©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inositol Phosphate Capping of the Nonreducing Termini of Lipoarabinomannan from Rapidly Growing Strains of Mycobacterium(*)

Kay-Hooi Khoo (1), Anne Dell (2), Howard R. Morris (2), Patrick J. Brennan (1), Delphi Chatterjee (1)(§)

From the (1) Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523 and (2) Department of Biochemistry, Imperial College, London SW7 2AZ, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies have demonstrated that the nonreducing termini of the lipoarabinomannan (LAM) from Mycobacterium tuberculosis are extensively capped with mannose residues, whereas those from a fast growing Mycobacterium sp., once thought to be an attenuated strain of M. tuberculosis, are not. The noncapped LAM, termed AraLAM, is known to be more potent than the mannose-capped LAM (ManLAM) in inducing functions associated with macrophage activation. Using a combination of chemical and enzymatic approaches coupled with fast atom bombardment-mass spectrometry analysis, we demonstrated that LAMs from all M. tuberculosis strains examined (Erdman, H37Ra, and H37Rv), as well as the attenuated Mycobacterium bovis BCG strain, are mannose-capped with the extent of capping varying between 40 and 70%. The nonreducing termini of LAM from Mycobacterium leprae were also found to be capped with mannoses but at a significantly lower level. A novel inositol phosphate capping motif was identified on a minor portion of the otherwise uncapped arabinan termini of LAMs from the fast growing Mycobacterium sp. and Mycobacterium smegmatis ATCC 14468 and mc155. In addition, an inositol phosphate tetra-arabinoside was isolated from among endoarabinase digestion products of AraLAM and was shown to induce tumor necrosis factor- production. Accordingly, we concluded that AraLAM is characteristic of some rapidly growing Mycobacterium spp. It is distinct from ManLAMs of M. tuberculosis, M. bovis BCG, and Mycobacterium leprae not only in the absence of mannose-capping but also in containing some terminal inositol phosphate substituents which may account for its particular potency in inducing macrophage activation.


INTRODUCTION

Lipoarabinomannan (LAM)() is a major component of the mycobacterial cell envelope widely implicated in the immunopathogenesis of leprosy and tuberculosis (Brennan et al., 1990). Both LAM and the structurally related lipomannan (LM) from Mycobacterium tuberculosis and Mycobacterium leprae contain an intact phosphatidylinositol unit (Hunter et al., 1986) and a branched mannan core, [6(Man12)Man1], directly attached to position 6 of the inositol of phosphatidylinositol (Hunter and Brennan, 1990; Chatterjee et al., 1991, 1992a, 1992b). The salient feature which distinguishes LAM from LM is the presence of an additional immunodominant arabinan that extends from the mannan core in an undefined manner.

Initial structural studies demonstrated that the nonreducing termini of the arabinan of LAM from the virulent M. tuberculosis Erdman strain, whether branched or linear, were capped extensively (>70%) with Man residues (termed ManLAM) (Chatterjee et al., 1992b) (Fig. S1). In contrast, those from an ``avirulent'' fast growing strain, which was believed at the time to be a rapidly growing M. tuberculosis H37Ra variant, were uncapped (termed AraLAM) (Chatterjee et al., 1991). It was then thought that these terminal Man residues might be responsible for the differences in the capacity of the two LAMs to induce functions associated with macrophage activation and thus be related to virulence (Chatterjee et al., 1992c; Roach et al., 1993, 1994). However, the implication of the mannose caps as virulence factors was not supported by subsequent observation that LAMs from both virulent M. tuberculosis H37Rv and avirulent Mycobacterium bovis BCG strains were mannose-capped (Prinzis et al., 1993; Venisse et al., 1993). Furthermore, doubts were raised concerning the identity of the highly passaged, fast growing strain from which AraLAM was first isolated with the observation that it clearly was not an authentic strain of M. tuberculosis H37Ra (Prinzis et al., 1993).


Figure S1: Scheme 1



Awareness of the subtle structural differences among LAMs produced by various Mycobacterium sp. and their varying capacity to modulate host immune responses prompted us to definitively map the recognized structural motifs of LAMs from several biologically important strains. We report here our FAB-MS and chemical analysis of the nonreducing termini of a variety of LAMs, showing conclusively the presence of a novel inositol phosphate capping function associated specifically with the biologically potent AraLAM. We also confirm that mannose capping is a common feature of LAMs from all strains of M. tuberculosis including the H37Ra strain and therefore may not be directly correlated with virulence.


EXPERIMENTAL PROCEDURES

Isolation and Purification of LAMs and LM, Endoarabinase Digestion, and Subsequent Purification

Growth of mycobacteria and the isolation and purification of LAMs from the rapidly growing Mycobacterium sp., M. tuberculosis (Erdman and H37Rv), Mycobacterium leprae and Mycobacterium smegmatis (ATCC 14468 and mc155) were performed as described (Chatterjee et al., 1991, 1992b). The M. smegmatis mc155 strain is an efficient plasmid transformation mutant and was cultured as described (Snapper et al., 1990) for use in electroporation. LAM from H37Ra was similarly isolated and purified from a culture given by Dr. William R. Jacobs (Albert Einstein College of Medicine of Yeshiva University, New York City). The selective growth of a soil microorganism, Cellulomonas sp., on an arabinogalactan-containing medium and the isolation and characterization of an extracellular endoarabinase have been described (McNeil et al., 1994). Deacylated AraLAM (5 mg) was incubated with the Cellulomonas sp. enzymes (500 µg in 1 ml), and aliquots were withdrawn after every 4 h for reducing sugar assay (Lever, 1972). The reaction was terminated by boiling for 2 min when the reducing sugar formation reached a plateau after 12 h. The entire digestion mixture was applied to a column of Bio-Gel P-6 (Bio-Rad) in water. Fractions (1 ml) were assayed for carbohydrate (Chatterjee et al., 1993) and pooled accordingly.

Affinity Chromatography with Concanavalin A

The voided peak, containing both Ino-P-Ara and the mannan core of AraLAM, was reconstituted in a buffer (20 mM Tris and 0.5 M NaCl, pH 7.4), applied to a column (1 ml) of concanavalin A-Sepharose (Pharmacia Biotech Inc.), and eluted with 2.5 ml of the starting buffer to obtain the unbound Ino-P-Ara. It was further eluted with 2.5 ml of the starting buffer containing 0.5 M -methylmannoside in order to recover the mannan core. The combined fractions were immediately applied to a Sephadex G-10 column (1 117 cm, Pharmacia) and eluted with water, and fractions prior to elution of salt were pooled. A portion of the pooled fractions was converted to alditol acetates and analyzed by GC to ensure the presence of arabinose or mannose.

Perdeuteroacetylation, Mild Trifluoroacetic Acid Hydrolysis, and Deuteroacetolysis

Perdeuteroacetylation of intact and partially degraded LM and LAM samples was performed in pyridine:d-acetic anhydride (1:1, v:v) at 80 °C for 2 h. LAM samples were subjected to mild acid hydrolysis with 40 mM aqueous trifluoroacetic acid (Rathburn, UK; gas phase sequencer grade) for 20 min at 100 °C after which the reagent was directly removed under a stream of nitrogen. For deuteroacetolysis, perdeuteroacetylated samples were treated with a d-acetic anhydride:d-acetic acid:d-sulfuric acid (10:10:1, v:v:v) mixture for 30-40 min at 40 °C. After quenching with water, the reaction mixture was neutralized with a few drops of 30% aqueous ammonia, loaded directly onto a reverse phase C18 Sep-Pak cartridge (Waters, Milford, MA), and eluted sequentially with 2 ml of 50 and 100% aqueous acetonitrile (Rathburn, high performance liquid chromatography grade) after initial washes with water and 15% acetonitrile. The collected fractions were evaporated to dryness and redissolved in methanol for FAB-MS analysis. Similar Sep-Pak conditions were employed for further purification of all peracetylated samples.

Diazomethane was generated from 1-methyl-3-nitro-1-nitrosoguanidine (Aldrich) in a 13 100-mm screw capped tube as opposed to the Mini Diazald apparatus. To the acetylated or acetolyzed samples, diazomethane in ether was added directly until the yellow color persisted. The reaction mixture was left at 0 °C for 30 min and allowed to dry at room temperature in the hood.

Permethylation, Glycosyl Composition, and Linkage Analysis

Arabinan samples were permethylated for FAB-MS analysis and linkage analysis by using powdered sodium hydroxide as described by Dell et al.(1994). For analysis of inositol, samples were hydrolyzed in 3 N HCl for 6 h at 100 °C. After evaporation under vacuum, the hydrolysates were derivatized with the Tri-Sil® reagent (Pierce) for about 30 min at room temperature and then extracted into hexane. Partially methylated alditol acetates for linkage analysis were prepared according to Albersheim et al.(1967). GC-MS analysis was carried out on a Fisons instrument MD800 fitted with a DB-5 capillary column (J& Scientific, Folsom, CA). Samples were dissolved in hexane and injected on-column at 90 °C. After an initial hold at 90 °C for 1 min, the oven temperature was increased to 290 °C at a rate of 8 °C/min for linkage analysis. For inositol analysis, the oven temperature was increased to 140 °C at 25 °C/min and then to 200 °C at 5 °C/min, and finally to 300 °C at 10 °C/min.

FAB-MS Analysis

FAB mass spectra were acquired using a VG Analytical ZAB-2SE FPD mass spectrometer fitted with a cesium ion gun operated at 20-25 kV. Data acquisition and processing were performed using the VG Analytical Opus® software. Monothioglycerol (Sigma) was used as matrix in both positive and negative ion modes. CAD MS-MS collisionally activated decomposition spectra were recorded using a Fisons VG analytical four sector ZAB-T mass spectrometer in the array detector mode. The array was calibrated using daughters of CsI clusters, and all experiments were carried out at 4-kV collision cell voltage (8-kV primary ion beam) using an argon gas pressure set to reduce the C (M + H) of a Substance P standard to two thirds of the normal peak height prior to gas introduction. Spectra were recorded by FAB ionization of samples in a monothioglycerol matrix (cesium ion gun) and transmission of the C parent ion of interest from MS1 into the collision cell. Array daughter spectra produced from the B/E scan of MS2 were summed over the period of ionization of the sample.

P NMR

The voided peak was deuterium exchanged and dissolved in 600 µl of [H]O. For P NMR under basic conditions, a drop of N[H]O[H] was added. One-dimensional NMR spectra were recorded on a Bruker Ace-300 spectrometer (McNeil et al., 1990). HPO (90%) was used as the external standard.


RESULTS

Strategy for Deuteroacetolysis/FAB-MS Analysis of LAMs

The selective susceptibility of the 16-mannosyl linkages of the LAM core to acetolysis provided a basis for applying time-course acetolysis/FAB-MS analysis to map the structural motifs of various LAMs (Tsai et al., 1986). In exploratory experiments, it was established that the mannan and the arabinan were extensively degraded to Man, Ara, and Ara in a 3-h acetolysis while the phosphatidylinositol unit was largely preserved. To optimize the yield of putative capped and/or modified oligosaccharides, the acetolysis reactions were monitored at intervals by FAB-MS analysis of sample aliquots after partial separation of the acetolyzed products on a reverse phase Sep-Pak cartridge using a two-step gradient of 50 and 100% acetonitrile. It was anticipated that any resulting charged species would be eluted in the earlier fraction together with mono- and disaccharides while larger neutral oligosaccharides would be recovered in the later fraction. Deuteroacetolysis on perdeuteroacetylated samples was performed throughout to avoid ambiguity in subsequent FAB-MS signal assignments. Normal acetolysis under identical experimental conditions was also carried out for selected samples to confirm assignments by observing mass shifts.

As described previously (Tsai et al., 1986), each glycan fragment produced by acetolysis can exist in several different forms depending on the extent of ring opening (Rosenfeld and Ballou, 1974). Thus, in the first 30-40 min, deuteroacetolyzed Man (where x = 1-8)() were observed primarily as (M + NH). After more prolonged reaction, additional molecular ion species were observed which can be assigned to the ring open structure (I), hereafter denoted as (+dAcO). Since the ring open structure can be formed at each noncleaved glycosidic linkage (II) in addition to the reducing end, more than one dAcO moiety may be contained in the molecular ions as well as in the A-type ions, resulting in multiples of 108-unit increments (Fig. S2). This phenomenon was most pronounced with the Ara containing fragments (where n = 1-6) to the extent that, even at the early time point, they were observed almost exclusively as dAcO adducts, where the total number of dAcO moieties incorporated was usually n - 1. As a consequence, the mass increment for the deuteroacetolyzed Ara series is 330 units (Ara + dAcO) and not 222 units (Ara).


Figure S2: Scheme 2



Nonreducing Terminal Mannose Cap Motifs

To define the structural difference among LAMs from a variety of sources, deuteroacetolysis/FAB-MS analysis was performed on LAMs from M. tuberculosis H37Ra, H37Rv, and Erdman strains, M. leprae (abbreviated as RaLAM, RvLAM, ErdLAM, and LepLAM, respectively, for ease of description), as well as AraLAM (Chatterjee et al., 1991). In addition, LM from M. tuberculosis Erdman (ErdLM) was also analyzed in parallel. The positive FAB-mass spectra of the 100% acetonitrile Sep-Pak fractions from 40-min deuteroacetolysates of ErdLM, AraLAM, ErdLAM, RvLAM, and LepLAM in the mass range of m/z 1190-1700 are reproduced in Fig. 1and the assignments of the major ions are listed in . The FAB-MS data showed that while Ara and Man are common motifs of all LAMs, ions corresponding to ManAra (where x` is 2 or 3, referring to the number of Man residues which constitute the individual mannose cap) were observed as dominant species only in the deuteroacetolysates of LAMs from the M. tuberculosis strains. Thus LAMs from Erdman, H37Rv, and H37Ra strains are all capped extensively with Man and Man, giving ManAra and ManAra, whereas AraLAM is not capped. This observation reinforced the evidence (Prinzis et al., 1993) that the rapidly growing strain from which AraLAM was derived (Chatterjee et al., 1991) is not bona fide M. tuberculosis H37Ra. This strain does have many of the characteristics of M. smegmatis including its distinct mycolic acids, although it could not be categorically identified as such. Mannose-capping is, however, not a phenomenon restricted to LAMs from M. tuberculosis. In addition to LAM from M. bovis BCG, which was reported to be mannose-capped (Prinzis et al., 1993; Venisse et al., 1993), our FAB data (Fig. 1E) indicated that LepLAM is also mannose-capped but apparently less so. Ions ascribed to ManAra (e.g. m/z 1272 for ManAra + dAcO) were evidently present but in minor abundance relative to other Man and Ara ions.


Figure 1: Positive FAB-mass spectra of the 100% acetonitrile Sep-Pak fraction of the deuteroacetolysates of ErdLM (A), AraLAM (B), ErdLAM (C), RvLAM (D), and LepLAM (E), in the mass range of m/z 1180-1700. FAB-mass spectrum of the deuteroacetolysates of RaLAM is identical to those of ErdLAM and RvLAM. Assignments of the major ions were listed in Table I. At lower masses, A-type oxonium ions for Ara, Ara, Ara, and Man, Man, Man, and molecular ions for Ara, Ara, and Man, Man were afforded by all LAM samples while those of ManAra were present in all LAMs except AraLAM. Above m/z 1700, the Man series extended to Man and Man, with a much weaker signal for Man.



These data were further corroborated by methylation analysis of RvLAM, RaLAM, and LepLAM (). In contrast to AraLAM, which yielded almost equal amounts of terminal Araf and 2-linked Araf but no 2-linked Manp, both RvLAM and RaLAM gave terminal Araf and 2-linked Araf in about a 1:2 ratio, as well as substantial amounts of 2-linked Manp. Since each 2-linked Araf should be substituted with a terminal Araf unless it is further capped with Manp1(2-Manp1), where x in this case is 0-3, the methylation analysis data therefore suggested that approximately half of the nonreducing terminal Araf of both RaLAM and RvLAM are capped with Man residues. The approximate 1:1 ratio of terminal Araf:2-linked Araf of LepLAM indicated that most of its Araf termini are not capped. However, the presence of some degree of mannose capping in LepLAM was evident from both the FAB-MS data and the presence of significant amounts of 2-linked Man. Assuming that the dominant cap is Manp12Manp and that all 2-linked Manp are constituents of the cap, the degree of mannose capping in LepLAM was estimated to be not more than 30%, considerably lower than RvLAM, RaLAM, and ErdLAM (70%).

Presence of Additional Structural Motifs

The deuteroacetolysis/FAB-MS data also attested to the presence of a common branched mannan core [6(Man12)Man1], the 16 bonds of which were acetolyzed much more readily than the 12 linkages. Thus, signals corresponding to Man, Man, Man, and Man were much stronger relative to those corresponding to Man, Man, and Man in all LAMs examined. LM possessed only the Man series (x = 1-8). In the 50% acetonitrile Sep-Pak fraction, a trace amount of Ara was present in the FAB-mass spectrum of deuteroacetolyzed ErdLM, the origin of which is not clear. Otherwise, the only other prominent signals present were the molecular ions and A-type fragment ions of Man and Man. In contrast, the FAB-mass spectra of the deuteroacetolysates of all LAMs were dominated by Ara and Ara in addition to Man and Man. The positive FAB-mass spectra of the 50% acetonitrile Sep-Pak fractions of the deuteroacetolysates of ErdLM, AraLAM, and ErdLAM in the mass range of m/z 650 to 1180 (Fig. 2) contained ions at m/z 720 and 725, corresponding, respectively, to (M + NH) and (M + Na) of Man. Their under-deuteroacetylated counterparts afforded the signals at m/z 675 and 680. In addition, Ara was observed as (M + dAcO + NH) and (M + dAcO + Na) at m/z 678 and 683, respectively.


Figure 2: Positive FAB-mass spectra of the 50% acetonitrile Sep-Pak fraction of the deuteroacetolysates of ErdLM (A), AraLAM (B), and ErdLAM (C), in the mass range of m/z 650 to 1180. All signals at lower masses were attributed to Ara, Ara, Man, and Man. The signal at m/z 690 in B corresponds to the oxonium ion of Ino-P-Ara. Signals at m/z 977 and 902 in C correspond to (M + Na) of under-deuteroacetylated Man and ManAra, respectively. Other signals at 45 units lower than those assigned correspond to under-deuteroacetylated species.



Evidence for a Novel Ino-P-Ara Motif in AraLAM

Strikingly, the FAB-mass spectrum of the deuteroacetolysates of AraLAM (Fig. 2B) was dominated by two signals not present in the corresponding spectra of all other LAM/LM samples analyzed. The mass difference of 330 units (222 + 108) between the signals at m/z 775 and m/z 1105 was indicative of an X-Ara series with the latter containing an additional Ara (222 units) residue and a dAcO adduct (108 units). In the negative ion mode, the same sample afforded two correspondingly prominent negative ion signals at m/z 751 and 1081, which were not found in the spectra of any other LAM. It thus appeared that a moiety of X-Ara yielded strong (M - H) ions in the negative ion mode while desorbing primarily as (M + Na) and (M - H + 2Na) in the positive ion mode (Fig. 2B). When the deuteroacetolyzed AraLAM was treated with diazomethane, the relevant positive ions were shifted 14 units higher while the negative ions were abolished, suggesting that the negative charge could be due to the presence of a phosphodiester which was methylated by diazomethane and hence lost its charge. Taken together, these data were consistent with Ino-P-Ara and [Ino-P-Ara (+dAcO)] as the two components unique to the deuteroacetolysates of AraLAM.

LAMs from different strains were subjected to mild trifluoroacetic acid hydrolysis under conditions which were expected to specifically hydrolyze furanosyl or glycosyl phosphate bonds (McConville and Blackwell, 1991). After perdeuteroacetylation, the partial hydrolysates were analyzed by FAB-MS in both positive and negative ion modes. For all mannose-capped LAMs, positive ions corresponding to ManAra and Ara but not Man were observed, indicating that only the arabinofuranosyl linkages were partially hydrolyzed. Specifically, RaLAM afforded major (M + Na) molecular ion signals at m/z 947, 1169, 1391, 1613 (ManAra, ManAra, ManAra, ManAra, respectively) and m/z 1244, 1466, 1688 (ManAra, ManAra, ManAra, respectively), in addition to m/z 353, 575, 797, 1019 (Ara, Ara, Ara, Ara, respectively), whereas AraLAM gave only the Ara series (data not shown).

In the negative ion mode, the partial hydrolysates of AraLAM gave prominent negative ion signals which were absent in the negative ion spectra of all other LAMs. After further separation of the hydrolysis fragments from the nonhydrolyzed phosphatidylinositol-mannan core, the negatively charged components from AraLAM afforded the negative FAB-mass spectrum shown in Fig. 3 . The major molecular ion at m/z 751 corresponded to (M - H) of Ino-P-Ara. At 222 units higher (m/z 973) was the (M - H) of Ino-P-Ara which, as expected, lacked the extra dAcO moiety found in the deuteroacetolyzed sample whose corresponding ion occurred at m/z 1081. In addition, Ino-P-Ara were also produced, affording a series of ions with mass intervals of 222 units. Upon complete hydrolysis of this sample, inositol was identified as its trimethylsilyl derivative in subsequent GC-electron impact-MS analysis (characteristic ions at m/z 318, 305, 217, 147, 73) where it eluted at a retention time identical to the trimethylsilyl derivative of an authentic myo-inositol standard. Information on the attachment point of the phosphodiester linkage was obtained by negative ion CAD MS-MS of the molecular ion for Ino-P-Ara at m/z 751 (Fig. 3, inset). A prominent fragment ion at m/z 627 was obtained, consistent with loss of deuteroacetyl substituents from positions 1 and 2 of the reducing end Ara via an established fragmentation pathway (Dell et al., 1990). Analogous fragment ions were observed in the CAD spectra of the Ara oligomers (see Fig. 4), suggesting that the phosphodiester moiety is not attached at position 2.


Figure 3: The negative FAB-mass spectrum of perdeuteroacetylated mild trifluoroacetic acid hydrolysates of AraLAM after Sep-Pak purification. The negatively charged Ino-P-Ara were eluted with 50% aqueous acetonitrile while the nonhydrolyzed mannan core was retained on the C18 cartridge. Signals observed correspond to (M - H) of Ino-P-Ara through to Ino-P-Ara, each accompanied by under-deuteroacetylated species. Inset, negative ion CAD MS-MS spectrum of the molecular ion at m/z 751. Signals at m/z 706 and 688 correspond to loss of a trideuteroacetyl and a trideuteroacetic acid moiety respectively. Other signals assigned to fragment ions resulting from collisional activation of m/z 751 are: HPO (m/z 97); (P-Ara) (m/z 364); (Ino-P) (m/z 484); and the ion at m/z 627 described in the text.




Figure 4: Positive ion CAD mass spectra of Ara after collisional activation of the (M + Na) molecular ion at m/z 1019 (A) and Ara after collisional activation of the (M + Na) molecular ion at m/z 1463 (B). Sequence and/or linkage informative fragment ions were assigned as shown schematically on the figures. Other secondary fragment ions were derived from loss of a trideuteroacetic acid moiety (minus 63 units) or multiple cleavages.



Localizing the Ino-P-Ara Motifs

To further define the location of the Ino-P-Ara structural motif and to ascertain whether it carries any other additional acid-labile moiety which might have been hydrolyzed or deuteroacetolyzed, deacylated AraLAM was digested with the Cellulomonas endoarabinase. This enzyme was previously shown to be highly selective for the 5-linked Araf residues which follow a branched 3,5-linked Araf unit and the fourth Araf residue from the nonreducing end in the case of the linear terminal motif (Chatterjee et al., 1993; McNeil et al., 1994). The endoarabinase-digested AraLAM was fractionated on a Bio-Gel P-6 column, pooled into void and fractions A, B, and C, and screened by FAB-MS after perdeuteroacetylation and permethylation (I). The dominant components in fractions A, B, and C were Ara, Ara, and Ara, respectively. In agreement with the known enzyme specificity, the Ara and Ara digestion products were shown to be branched and linear, respectively, by methylation analysis (). This was further confirmed by tandem mass spectrometric analysis. The CAD spectrum of Ara [m/z 1019 for (M + Na)] afforded sodiated reducing terminal ring cleavage ions at m/z 336, 558, and 780, corresponding to Ara, Ara, and Ara, respectively (Fig. 4A). More importantly, glycosidic cleavage concomitant with elimination of the 2-O-substituent yielded the nonreducing terminal cleavage ions at m/z 895 (Ara) and 673 (Ara) but not 451 (Ara), thus confirming the known Ara12Ara linkage at the nonreducing end of the linear Ara chain.

CAD on Ara at m/z 1463 afforded analogous fragment ions (Fig. 4B). Significantly, sodiated ring cleavage ions were observed only for Ara, Ara, and Ara at m/z 336, 1002, and 1224, respectively, and not for Ara or Ara. In addition, strong nonreducing terminal ions resulting from glycosidic cleavage concomitant with elimination of the 2-O-substituent were observed only for Ara (at m/z 1117) and Ara (at m/z 1339). The CAD spectrum afforded by Ara therefore firmly demonstrated that the major Ara component produced by endoarabinase digestion corresponded to the previously characterized branched structure.

The Ino-P-Ara motifs coeluted with the mannan core in the voided peak, presumably due to the negative charge they carried. The presence of two different families of phosphodiester-containing oligomers in the voided peak was further supported by P NMR experiments. In contrast to the spectrum afforded by the mannan core similarly isolated from ErdLAM, which contained only a single phosphodiester signal at 0.68 at neutral pH, the NMR spectrum afforded by the void fraction from AraLAM showed two distinct signals of phosphodiester at 0.71 and 0.45 ppm. At basic pH, the chemical shifts of the phosphodiester remained unchanged, indicating that both the phosphates were in the form of phosphodiester, one belonging to the Man-Ino-P-glycerol from the mannan core ( 0.71) and the other attributable to Ino-P-Ara.

In the negative FAB-mass spectrum of the perdeuteroacetyl derivatives, (M - H) molecular ion signals were present at m/z 1417, 1639, 1862, 2084, and 2306, corresponding to Ino-P-Ara, Ino-P-Ara, Ino-P-Ara, Ino-P-Ara, and Ino-P-Ara, respectively (Fig. 5). Interestingly, although the larger Ino-P-Ara were also produced, these were clearly very minor components relative to the predominant Ino-P-Ara. In the positive ion mode, the Ino-P-Ara series afforded both (M + NH) and (M + Na) molecular ions (data not shown). Significantly, despite the intense molecular ion signals of Ino-P-Ara, A-type oxonium ions of Ara and Ara were not prominent among the matrix noise. Instead, a strong ion was present at m/z 690, corresponding to the A-type ion Ino-P-Ara. Following diazomethane treatment, this ion shifted to m/z 704. Similarly, all molecular ions were shifted 14 units higher, consistent with monomethylation of a phosphodiester moiety. In addition, the permethyl derivatives of Ino-P-Ara afforded a major (M + NH) molecular ion at m/z 1016 and a strong A-type ion at m/z 487, corresponding to Ino-P-Ara and Ino-P-Ara, respectively, with all hydroxyl groups including that of the phosphodiester moiety being fully methylated. Taken together, the data were fully consistent with a novel inositol phosphate moiety capping a portion of the nonreducing termini of the linear arabinan chain which, upon digestion with endoarabinase, yielded predominantly Ino-P-Ara.


Figure 5: The negative FAB-mass spectrum of perdeuteroacetylated Ino-P-Ara recovered from the endoarabinase digestion products of AraLAM. Signals at 45 units lower than those assigned as (M - H) of Ino-P-Ara correspond to under-deuteroacetylated species. Inset, schematic representation of the provisional structure for Ino-P-Ara which afforded the ion at m/z 1417. The glycosyl linkage positions for the arabinan were not defined. Linkage analysis of the purified Ino-P-Ara sample gave only 2-linked and 5-linked Araf. The absence of terminal Ara is consistent with the Ino-P being attached to the nonreducing arabinose. The attachment point for the phosphate is likely to be position 5 in view of the CAD MS/MS experiment which indicated that deuteroacetylated Ino-P-Ara has a deuteroacetyl substituent at position 2. However, attachment to position 3 cannot be ruled out.



LAMs from M. smegmatis

The identification of the Ino-P-Ara motif on AraLAM from an unknown, rapidly growing Mycobacterium sp. prompted us to examine LAMs from the speciated, fast growing M. smegmatis (ATCC 14468 and mc155). Methylation analysis indicated that they are not mannose-capped (). After deuteroacetolysis, the same positive and negative ions assigned to Ino-P-Ara (m/z 775 and 751, respectively) and [Ino-P-Ara (+dAcO)] (m/z 1105 and 1081, respectively) were produced. Mild trifluoroacetic acid hydrolysis followed by perdeuteroacetylation and C18 Sep-pak separation yielded the same series of negative ions attributed to Ino-P-Ara. Finally, endoarabinase digestion of LAM from the mc155 strain followed by Bio-Gel P-6 fractionation, perdeuteroacetylation, and FAB-MS screening afforded the same m/z 1417 ion ((M - H) of Ino-P-Ara) as the dominant negative ion in the void fraction. Although rigorous biochemical characterization was not performed, the FAB-MS data strongly indicated that the same inositol phosphate capping motif as that characterized for AraLAM is also present on LAM from M. smegmatis.

Biological Activity of Ino-P-Ara from AraLAM

To obtain the Ino-P-Ara components produced by endoarabinase digestion of AraLAM for biological studies, the voided peak from Bio-Gel P-6 column was passed through a concanavalin A-Sepharose affinity column to remove the mannan core. The potency of Ino-P-Ara in eliciting TNF- production in murine bone marrow-derived macrophages was compared to that of intact AraLAM and lipopolysaccharide. The assay involved reverse transcription of stimulus-induced mRNA for TNF-, followed by PCR amplification of its specific sequences (Wynn et al., 1993). It was found that the potency of AraLAM at 1 µg was 30% lower than that of an equal amount of lipopolysaccharide, whereas that of Ino-P-Ara was 20% higher (data not shown). The inositol phosphate-capped Ara termini in AraLAM may thus be responsible for its known potency in elevated level of TNF- induction (Chatterjee et al., 1992c).


DISCUSSION

As a complement to our earlier work on LAM involving detailed chemical analysis of small fragments derived from partial hydrolysis of per-O-alkylated LAMs (Chatterjee et al., 1991, 1992b; Prinzis et al., 1993), alternative ways of characterizing the nonreducing termini of LAM isolated from various strains of Mycobacterium have now been developed based on FAB-MS analysis of endoarabinase digestion and acetolysis products.

Peracetylation of a variety of glycans under basic conditions retains most of the common non-glycosyl substituents, notably acyl, phosphate, and sulfate (Dell et al., 1994). Direct FAB-MS analysis of derivatized sample prior to further purification allows rapid determination of the exact molecular weight and hence the composition of each component present in a mixture without prior knowledge of its chemical nature. Despite being more complicated in spectra interpretation due to the +dAcO ring open forms, FAB-MS analysis of the acetolysates or deuteroacetolysates of LAMs has the advantage of simultaneously defining both nonreducing termini of the differentially capped arabinan and the mannan core. In a time course experiment, an early time point can be chosen to optimize the recovery of Ara-containing fragments and limit the acetolysis of the mannan core specifically to Man16Man linkages while more extended acetolysis affords fragments of the mannosyl phosphatidylinositol anchor (Khoo et al., 1995). Any additional acyl or glycopyranosyl substituent(s) on the arabinan and mannan core will be uncovered by this approach. Indeed, we have preliminary evidence for the presence of additional negatively charged substituents on the core, the identity of which is currently under investigation.

In this study, we reported the identification of a unique inositol phosphate capping motif apparently confined to LAMs from rapidly growing mycobacteria which are devoid of the mannose cap commonly present on LAMs from M. tuberculosis. Our results suggested that the inositol phosphate cap is probably restricted to a portion of the linear arabinan termini while the unsubstituted branched arabinan termini, which are probably the immunodominant epitopes, constitute the bulk of AraLAM. It should be emphasized that inositol phosphate caps only a minor portion of the arabinan termini, the majority of which are exposed as evident from the terminal Ara:2-Ara ratio of 7:9 in our methylation analysis of AraLAM. In retrospect, this novel entity may actually correspond to the ``base-labile inositol phosphate'' described previously by Hunter et al.(1990). The low abundance of this motif also presents considerable challenge to its complete characterization. Preliminary linkage and CAD MS/MS data suggested that the inositol phosphate is linked to position 5 of the terminal -Araf residue, replacing the Man or Man unit that typifies ManLAM. Incidentally, this is also the point of attachment of the mycolyl substituent in the arabinogalactan-peptidoglycan complex of mycobacterial cell walls (McNeil et al., 1991).

It is now clear that AraLAM from the dubious, rapidly growing Mycobacterium sp. is distinctively different from LAM from a genuine M. tuberculosis H37Ra strain which is of the ManLAM type. In addition, LAM from well known strains of the rapidly growing M. smegmatis was shown to be similar to AraLAM and that the same Ino-P-Ara motif was identified in its acid hydrolysates, deuteroacetolysates, and endoarabinase digestion products. Although the rapidly growing strain which produces AraLAM was originally believed to be an aberrant M. tuberculosis H37Ra, it now seems possible that it is M. smegmatis. Alternatively, it may have acquired these characteristics of M. smegmatis or rapidly growing species during subculturing through preferential growth of a contaminating rapid growing species. A mix-up in cultures may also have happened.

The earlier studies on the greater potency of AraLAM over ManLAM in inducing a macrophage TNF- response (Chatterjee et al., 1992c) were recently extended to examination of the expression of early response genes in murine bone marrow-derived macrophages (Roach et al., 1993). Both AraLAM and ManLAM elicited immediate early gene (c-fos, JE, KC) responses. However, only AraLAM, like lipopolysaccharide, induced both TNF- secretion and a potentially lethal TNF-dependent-NO response (Roach et al., 1995). From these studies, it appeared that LAMs from strains of M. tuberculosis, through mannose-capping, are designed to avoid stimulation of macrophage early gene expression which will lead to the induction of a potentially lethal TNF-dependent NO response. However, the present finding that LAM from avirulent M. tuberculosis H37Ra as well as that from the vaccine strain M. bovis BCG (Prinzis et al., 1993; Venisse et al., 1993) are both mannose-capped indicates that any putative role for LAM in the variable virulence of the strains of the M. tuberculosis complex is related to factor(s) other than mannose capping. It has recently been shown that mannose-capping on LAM may be involved in the uptake of mycobacteria by direct binding to the mannose receptors of macrophages (Schlesinger, 1993; Schlesinger et al., 1994). Since all M. tuberculosis strains produce mannose-capped LAM, it is possible that the mannose caps are essential for their initial binding to macrophages. Virulence and pathogenesis of strains would then be dependent on events subsequent to this binding.

The structural studies reported in this work provide a foundation for future research directed toward probing the function of inositol phosphate capping of the terminal Araf residues. For instance, it will be interesting to further determine whether this particular modification of LAM contributes toward induction of different cytokines other than TNF, to ascertain if it is responsible for inducing early gene responses, and to determine whether it can elicit a second messenger within target cells. On the other hand, the dubious origin of the strain from which AraLAM was derived and the presence of an analogous entity in M. smegmatis do impose searching questions pertaining to the significance of AraLAM as a valid experimental probe in analyzing the basis of the pathogenesis of tuberculosis.

  
Table: Positive ions observed in the FAB-mass spectra of the 100% acetonitrile Sep-Pak fraction of the deuteroacetolysates of LAMs in the mass range of m/z 1180-1700 (Fig. 1)

The m/z values tabulated are based on C-monoisotopic nominal masses calculated while those labeled in Fig. 1 are computer-assigned accurate masses, rounded out to the nearest integer.


  
Table: 0p4in From Chatterjee et al. (1991).(119)

  
Table: Major components identified by FAB-MS screening of perdeuteroacetylated samples from the pooled Bio-Gel P-6 fractions of endoarabinase digested AraLAM


  
Table: Methylation analysis of the pooled Bio-Gel P-6 fractions of endoarabinase digested AraLAM



FOOTNOTES

*
This work was supported by National Institutes of Health NIAID Grant AI 18357 (to P. J. B.) and Contract AI-05074 (to Colorado State University), a Medical Research Council Programme Grant, a Wellcome Trust Grant 030826 (to H. R. M. and A. D.), Wellcome Trust Prize Fellowship (036485/Z/92/Z), and Heiser Research in Leprosy and Tuberculosis Fellowship (to K.-H. K.). The CAD MS-MS work was supported by the EC Large Scale Installation Plan (EC/CNR Contract ERBGEI CT920045 REP457) located in CNR-Napoli (Italy). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 1-970-491-7495; Fax: 1-970-491-1815; E-mail: delphi@lamar.colostate.edu.

The abbreviations used are: LAM, lipoarabinomannan; Araf, arabinofuranose; Ara, Ara, Ara, etc., a single arabinose, an arabinobiose, an arabinotriose, etc.; Manp, mannopyranose; Man, Man, Man, etc., a single mannose, a mannobiose, a mannotriose, etc.; LM, lipomannan; AraLAM, LAM from the rapidly growing Mycobacterium sp.; ErdLM and ErdLAM, LM and LAM from M. tuberculosis Erdman; RvLAM and RaLAM, LAM from M. tuberculosis H37Rv and H37Ra; CAD, collision-activated dissociation; FAB, fast atom bombardment; GC, gas chromatography; Ino-P, inositol phosphate; MS, mass spectrometry; TNF, tumor necrosis factor.

The letters in the subscript for Man and Ara indicate the number of glycosyl residues present in a given oligomer and should not be confused with glycosyl linkage. The size range of the oligomers observed are indicated in parentheses in the text.


ACKNOWLEDGEMENTS

We gratefully acknowledge the cooperation of Prof. A. Malorni and S. Howe in conducting the MS/MS experiments in CNR-Napoli (Italy); and the skillful technical assistance of Tyler Lahusen. We thank Marilyn Hein for preparing the manuscript.


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