5-methylthiopentose: a new substituent on lipoarabinomannan in Mycobacterium tuberculosis1

doi:10.1006/jmbi.2001.5317 available online at http://www.idealibrary.com on

J. Mol. Biol. (2002) 316, 89±100

5-Methylthiopentose: a New Substituent on Lipoarabinomannan in Mycobacterium tuberculosis Achim Treumann1, Feng Xidong2, Liam McDonnell2, Peter J. Derrick2 Alison E. Ashcroft1, Delphi Chatterjee3 and Steve W. Homans1* 1

Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds Leeds LS2 9JT, UK 2

Institute of Mass Spectrometry and Department of Chemistry University of Warwick Coventry CV4 7AL, UK 3 Department of Microbiology Colorado State University, Fort Collins, CO 80523-1677, USA

We have identi®ed and characterised in several strains of Mycobacterium tuberculosis a new 5-methylthiopentose substituent on lipoarabinomannan (LAM). The 5-methylthiopentose was initially observed in heteronuclear 1 H-13C-NMR spectra of intact, 13C-enriched LAM. Oligosaccharides carrying this substituent were released from 13C-enriched LAM and from unlabelled LAM using an endo-arabinanase from Cellulomonas gellida. The presence of the methylthio group in these oligosaccharides was established using NMR, high-resolution Fourier-transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry using a QTOF mass spectrometer. The 5-methylthiopentose is linked to a terminal mannose in the cap structures of these oligosaccharides as evidenced by tandem mass spectrometry and by NMR. We suggest interference with the signal transduction mechanisms of infected macrophages as a possible function for this newly discovered LAM substituent. # 2002 Elsevier Science Ltd.

*Corresponding author

Keywords: lipoarabinomannan; Mycobacterium; cell wall; NMR; FTICR

Introduction Lipoarabinomannan (LAM) is an important component of the mycobacterial cell wall and several recent reviews have highlighted the relevance of this polysaccharide for the pathogenesis of tuberculosis.1 ± 4 Building on the foundations of some early structural work,5,6 the main features of the structure of LAM were outlined in the 1990 s and are summarised in Figure 1. LAM is attached to a lipophilic region of the mycobacterial cell wall through a phosphatidyl inositol anchor carrying up to four acyl chains attached to glycerol, inositol or to a mannosyl residue, which is linked to the C2 of inositol.7,8 This Abbreviations used: LAM, lipoarabinomannan; ManLAM, mannosyl-capped LAM; AraLAM, phosphatidylinositol-capped LAM; FTICR-MS, Fourier transform ion cyclotron resonance mass spectrometry; QTOF-MS, quadrupole time-of-¯ight mass spectrometry; OS, octyl sepharose; HIC, hydrophobic interaction chromatography; CID, collision induced dissociation; 5-MTP, 5-methylthiopentose; 5-MSP, 5sulphoxythiopentose; SORI-CID, sustained off-resonance irradiation collision-induced dissociation; GAS, glycerolalanine-salt; JBAM, Jack bean a-mannosidase. E-mail address of the corresponding author: [email protected] 0022-2836/02/010089±12 $35.00/0

lipid anchor carries the mannan core of LAM consisting of a Mana1-6 backbone, to which Man residues are attached in a1-2 linkages.9,10 Either one or several D-arabinofuranan side-chains can be found in unknown linkage position to the mannan core. The arabinan chains have an a1-5 backbone and numerous internal 3,5aAraf branching points. At the non-reducing termini, these side-chains carry a b-arabinofuranose, which is extensively capped in slow growing mycobacterial species with a (Mana1-2)nMana1-5 (0 4 n 4 2) motif.11 A small number of the branching a3,5Araf residues is substituted with ester-linked succinate, linked to the C-2.12 Many attempts were made to use the structural information about LAM to improve the understanding of the functions of this glycoconjugate. Quite early on it was recognised that many biological responses to LAM can be abolished through alkaline hydrolysis.13 ± 15 This observation is generally related to the hydrolysis of the lipid anchor. However, this could also be caused by the abrogation of other alkali-labile substituents. A major breakthrough was the discovery that mannosyl-capped LAM from slow growing mycobacterial species (ManLAM) differs from phosphatidylinositol-capped LAM (AraLAM) in its ability to induce the production of cytokines.14,16 AraLAM was a stronger inducer of cytokines than Man# 2002 Elsevier Science Ltd.

90

5-Methylthiopentose in Lipoarabinomannan

Figure 1. Schematic representation of the structure of LAM. The number of arabinan side-chains and their attachment point to the mannan core are not known. Also, the precise arrangement of the branching points in the arabinan is not known. Several branching aAraf residues are substituted with a succinyl ester. The predominant fatty acyl chains on the phosphatidylinositol are palmitate (C16:0) and 10-methyloctadecanoate (tuberculostearate, C19) with smaller amounts of C14:0, C17:0, methylC17:0 and C18:0. The inositol and the mannosyl residue linked to the C-2 of the inositol can carry further acyl chains.

LAM, suggesting that the mannose capping has evolved in pathogenic mycobacterial species as part of their survival strategy inside the macrophage. These advances make it only more evident that a clearer understanding of the structure of LAM is needed to obtain a thorough appreciation of the role of this glycoconjugate in the pathogenesis of tuberculosis. Here, we report the application of 13 C-labelling combined with high-resolution heteronuclear NMR spectroscopy and state of the art mass spectrometric methods to the structural analysis of LAM. These approaches have led us to the discovery of a methylthiopentose as a new structural component with far-reaching functional implications at the non-reducing end of LAM.

Results LAM can be separated into different fractions by HIC, all of which contain small amounts of an unusual sugar We grew Mycobacterium tuberculosis H37Ra in media containing [U-13C]glucose (see Materials and Methods) to arrive at a more detailed picture of the structure of LAM. Quadrupole-time-of-¯ight mass spectrometry (QTOF-MS) spectra of oligosaccharides obtained from endo-arabinanase digestions of the 13C-enriched preparation of LAM indicated a random degree of labelling of about 50 % in mannose and arabinose alike (data not shown). LAM was separated from lipomannan using gel permeation chromatography as described previously.17 To obtain a preparation of higher purity, the glycoconjugate was subjected to hydrophobic interaction chromatography (HIC) on octyl Sepharose (OS) (Figure 2). As shown previously by Leopold and Fischer18, this procedure separates LAM into fractions differing in their degree of acylation. In contrast to these authors, however, we observed one large fraction of LAM, which did not bind to

OS at all (fraction A) under the initial binding conditions (5 % n-propanol, 100 mM NH4OAc) and two fractions that eluted with a linear gradient of n-propanol (fractions B and C). Fractions A and B were pooled for further investigations and are referred to as OS-non-binding and OS-binding LAM, respectively. Three fractions, eluting at 5 % n-propanol (notbinding), 18 % n-propanol and 40 % n-propanol, were also observed for batches of LAM puri®ed from M. tuberculosis H37Ra grown in glycerolalanine-salts (GAS) broth and for LAM puri®ed from other strains of M. tuberculosis. We do not have an explanation for this apparent inconsistency and we are currently investigating structural differences between the fractions of LAM that do and do not bind to the HIC column. The availability of 13C-enriched LAM enabled us to perform a more detailed assignment of LAM

Figure 2. Hydrophobic interaction chromatography of H37Ra LAM. The thick line with square symbols represents the carbohydrate content per fraction (left yaxis), the thin line with open circles represents the measured n-propanol content.

91

5-Methylthiopentose in Lipoarabinomannan

NMR-spectra than had previously been possible. Here, we will describe a new, minor component of LAM that had so far escaped the attention of researchers due to its low abundance. In the anomeric region of 1H-13C-HSQC spectra of OS-binding LAM and of non-OS-binding LAM, a pair of down®eld-shifted peaks could not be assigned to any of the known components of LAM (Figure 3). TOCSY and HCCH-TOCSY experiments showed that these resonances were part of two coupling systems that showed the characteristics of pentofuranoses, but the 1H and 13C-resonances of

the methylene groups (corresponding to the C-5/ H-5 of these pentofuranoses) were strongly shifted to a higher ®eld (Figure 4, Table 1). This showed clearly that these coupling systems belonged to two very similar sugars that were neither arabinose nor mannose. Investigation of the anomeric region of 1D1 H-NMR spectra from various preparations of ManLAM con®rmed the presence of these sugars in all the preparations we examined thus far. Integration of the anomeric peaks indicated the presence of one of these sugars per 60-100 Araf and Man residues, corresponding to one glycosyl residue per molecule of LAM. Oligosaccharides containing the unusual sugar are liberated from LAM using an endo-arabinanase Intact 13C-enriched H37Ra-LAM ([13C]RaLAM) was exhaustively digested with endo-arabinanase from Cellulomonas sp.9 The digestion products were analysed by BioGel P4 gel permeation chromatography (Figure 5). Peaks were pooled as indicated on Figure 5 and the pools were analysed by NMR. The pentofuranose derivative was found in oligosaccharides with a hydrodynamic volume of more

Figure 3. Partial 1H-13C-HSQC spectra (anomeric region) of 13C-enriched H37Ra LAM from the octylSepharose non-binding fraction (a) and from the octylSepharose-binding fraction (b). The cross-peaks corresponding to 5-methylsulphfoxypentose and to 5methylthiopentose are labelled MSP and MTP, respectively.

Figure 4. Coupling systems corresponding to the 5deoxy-5-methyl-5-thio-pentofuranose. Cross-peaks derived from 5-methylsulphoxypentose and 5methylthiopentose are labelled MSP and MTP, respectively. (a) Partial HCCH-TOCSY spectrum of 13Cenriched H37Ra LAM. (b) Partial TOCSY spectrum of CSU20 LAM.

92

5-Methylthiopentose in Lipoarabinomannan

Table 1. Chemical shifts for 5-methylthiopentose (5-MTP) and 5-methylsulphoxypentose (5-MSP) in intact lipoarabinomannan Chemical shift (ppm) MTP MSP-1 MSP-2

C1

C2

C3

C4

C5

H1

H2

H3

H4

H5a

H5b

105.2 105.4 105.4

79.4 79.4 79.3

78.3 78.5 78.5

80.5 76.5 75.6

35.8 55.6 57.1

5.40 5.44 5.45

4.21 4.20 4.22

4.26 4.34 4.34

4.38 4.65 4.61

2.80 3.28 3.12

2.68 3.08 3.12

than six glucose units (Figure 5), but not in the mannan core (data not shown), showing that it was linked to the outer arabinan chains of LAM. For mass-spectrometric analysis non-labelled H37Rv-LAM was digested with Cellulomonas endoarabinanase and the digestion products separated by BioGel-P4. 1D 1H-NMR analysis revealed the presence of a 5-MSP-containing oligosaccharide in a heterogeneous fraction eluting at 7.8 glucose units (Figure 6(a)-(c)). The unusual sugar contains a methylsulphoxide group Mass spectrometric analysis of the 7.8 glucose unit fraction of RvLAM revealed the presence of two main components with pseudomolecular [M ‡ H]‡ at m/z 1135 and at m/z 1049 and their Na‡-adducts at m/z 1157 and m/z 1071, respectively (Figure 7(a)). The CID spectrum of the ion at m/z 1135 displayed a series of ions at m/z 1117.4, 985.3, 853.3, 721.2, 589.2, 457.2, 325.1 and 163.1 (Figure 7(b)). It is known that [M ‡ H]‡ of underivatised oligosaccharides tend to fragment at the glycosidic bond in low-energy collision conditions.19 Therefore, it is reasonable to assign this ion series to the B-ions20 generated from a parent ion with the structure Man2Ara6, which is known

to be one of the main non-reducing terminal structures of LAM.9 The CID spectrum of the second molecular species of the 7.8 glucose unit fraction of the RvLAM endo-arabinanase digest displayed a series of ions at m/z 1031.3, 899.3, 767.3, 635.2, 503.2, 341.1 and 179.0 (Figure 7(c)). Assuming that this series corresponded also to a series of B-ions, we assigned this spectrum to an oligosaccharide with the structure MSP-Man2-Ara4.The B1-ion caused by the structural element MSP was detected at m/z 179, indicating a molecular mass for the corresponding monosaccharide of 196. Further information was obtained from the CID spectrum of the Na‡-adduct at m/z 1071. A characteristic fragment ion was observed at m/z 1007.3. This corresponds to a neutral loss of 64, which has been characterised as being diagnostic for the presence of oxidised methionine in peptides. The elimination of methanesulphenic acid (CH3SOH) enables one to distinguish oxidised methionine from the isobaric phenylalanine in peptide CID spectra.21,22 This suggested the presence of the structural element CH3-SO- as part of the unknown sugar and led us to propose that the oligosaccharide giving rise to a pseudomolecular ion at m/z 1049 would have a 5-deoxy-5-methyl-5-sulphoxypentofuranose at its non-reducing end.

Figure 5. P4 chromatogram of the oligosaccharides released from 13 C-enriched H37Ra LAM by endoarabinanase from Cellulomonas sp. Fractions were pooled as indicated by vertical lines and analysed by 1 H-13C-HSQC. Fractions shaded in grey contain oligosaccharides displaying cross-peaks at the characteristic resonances of 5-methylthiopentose or 5-methylsulphoxypentose. The numbered dots above the chromatogram indicate the elution positions of an external standard of a partial dextran hydrolysate.

5-Methylthiopentose in Lipoarabinomannan

Figure 6. Puri®cation of an oligosaccharide fragment from H37Rv LAM containing 5-methylsulphoxypentose. The triplet characteristic of the oxidised 5-methylsulphoxypentose is indicated with an arrow in the NMR spectra. (a) 1D 1H-NMR spectrum of H37Rv LAM. (b) Partial BioGel P4 chromatogram of the products of the endo-arabinanase digestion of H37RvLAM. Pooled fractions are hatched. The elution positions of glucose oligomers are indicated above the chromatogram. (c) Partial 1D 1H-NMR spectrum (anomeric region) of the pool shown in (b). (d) Partial BioGel P4 chromatogram of the products of the Jack Bean a-mannosidase digestion of the pool shown in (b). Pooled fractions are hatched. (e) Partial 1D 1H-NMR spectrum (anomeric region) of the pool shown in (d).

We puri®ed the 5-deoxy-5-methylsulphoxy-pentofuranose-containing oligosaccharide by treating the 7.8 glucose unit fraction with Jack Bean a-mannosidase, an exoglycosidase that removes nonreducing terminal Man from oligosaccharides, and subsequent separation of the digestion products by size exclusion chromatography on BioGel P4 (Figure 6(d)). The 1D-1H-NMR spectrum of the a-mannosidase-resistant material showed that this fraction did contain the sugar responsible for the diagnostic peak at 5.46 ppm (Figure 6(e)). A nanospray-FTICR mass spectrum of the puri®ed oligosaccharide, recorded with a wide mass window, showed only the Na‡-adduct at m/z

93 1071.30351 (Figure 8(a)). The Na‡-adduct of an oligosaccharide with the proposed structure and a sum formula of C38H64O31S1Na1 would give a theoretical molecular mass of 1071.30445. The difference between theoretical prediction and experiment is 0.89 ppm, providing good evidence for the proposed sum formula. The experimental isotope pattern is almost identical to the theoretical prediction (Figure 8(a), inset). Unequivocal evidence for the presence of a sulphur atom in this oligosaccharide is presented in an FTICR experiment with a smaller mass window, allowing for higher resolution, shown in Figure 8(b). The inset shows two isotopic peaks at m/z 1073 [M ‡ Na ‡ 2]‡. The larger one corresponds to the isotopic peaks caused by the 13C2 (18O034S0) isotope (m/z 1073.31170, abundance 8.22 % relative to the monoisotopic ion at m/z 1071) and the 18O1(13C034S0) isotope (m/z 1073.30924, abundance 6.37 % relative to the monoisotopic ion at m/z 1071) and the smaller one corresponds to the peak caused by the 34S1(13C18 0 O0) isotope (m/z 1073.30079, abundance 4.52 % relative to the monoisotopic ion at m/z 1071). Despite differing only by 0.0085 mass units, the isotopic peak caused by the presence of 34S could be resolved from the peak corresponding to its 13C- or 18O-containing isotopomers. From the relative intensity of the 34S-isotopomer peak to the monoisotopic peak at m/z 1071, the number of sulphur atoms per oligosaccharide was calculated to be 1.095. These results clearly con®rmed the presence of one sulphur atom per molecule. Finally, when the oligosaccharide was fragmented using sustained off-resonance irradiationcollision-induced dissociation (SORI-CID), one fragment ion was observed in the high mass region at m/z 1007.30703 (Figure 8(c)). The accurate mass of this fragment ion yielded an atomic composition of C37H60O30Na1 (theoretical m/z ˆ 1007.30671), con®rming that the neutral loss of 64, which we observed already in the QTOF-MS-MS spectrum, was indeed caused by the loss of methanesulphenic acid. The mass spectrometric data show that the coupling system labelled MSP in Figure 3 is a methylsulphoxypentose. This is consistent with the measured 1H and 13C chemical shifts in NMR spectra. Also consistent with the NMR data is the assignment of the other coupling system (MTP) to a 5-deoxy-5-methyl-5-thiopentose. We suggest that 5-MSP is a non-enzymatic oxidation product of 5MTP. This is supported by the observation that 5MSP is actually represented by two very similar coupling systems in the spectra, resulting in an apparent triplet for the anomeric 1H-resonance of 5-MSP in 1D-1H-NMR spectra (Figure 4). These two coupling systems stand for the two stereoisomers that are formed by the introduction of a new stereocentre upon oxidation of the thioether. The presence of both stereoisomers in equal proportions in all LAM preparations and fragments thereof that we have looked at so far, suggests that

94

5-Methylthiopentose in Lipoarabinomannan

Figure 7. QTOF-MS-MS analysis of the 7.8 glucose unit fraction of endo-arabinanase-digested H37Rv LAM. (a) QTOF-MS spectrum of the 7.8 glucose unit fraction of endo-arabinanase-digested H37Rv LAM. (b) QTOF-MSMS daughter ion spectrum of the m/z 1135.4 pseudomolecular ion from (a). (c) QTOF-MSMS daughter ion spectrum of the m/z 1049.3 pseudomolecular ion from (a). (d) QTOF-MSMS daughter ion spectrum of the m/z 1071.3 pseudomolecular ion from (a). The daughter ions at m/z 1039.3 (c) and m/z 1053.3 (d), respectively, are [M ‡ 4] ‡ that arise frequently in QTOF/tandem-MS experiments of pseudomolecular [M ‡ H] ‡ of carbohydrates. They are not present in the QTOF-MS spectrum (compare (a)), indicating that they are derived from the parent ions at 1035.3 and 1049.3, respectively. We have currently no explanation for their origin.

5-Methylthiopentose in Lipoarabinomannan

95

Figure 8. FTICR mass spectra of the a-mannosidase-resistant oligosaccharide from the 7.8 glucose unit fraction of endo-arabinanasedigested H37Rv LAM. (a) Wide mass range spectrum, transient duration ˆ 0.89 second. The inset shows a magni®cation of the region between m/z 1070 and m/z 1075 and the calculated isotope pattern in this region for the ion with the composition C38H64O31S1Na1. (b) Higher resolution spectrum, transient duration ˆ 1.76 seconds. The inset shows a magni®cation of the region between m/z 1073.24 and m/z 1073.4 and the calculated isotope pattern in this region for an ion with the composition C38H64O31S1Na1. (c) Partial SORICID mass spectrum of the m/z 1071 pseudomolecular ion from (a).

the oxidation is spontaneous and non-enzymatic. It has been known for a long time that methylthio sugars are easily oxidised to their sulphoxyderivatives.23 ± 25 5-MTP is linked to a capping mannose in LAM Two experiments have already been presented, which indicate that the 5-methylthiopentose is linked to a mannose cap in LAM: (a) The oligosaccharide containing 5-MSP is resistant to digestion with a-mannosidase, indicating that the non-reducing terminal mannose of this oligosaccharide is either substituted or that access to this sugar by the enzyme is sterically hindered.

(b) The B-series in the QTOF nanospray-CID spectrum of the [M ‡ H‡]-pseudomolecular ion of the oligosaccharide containing 5-MSP ends in the B1-ion of 5-MSP (m/z 179), indicating that this sugar is at the non-reducing end of the oligosaccharide. This is supported by the linkage information that can be obtained using NMR spectroscopy (Figure 9). In a multiple-bond HMBC spectrum, the anomeric 1H and 13C atoms of 5-MTP at 5.40 ppm and 105.2 ppm correlate with a quartet that can be observed at 3.77 ppm/77.0 ppm (Figure 9(a)-(c)). An HCCH-TOCSY experiment connects this quartet with the anomeric of a coupling system with an anomeric proton resonating at

96

5-Methylthiopentose in Lipoarabinomannan

Figure 9. Through-bond connectivity of 5-methylthiopentose. (a) and (d) Partial 2D 1H-13C-NMR multiple bond HMBC spectra of 13C-enriched H37Ra LAM. (b) and (c) Partial 2D 1H-13C-NMR HSQC spectra of 13C-enriched H37Ra LAM.

5.03 ppm (data not shown), overlapping with the anomeric 1H and 13C resonances of terminal Man in Man caps. A ROESY experiment performed on CSU20 LAM correlated the H1 of 5-MTP to the H2 at 4.21 ppm (Figure 10(a)). Three ROESY cross-peaks at higher ®eld cannot be observed in a 1H-TOCSY spectrum that was acquired with the same sample (Figure 10(b)) and belong thus to a different coupling system. These three ROESY cross-peaks were correlated with a coupling system with an anomeric proton resonating at 5.03 ppm (Figure 10(c)). There is no evidence for any arabinofuranosylderived coupling systems that would overlap with the anomeric protons of the mannosyl residues resonating at this chemical shift. This suggests that 5-MTP is linked to a mannosyl residue on a non-reducing terminus of LAM and that the linkage of 5-MTP does not detectably affect the chemical shift of the anomeric 1H and 13 C of the Man it is linked to.

Discussion We present here evidence for a new structural component of LAM in M. tuberculosis. 5-deoxy-5methyl-5-thio-pentofuranose is present in LAM in very low concentrations (one molecule of 5-MTP or 5-MSP per molecule of LAM) on the non-reducing termini of the molecule as a substituent of a mannosyl cap. The discovery and characterisation of this lowabundance structural element of LAM was greatly facilitated through [13C]enrichment by growing M. tuberculosis on media containing 13C-glucose as

the only carbon source. This gave us the necessary sensitivity to perform heteronuclear experiments to assign 13C chemical shifts of the 5-methylthiopentose in the strongly overlapping NMR spectra of LAM. The increased sensitivity was even more important when we started to look at the digestion products of LAM with endo-arabinanase, where the available amounts of material became a lot smaller. The 13C-labelling strategy used also provided a convenient ®lter that permitted us to differentiate between 1H signals that were derived from LAM carbohydrates and other 1H-signals (e.g. those due to contamination from the enzyme preparations used), as only signals from protons attached to 13 C-atoms will be detected in heteronuclear 1Hdetected 1H-13C-NMR experiments. The high resolving power and the mass accuracy of high-®eld FTICR-MS provided unequivocal proof of the presence of a sulphur atom in the puri®ed oligosaccharide. Sulphur does not have any NMR-active isotopes and its mass of 32 amu makes it dif®cult to distinguish its presence from that of two oxygen atoms in low-resolution mass spectra. The high mass accuracy (deviation of < 1 ppm from calculated values) that was obtained with the use of an external standard allowed the composition of each peak to be assigned with con®dence. The ability to resolve two isotopomers that differed by only 0.01 amu was crucial in providing clear evidence for this unexpected structural element. This is the ®rst time that a 5-methylthiopentose has been observed as a structural building block of an oligosaccharide. 5-Deoxy-5-methyl-5-thio-ribofuranose is well known as a component and as a

97

5-Methylthiopentose in Lipoarabinomannan

tor of methyltransferases28,29 and of the FGF-receptor tyrosine kinase.30,31 It is possible, that 5-MTP can also exert an inhibitory effect on a regulatory enzyme in macrophages, interrupting a signal transduction chain and thus contributing to the pathogen's survival within the macrophage. It is known that LAM has a modulatory effect on the immune system3,4 and 5-MTP could be a key instrument through which mycobacteria can exert this modulatory effect.

Materials and Methods Reagents Aluminium-backed thin layer chromatography sheets were from Merck (UK); Jack Bean a-mannosidase was from Glyko (UK); [13C]glucose was from Martek (US); 2 H2O (99.95 %) was from Aldrich (UK) or from Goss (UK). All other reagents and solvents were of the highest purity commercially available. Cell culture, media and growth conditions Bacterial strains and culture conditions

Figure 10. Through-space connectivity of 5methylthiopentose. (a) Partial ROESY spectrum of CSU20 LAM. (b) and (c) Partial TOCSY spectra (130 ms mixing time) of CSU20 LAM.

metabolic product of 50 -methyl-thio-adenosin, which is a universal metabolite.26 It is likely that the 5-methylthiopentose in LAM is synthesised through 5-methylribose as an intermediate. We report data on the 5-methylthiopentose derived from LAM from several different mannosyl-capped M. tuberculosis strains, the non-virulent laboratory strain H37Ra, the well-characterised virulent laboratory strain H37Rv and CSU20, a clinical isolate. Since then, we have also noticed the presence of this sugar in several other strains, including several clinical isolates. This widespread presence suggests an important biological function for this unusual sugar. One possible function could be related to the observation that 5-MTP appears to be very easily oxidised to the sulphoxide derivative 5-MSP. Consequently, this sugar might be responsible for the anti-oxidative properties of LAM that have been reported.27 However, one would assume, that an antioxidant would be more abundant on the mycobacterial surface than this sugar which is only a very minor constituent of LAM. An alternative possible function is related to 5MTP's structural relative, 50 -methyl-thio-adenosine (5-MTA). 5-MTA has been reported to be an inhibi-

Frozen stocks of M. tuberculosis, H37Rv and a clinical isolate CSU 20 were plated on Middlebrook 7H11 agar plates (Difco) with oleic acid/albumin/dextrose/catalase enrichment supplement. Isolated colonies were picked and transferred to 5 ml of GAS (glycerol-alanine salts) broth32 and grown for two weeks at 37  C on a shaker (starter culture). Large-scale cultures were initiated by using 1 ml of GAS broth from the starter cultures to inoculate 50 ml of fresh GAS broth. After seven days at 37  C, 1 ml of the GAS culture was transferred to 400 ml medium and grown at 37  C until late log phase. About 8-10 g of cells was obtained from two litres of culture. For growth of M. tuberculosis, H37Ra in [U-13C]glucose, glycerol was eliminated from the GAS medium and replenished with lower concentration of normal glucose at ®rst. When growth was perceived, glucose was replaced with [U-13C]glucose gradually to a concentration of 1 g/litre of broth. Typically, this process took six months from the starting date of replenishing glycerol with glucose. About 7.4 g of wet cells was obtained from two litres of culture. Extraction of LAM Harvested cells were delipidated with chloroform:methanol:water (10:10:3 by vol.) two times for two hours at room temperature. The delipidated cells were dried under a nitrogen stream to eliminate organic solvents. The dried residual biomass was re-suspended in breaking buffer and disrupted mechanically using a French Press to produce 90 % breakage of the cells. Breaking buffer for the cell disruption contained a protease inhibitor cocktail (pepstatin A, PMSF, leupeptine), DNAse, and RNAse (Sigma) in phosphate buffered saline. Triton X-114 (from a 32 % stock) (Sigma) was added to the lysed cells to a ®nal concentration of 8 % (v/v) and, after cooling on ice, the solution was rocked at 4  C overnight. The cellular debris was removed by centrifugation at 27,000 g for one hour at 4  C, and the supernatant was incubated at 37  C to induce biphasic separation.33,34 The upper aqueous layer was mixed with the cellular debris and re-extracted as described above. The detergent layers

98 were combined and the lipoglycan precipitated by the addition of nine volumes of cold ethanol (95 %, ÿ20  C). The precipitate was collected and lyophilized. The lyophilized material was suspended in a minimal amount of water and partitioned with phenol. The solution was dialysed and lyophilised once more prior to gel ®ltration chromatography as has been described.17 Lyophilised LAM was redissolved in 500 ml of a buffer containing 100 mM ammonium acetate and 5 % n-propanol (buffer A) and applied at 2 ml/hour to an octyl Sepharose (Sigma) column (100 mm  10 mm) that had been equilibrated in the same buffer. After washing at 2 ml/hour with 10 ml of buffer A, the column was eluted with 100 ml of a linear gradient from buffer A to 80 % n-propanol in water at 6 ml/hour. The carbohydrate content of collected fractions was monitored by spotting 1 ml aliquots from each fraction and from a dextran standard of known concentration onto a thin layer chromatography plate and spraying with orcinol reagent. n-Propanol content in non-carbohydratecontaining fractions was monitored using an Abbe refractometer (Bellingham and Stanley, England). Carbohydrate-containing fractions were pooled, n-propanol was evaporated in a Vapor-mix S10 shaking evaporator (Eyela, Japan) and the NH4OAc was removed by repeated lyophilisation. Fractions that had bound to the octyl Sepharose were suf®ciently pure for further analysis. LAM-containing fractions from the ¯owthrough were further desalted by chromatography over a BioGel P4 (BioRad) column (1000 mm  15 mm) at 55  C where intact LAM ran in the void volume. Endo-arabinanase digestion of LAM Up to 8 mg of LAM was lyophilised. 30 ml of an endo-arabinanase preparation from Cellulomonas gellida, prepared as described17 was added and the digest was incubated at 37  C. Other 30 ml aliquots of the enzyme preparation were added after ten hours and again after 14 hours. The incubation was ®nished after 20 hours by boiling the tube for ten minutes. The digestion products were applied to a BioGel P4 column (1000 mm  15 mm, 55  C, 10 ml/hour) and eluted with water at 6 ml. The elution pro®le was followed using a refractive index (RI) detector (Erma, Japan). Before and after the digest a set of glucose oligomers (prepared following35) were separated as an external standard on the same column under identical conditions.

5-Methylthiopentose in Lipoarabinomannan volume was either 650 ml in 0.5 mm NMR tubes or 250 ml in 0.5 mm symmetrical NMR microtubes (Shigemi, US). All spectra were acquired after several rounds of lyophilisation with 2H2O. Processing of all two-dimensional data was performed using the NMRPipe suite36 and the processed spectra were analysed using NMRView.37 Typically time-domain data were multiplied with a cosine window function and zero-®lled to twice the number of data points before Fourier transform. Heteronuclear two-dimensional 1H-13C chemical shift correlations were measured in the 1H-detected mode via single quantum coherence (HSQC). A constant time HSQC spectrum for OS non-binding LAM was collected at 600 MHz with a constant time period (2T) of 44 ms. GARP decoupling38 was employed in the 13C domain during the t2 period for all HSQC spectra. Data sets contained 256 t1 (13C)  1024 t2 (1H) complex points with spectral widths of 10,000 Hz (13C) and 3100 Hz (1H). A total of 16 transients were accumulated with one second relaxation delay between scans. A constant time HCCH-TOCSY was recorded at 500 MHz under essentially the same conditions as described.39 Data sets contained 224 t1 (13C)  256 t2 (1H) complex points with spectral widths of 9523 Hz (13C) and 1155 Hz (1H). A total of 104 transients were accumulated with one second relaxation delay between scans. A TOCSY experiment was acquired at 600 MHz with 130 ms mixing time. Data sets contained 256 t1  1024 t2 complex points with spectral widths of 3100 Hz in both domains. A total of 104 transients were accumulated with one second relaxation delay between scans. An HMBC experiment was recorded at 500 MHz with a data set of 256 t1  512 t2 complex points with spectral widths of 1644 Hz in the 1H-domain and 14925 Hz in the 13C-domain. A total of 256 transients were accumulated with one second relaxation delay between scans. A ROESY experiment was acquired at 600 MHz with 200 ms mixing time. Data sets contained 256 t1  1024 t2 complex points with spectral widths of 3100 Hz in both domains. A total of 64 transients were accumulated with one second relaxation delay between scans. One-dimensional proton spectra were recorded using a spectral width of 3100 Hz and a dataset of 2048 complex points. A total of eight transients were accumulated for intact H37Rv LAM and 4096 transients were recorded for endo-arabinanase generated fragments thereof. Spectra were processed using VNMR software (Varian, Germany).

Jack Bean a -mannosidase (JBAM) digestion 200 mg of the eight glucose unit (gu) endo-arabinanase fragment were digested for 19 hours at 37  C with 0.28 units of JBAM in a buffer containing 100 mM sodium acetate (pH 5.0), 10 mM ZnCl2, 20 mM citrate/phosphate buffer, 250 mg/ml bovine serum albumin. The incubation was ®nished by boiling the mixture for ten minutes. Digestion products were separated on a BioGel P4 column as outlined for endo-arabinanase digestion products. NMR spectroscopy All spectra were recorded at 303 K on a Varian Unity INOVA systems at 500 MHz or 600 MHz for 1H and 125 MHz or 150 MHz for 13C. Spectra were acquired from puri®ed LAM samples or endo-arabinanase fragments at concentrations of 0.2 mg-7 mg in 99.96 % 2H2O. Sample

Mass spectrometry Q-TOF mass spectra were acquired using a Micromass Q-TOF mass spectrometer (Micromass, UK). Samples were dissolved in 50 % (v/v) methanol/water and 2 ml aliquots were infused into the mass spectrometer using the nano¯ow probe. The needle voltage was 900 V and the ion source was maintained at 30  C. Argon at 10 psi was the collision gas and the collision energy was adjusted to give approximately equal fragment ion intensities across the whole mass range of the spectrum. To this purpose, for some spectra scans were averaged that had been acquired using different collision energies. Spectra were accumulated until a satisfactory signal/ noise ration had been obtained. Spectra were acquired and processed using Micromass MassLynx software.

99

5-Methylthiopentose in Lipoarabinomannan FTICR mass spectrometry. Fourier transform ion cyclotron resonance (FTICR) mass spectra40 were obtained using a 9.4 T Bruker BioAPEX II mass spectrometer (Bruker, USA) equipped with an external electrospray ion source (Analytica, USA).41 Samples were dissolved in 50 % (v/v) methanol/water to approximately 50 pmol/ml, 2-3 ml aliquots were then infused into the mass spectrometer using a nanospray modi®cation of the electrospray ion source. All FTICR transients were recorded in broadband mode, using frequency sweep excitation, direct detection and a 512k data set. Initially spectra were acquired with a wide mass range to allow for the detection of all molecular species in the sample (Figure 8(a)). Later, the transient length was increased through reduction of the mass range in order to enhance resolution. An external calibrant (Gramicidin S) was used to calibrate all mass spectra, in addition the parent ion peak in the tandem mass spectrometry experiment was used as an internal lock mass.42 Tandem mass spectrometry was performed using a correlated sweep to isolate the parent ion and sustained off-resonance irradiation collision-induced dissociation (SORI-CID)43 to activate the ion. Brie¯y, nitrogen was pulsed into the cell (peak cell pressure ˆ 10ÿ6 mbar), after which an rf ®eld (ESORI ˆ 118 V/m) at a frequency 1000 Hz greater than the cyclotron frequency of the parent ion was applied for 0.5 second. Finally, a four seconds reaction delay was used to allow the activated ions to fragment and to allow the cell pressure to decrease to <2  10ÿ9 mbar. Data acquisition and data processing was performed using Bruker's XMASS (Version 5.0.6) software. The term ``monoisotopic'' refers to the isotopomer consisting of a single isotope of each atom, namely 12C 1 H 16O 32S. Finally, the difference between the experimental and the theoretical mass is reported as a relative error in parts per million (ppm).

Acknowledgements The authors thank Jordi Torrelles and Nannan Zhang, Colorado State University, Ft Collins for providing LAM from the M. tuberculosis strains CSU20 and H37Rv. This work was supported by the Wellcome Trust, grant no. 049856 (to S.W.H.) and in part by grant AI-37139 from the NIAID, National Institutes of Health (to D.C.).

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

References 1. Chatterjee, D. (1997). The mycobacterial cell wall: structure, biosynthesis and sites of drug action. Curr. Opin. Chem. Biol. 1, 579-588. 2. DaffeÂ, M. & Draper, P. (1998). The envelope layers of mycobacteria with reference to their pathogenicity. Advan. Microb. Phys. 39, 131-203. 3. Vercellone, A., Nigou, J. & Puzo, G. (1998). Relationships between the structure and the roles of lipoarabinomannans and related glycoconjugates in tuberculosis pathogenesis. Front. Biosci. 3, e149-e163. 4. Strohmeier, G. R. & Fenton, M. J. (1999). Roles of lipoarabinomannan in the pathogenesis of tuberculosis. Microbes Infect. 1, 709-717. 5. Weber, P. L. & Gray, G. R. (1979). Structural and immunochemical characterization of the acidic arabi-

16.

17.

18. 19.

nomannan of Mycobacterium smegmatis. Carbohydrate Res. 74, 259-278. Hunter, S. W., Gaylord, H. & Brennan, P. J. (1986). Structure and antigenicity of the phosphorylated lipopolysaccharide antigens from the leprosy and tubercle-bacilli. J. Biol. Chem. 261, 2345-2351. Khoo, K. H., Dell, A., Morris, H. R., Brennan, P. J. & Chatterjee, D. (1995). Structural de®nition of acylated phosphatidylinositol mannosides from Mycobacterium tuberculosis - de®nition of a common anchor for lipomannan and lipoarabinomannan. Glycobiology 5, 117-127. Nigou, J., Gilleron, M. & Puzo, G. (1999). Lipoarabinomannans: characterization of the multiacylated forms of the phosphatidyl-myo-inositol anchor by NMR spectroscopy. Biochem. J. 337, 453-460. Chatterjee, D., Khoo, K. H., McNeil, M. R., Dell, A., Morris, H. R. & Brennan, P. J. (1993). Structural de®nition of the nonreducing termini of mannosecapped lam from Mycobacterium tuberculosis through selective enzymatic degradation and fast-atom-bombardment mass-spectrometry. Glycobiol. 3, 497-506. Venisse, A., Riviere, M., Vercauteren, J. & Puzo, G. (1995). Structural-analysis of the mannan region of lipoarabinomannan from Mycobacterium-bovis Bcg heterogeneity in phosphorylation state. J. Biol. Chem. 270, 15012-15021. Chatterjee, D., Lowell, K., Rivoire, B., McNeil, M. R. & Brennan, P. J. (1992). Lipoarabinomannan of Mycobacterium tuberculosis - capping with mannosyl residues in some strains. J. Biol. Chem. 267, 62346239. Delmas, C., Gilleron, M., Brando, T., Vercellone, A., Gheorghiu, M., Riviere, M. & Puzo, G. (1997). Comparative structural study of the mannosylatedlipoarabinomannans from Mycobacterium bovis BCG vaccine strains: characterization and localization of succinates. Glycobiol. 7, 811-817. Sibley, L. D., Hunter, S. W., Brennan, P. J. & Krahenbuhl, J. L. (1988). Mycobacterial lipoarabinomannan inhibits g-interferon-mediated activation of macrophages. Infect. Immun. 56, 1232-1236. Barnes, P. F., Chatterjee, D., Abrams, J. S., Lu, S. H., Wang, E. & Yamamura, M. et al. (1992). Cytokine production induced by Mycobacterium tuberculosis lipoarabinomannan - relationship to chemical-structure. J. Immunol. 149, 541-547. Nigou, J., Gilleron, M., Cahuzac, B., Bounery, J. D., Herold, M., Thurnher, M. & Puzo, G. (1997). The phosphatidyl-myo-inositol anchor of the lipoarabinomannans from Mycobacterium bovis bacillus Calmette Guerin - heterogeneity, structure, and role in the regulation of cytokine secretion. J. Biol. Chem. 272, 23094-23103. Chatterjee, D., Roberts, A. D., Lowell, K., Brennan, P. J. & Orme, I. M. (1992). Structural basis of capacity of lipoarabinomannan to induce secretion of tumor necrosis factor. Infect. Immun. 60, 12491253. Khoo, K. H., Tang, J. B. & Chatterjee, D. (2001). Variation in mannose-capped terminal arabinan motifs of lipoarabinomannans from clinical isolates of Mycobacterium tuberculosis and Mycobacterium avium complex. J. Biol. Chem. 276, 3863-3871. Leopold, K. & Fischer, W. (1993). Molecular analysis of the lipoglycans of Mycobacterium tuberculosis. Anal. Biochem. 208, 57-64. Harvey, D. J. (2000). Collision-induced fragmentation of underivatized N-linked carbohydrates

100

20.

21.

22.

23. 24.

25.

26.

27.

28.

29. 30.

31.

5-Methylthiopentose in Lipoarabinomannan

ionized by electrospray. J. Mass Spectrom. 35, 11781190. Domon, B. & Costello, C. E. (1988). A systematic nomenclature for carbohydrate fragmentations in FAB-MS-MS spectra of glycoconjugates. Glycoconj. J. 5, 397-409. Jiang, X. Y., Smith, J. B. & Abraham, E. C. (1996). Identi®cation of a MS-MS fragment diagnostic for methionine sulfoxide. J. Mass Spectrom. 31, 13091310. Lagerwerf, F. M., van de Weert, M., Heerma, W. & Haverkamp, J. (1996). Identi®cation of oxidized methionine in peptides. Rapid Comm. Mass Spectrom. 10, 1905-1910. Schlenk, F., Zydek-Cwick, C. R. & Hutson, N. K. (1971). Enzymatic deamination of adenosine sulfur compounds. Arch. Biochem. Biophys. 142, 144-149. Schroeder, H. R., Barnes, C. J., Bohinski, R. C., Mumma, R. O. & Mallette, M. F. (1972). Isolation and identi®cation of 5-methylthioribose from Escherichia coli B. Biochim. Biophys. Acta, 273, 254-264. Ferro, A. J., Barrett, A. & Shapiro, S. K. (1978). 5-Methylthioribose kinase. A new enzyme involved in the formation of methionine from 5-methylthioribose. J. Biol. Chem. 253, 6021-6025. Williams-Ashman, H. G., Seidenfeld, J. & Galletti, P. (1982). Trends in the biochemical pharmacology of 50 -deoxy-50 -methylthioadenosine. Biochem. Pharmacol. 31, 277-288. Chan, J., Fan, X. D., Hunter, S. W., Brennan, P. J. & Bloom, B. R. (1991). Lipoarabinomannan, a possible virulence factor involved in persistence of Mycobacterium tuberculosis within macrophages. Infect. Immunol. 59, 1755-1761. Enouf, J., Lawrence, F., Tempete, C., Robert-Gero, M. & Lederer, E. (1979). Relationship between inhibition of protein methylase I and inhibition of Rous sarcoma virus-induced cell transformation. Cancer Res. 39, 4497-4502. Chelsky, D., Sobotka, C. & O'Neill, C. L. (1989). Lamin B methylation and assembly into the nuclear envelope. J. Biol. Chem. 264, 7637-7643. Maher, P. A. (1993). Inhibition of the tyrosine kinase activity of the ®broblast growth factor receptor by the methyltransferase inhibitor 50 -methylthioadenosine. J. Biol. Chem. 268, 4244-4249. Joy, A., Moffett, J., Neary, K., Mordechai, E., Stachowiak, E. K., Coons, S. et al. (1997). Nuclear accumulation of FGF-2 is associated with proliferation of human astrocytes and glioma cells. Oncogene, 14, 171-183.

32. Takayama, K., Schnoes, H. K., Armstrong, E. L. & Boyle, R. W. (1975). Site of inhibitory action of isoniazid in the synthesis of mycolic acids in Mycobacterium tuberculosis. J. Lipid Res. 16, 308-317. 33. Bordier, C. (1981). Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256, 1604-1607. 34. Severn, W. B., Jones, A. M., Kittelberger, R., deLisle, G. W. & Atkinson, P. H. (1997). Improved procedure for the isolation and puri®cation of lipoarabinomannan from Mycobacterium bovis strain AN5. J. Microbiol. Methods, 28, 123-130. 35. Ferguson, M. A. J. (1992). Chemical and enzymic analysis of glycosylphosphatidylinositol anchors. In Lipid Modi®cations of Proteins: a Practical Approach (Turner, A. J. & Hooper, N., eds), pp. 191-230, IRL Press, Oxford. 36. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J. & Bax, A. (1995). NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR, 6, 277-293. 37. Johnson, B. A. & Blevins, R. A. (1994). NMRView a computer program for the visualisation and analysis of NMR data. J. Biomol. NMR, 4, 603-614. 38. Shaka, A. J., Barker, P. B. & Freeman, R. (1985). Computer-optimized decoupling scheme for wideband applications and low-level operation. J. Magn. Reson. 64, 547-552. 39. Yu, L., Goldman, R., Sullivan, P., Walker, G. F. & Fesik, S. W. (1993). Heteronuclear NMR studies of 13 C-labeled yeast cell wall beta-glucan oligosaccharides. J. Biomol. NMR, 3, 429-441. 40. Comisarow, M. B. & Marshall, A. G. (1974). Fourier transform ion cyclotron resonance spectroscopy. Chem. Phys. Letters, 26, 489-490. 41. Palmblad, M., Hakansson, K., Hankansson, P., Feng, X., Cooper, H., Giannakopulos, A. E. et al. (2000). A 9.4 T Fourier transform ion cyclotron resonance mass spectrometer: description and performance. Eur. J. Mass Spectrom. 6, 267-275. 42. Burton, R. D., Matuszak, K. P., Watson, C. H. & Eyler, J. R. (1999). Exact mass measurement using a 7 Tesla Fourier transform ion cyclotron resonance mass spectrometer in a good laboratory practicesregulated environment. J. Am. Soc. Mass Spectrom. 10, 1291-1297. 43. Gauthier, J. W., Trautman, T. R. & Jacobsen, D. B. (1991). Sustained off-resonance irradiation for collision-activated dissociation involving Fourier transform mass spectrometry. Collision-activated dissociation technique that emulates infrared multiphoton dissociation. Anal. Chim. Acta, 246, 211-225.

Edited by J. Karn (Received 1 October 2001; received in revised form 3 December 2001; accepted 6 December 2001)