycosides. For assessment of their role in antibody binding, competitive ELISA was conducted. ELISA was carried out as described above except that 100 µl CS-35 (diluted 1:1000) plus varying concentrations of competitor solutions in blocking buffer were mixed and incubated at room temperature for 30 min prior to applying to the wells of the microtitre plate coated with AG or LAM. Bound CS-35 was detected as described above. The IC50 is defined as the concentration of an oligosaccharide required to achieve 50% inhibition of the binding of mAb CS-35 to the immobilized AG. Values for controls with no competitor were taken as 0% inhibition of antibody binding and values from controls with no antibody represented 100% inhibition of binding.
Preparation of AG and LAM from Mycobacterium smegmatis mc2155 and M. tuberculosis H37Rv.
M. smegmatis cells were grown to mid-exponential phase, harvested by centrifugation, washed with saline, lyophilized and extracted three times with CHCl3CH3OH (2:1, v/v) at 50 °C for 2 h to delipidate the cells. The lipid extracts were subject to a partitioning step (Mikusova et al., 1995 ). LAM and LM were removed from the delipidated cells by repeated refluxing in 50% aqueous ethanol. The ethanol extracts were combined with the aqueous phase from the lipid extracts, evaporated to dryness, and partitioned between hot phenol and water, resulting in partially purified preparations of LAM mixed with LM. The aqueous layer containing the majority of cellular LAM and LM was freeze-dried and used for SDS-PAGE. The final insoluble material, which contained mycolylarabinogalactanpeptidoglycan covalent complex (mAGP) was further extracted with 2% SDS in PBS to remove proteins (Hirschfield et al., 1990
).
In the case of M. tuberculosis, after delipidation, cells were disrupted mechanically using a French pressure cell (American Instrument Company) at a pressure of 1000 p.s.i. (6·9 MPa) The suspension after cell breakage was centrifuged at 27000 g for 30 min at 4 °C and the pellet was treated with SDS. SDS was removed by sequential washing with PBS, water, and finally with acetone. The insoluble mAGP was then treated with 1 M NaOH for 16 h at 80 °C, followed by neutralization and dialysis to prepare the soluble arabinogalactan. The freeze-dried material was used to prepare alditol acetates for GC-MS analysis as previously described (Frehel & Leduc, 1987 ). The AG from embA, embB and embC knock-out mutants of M. smegmatis (Escuyer et al., 2001
) were prepared as described above.
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RESULTS |
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DISCUSSION |
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The epitope on AG recognized by CS-35 was further characterized using a panel of synthetic oligosaccharides (DSouza & Lowary, 2000 ). Since attempts to bind the oligosaccharides directly to insoluble supports (either nylon membranes or plastic microtitre plates) failed, the relative affinity of mAb CS-35 for the various oligosaccharides was investigated in competitive ligand-binding studies against AG and AraLAM. As a control, competitive ELISA was performed in which AG or AraLAM was used as the immobilized antigen and various saccharides were used to competitively inhibit the binding of CS-35 to immobilized antigen. Some of the inhibitors, as well as AG and AraLAM, competitively inhibited the binding of CS-35 to immobilized AG, albeit with different efficiencies. The most effective competitor was AG (Fig. 7
). AraLAM was a little less effective, whereas YHF-2-1 and YHF-3 were approximately 10-fold and 100-fold less effective as competitors, respectively. YHF-5, at the highest concentrations tested, 500 and 1000 µg ml-1, did not compete with AG for binding to CS-35.
Several lines of evidence indicate that, although the terminal arabinosyl residues are a dominant part of the epitope recognized by mAb CS-35, it is not the only structural feature in AG recognized by the monoclonal antibody. The ability of arabinose-containing oligosaccharides to compete for the CS-35 site increased over 1 order of magnitude as the size of the oligosaccharide increased. Such size dependence would not be exhibited if the antibodies were recognizing only the terminal arabinosyl residues. Two possible explanations are offered for the difference in the behaviour of polymeric AG and oligosaccharides in the competitive binding assays. First, the superior ability of intact AG compared with AG oligosaccharides to compete for the antibody-combining site could be a reflection of the antibodys avidity for a multivalent antigen. Alternatively, mAb CS-35 may recognize a conformation that is favoured in polymeric AG but that is only infrequently adopted by AG oligosaccharides of the sizes tested in this report. Levy et al. (1991) have shown that the most energetically favoured conformation of oligomers is the one in which the backbone adopts a twisted conformation, which is not conducive to the formation of hydrogen bonds between the backbone chains.
The binding and ligand competition experiments may provide additional insight into the nature of the polysaccharide binding site on mAb CS-35. We propose that the binding site consists of a groove, into which the polysaccharide chain fits, with one or more pockets that accommodate the terminal arabinosyl residues. Groove-and pocket-type binding sites on antibodies have been previously proposed based on results from ligand-binding and molecular-modelling studies (Cisar et al., 1975 ; Glaudemans, 1987
; Oomen et al., 1991
). X-ray crystallographic evidence of a pocket-type site on a monoclonal antibody against the bacterial O-antigen oligosaccharides has been obtained (Cygler et al., 1991
). We hypothesize that the oligosaccharide, YHF-2-1 (Fig. 6
), with two terminal arabinosyl arrangements, interacts with both the pocket and groove, thereby interfering with the binding of polysaccharide.
The epitope recognized by CS-35 is different from the galactose epitopes recognized by human intelectin (Tsuji et al., 2001 ). Human intelectin is a secretory glycoprotein consisting of 295 amino acids and N-linked oligosaccharides. It has affinities to D-pentoses and D-galactofuranosyl residues in the presence of Ca2+ and recognizes the arabinogalactan of Nocardia containing D-galactofuranosyl residues. The fact that reactivity of mAb CS-35 with AG decreased upon removal of arabinosyl residues and binds to synthetic arabinose oligosaccharides indicates that CS-35 recognizes a different epitope than does human intelectin.
Interesting spatial and developmental patterns of arabinogalactan epitope localization have been observed in different plant tissues using several antiarabinogalactan antibodies (Knox et al., 1991 ; Kikuchi et al., 1993
; Pennell et al., 1992
). Interpretation of these localization patterns at the molecular level is hampered by the lack of information on the epitope recognized by the antibodies used in these studies. The type of epitope characterization reported here for mAb CS-35 will now allow molecular definition of antibodyantigen interaction and thereby help with the interpretation of the spatial arrangement of AG in the mycobacterial cell wall. This information should also enhance the value of these antibodies for the screening of cell-wall mutants of mycobacteria lacking AG and AraLAM motifs and the recognition of biosynthetic intermediates of cell-wall polysaccharides unique to mycobacteria.
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ACKNOWLEDGEMENTS |
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Received 21 March 2002;
revised 26 June 2002;
accepted 28 June 2002.