Lipoarabinomannan MULTIGLYCOSYLATED FORM OF THE MYCOBACTERIAL MANNOSYLPHOSPHATIDYLINOSITOLS*

The lipopolysaccharides of mycobacteria, lipoarabi- nomannan (LAM) and lipomannan (LM), of key importance in host-pathogen interaction, were recently shown to contain a phosphatidylinositol “anchoring domain.” We now have established that LAM and LM are based on the phosphatidylinositol mannosides, the characteristic glycophospholipids of mycobacteria. Digestion of the arabinose-free LM with an endo-al+ 6-mannosidase yielded evidence for the presence of the l-(sn-glycerol-3-phospho)-~-myo-inositol-2,6-bis-a- D-mannopyranoside unit, indistinguishable from that derived from phosphatidylinositol dimannoside. This same inositol substitution pattern was shown to be present in LAM by methylation analysis before and after dephosphorylation. Positions C-2 and C-6 of the inositol unit of LAM are occupied by mannosyl residues and C-1 by a phosphoryl group. Partial acid hydrolysis of per-0-methylated LAM and comparison by gas chro-matography-mass spectrometry of the resulting deriv- atized oligosaccharides with like products from phosphatidylinositol hexamannoside demonstrated that the C-6 of inositol is the point of attachment of the mannan core The temperature gradient programs for resolution of alditol acetates and per-0-alkylated oligosaccharides have been described (19). The Hewlett-Packard 5970 mass selection detector in the electron impact or selective ion mode was used to record masses. FAB/MS was performed on an VG 707 extra high frequency mass spectrometer (19). A 300 MHz Bruker spectrometer was employed for 'H and I3C NMR studies; chemical shifts are quoted with reference to acetone. A 500 MHz Bruker spectrometer was used for "P NMR, and chemical shifts were related to external 80% H,PO,. Thus, in this scheme, the Et(O-Cz[ZH]5) group points to a position of prior glycosidic linkage. All Man residues are a-D-hlanp. Evidence for the presence of the Ins-containing fragments 1,4, 8, 10, and 13 in LAM is discussed in the text. None of the fragments from IM6 that contained linear 2-linked Man residues (ix. 2, 6, 7, 12) were found in LAM. m HP-1

of mycobacterial protein antigens by antigen-presenting cells (4) and may abrogate T-cell activation, further ensuring persistence of pathogen within the host. LAM also induces the production of tumor necrosis factor (5, 6) and thus may be responsible for mediating granuloma formation, nerve damage, and tissue necrosis. So profound are some of its biological effects that they have been attributed to endotoxic-like activity or actual endotoxin (7). LAM is considered a mycobacterial virulence factor. The molecule has been studied in the past (8). However, only recently was it realized that LAM, and its arabinose-free relative, LM, contain phosphatidylinositol (9, lo), and thus are the first prokaryotic versions of the biologically important phosphatidylinositolglycans (11). We now report that LAM and LM are based on the phosphatidylinositol mannosides (PIM), a group of distinct mycobacterial glycophospholipids, known since the 1940s and fully described by Ballou and colleagues (12,13) in the 1960s. Consequently, the profound biological functions of LAM in terms of hostpathogen relationships can now be matched with thoughts on its biogenesis and its physiological implications for mycobacteria. EXPERIMENTALPROCEDURES Preparation of LAM, LM, PZMs, and the Deacylated Derivatives-Preparations containing the majority of cellular LAM, LM, and the more polar of the PIMs were obtained by disruption of armadilloderived, delipidated Mycobacterium leprae or cultured Mycobacterium tuberculosis H37Ra followed by extraction with 50% ethanol and partitioning between phenol and water (9). The freeze-dried aqueous layer was suspended in a buffer containing 0.2 M NaC1,0.25% deoxycholate, 1 mM EDTA, 0.02% NaN3, and 10 mM Tris, pH 8.0, and applied to a column (1.5 X 90 cm) of Sephacryl S-200 which was developed with the same buffer. Polyacrylamide gel electrophoresis, when combined with a periodate-containing silver stain (9), provided an excellent gauge of purification. Detergent was removed from fractions as described (14). PIMz was purified from the earlier CHC13/ CH3OH extracts as described (15). Deacylated versions of all products were obtained by treatment with 0.1 N NaOH and appropriate purification (10,12 Ins-LM was generated as described (lo), and complete separation of the two was achieved by chromatography on a column of DEAE-Sephacel in 0.01 M Tris-HC1, pH 7.4, containing 1% Triton X-100 (10).
[3H]Ins-dLM was generated by treating the parent product with 0.1 N NaOH and purification on a column of Bio-Gel P-2 in 0.1 N CH3COOH (10). Enzymatic Digestion of LM-[3H]Ins-dLM (25 mg) was digested with exo-a-mannosidase as described (LO). The reaction mixture was boiled, centrifuged, and applied to a column (1 X 100 cm) of Bio-Gel P-10 in 0.1 N CH3COOH (10). The [3H]Ins-~~ntainingfractions were collected, combined, dried (22 mg), and further digested for another 72 h with endo-a(1+6)-mannosidase as described by Nakajima et al. (16). The product was applied three times to a 2-ml column of mixed bed resin each time recovering the [3H]Ins-~~ntaining material with 2 N HCOOH.

6228
The HF was removed by continuous evacuation over KOH and the products applied to columns of Bio-Gel (P-2 for PIM and P-100 for LAM and LM). Fractions were assayed for carbohydrate, organic-, and inorganic-P, and the carbohydrate-containing P-free fractions were dried prior to further analysis.
Sugar Compositional and Linkage Analysis-Products of the deacylation and dephosphorylation steps were freeze-dried, methylated with CH3I (17), and applied to cartridges of SEP-PAK (Waters Associates, Inc., Milford, MA) for removal of reaction products (14). The CH&N and C2H50H eluates were combined and samples converted to the respective alditol acetates for GC/MS analysis (14). In addition, the intact native LAM, PIM2, and PIM6 were methylated under neutral conditions with tert-butylpyridine and methyltrifluoromethane sulfonate under Argon for 2 h at 50 "C (18). Per-Me-LAM was dialyzed exhaustively against H 2 0 and lyophilized. The per-Me-PIM, and -PIM, were purified on columns of Sephadex LH-20 (17).
GCIMS, FABIMS, and NMR-GC/MS was performed on HP-1 (Hewlett-Packard, Avondale, PA) or DB-23 (J&W Scientific, Rancho Cordova, CA) 12-m capillary columns. The temperature gradient programs for resolution of alditol acetates and per-0-alkylated oligosaccharides have been described (19). The Hewlett-Packard 5970 mass selection detector in the electron impact or selective ion mode was used to record masses. FAB/MS was performed on an VG 707 extra high frequency mass spectrometer (19). A 300 MHz Bruker spectrometer was employed for 'H and I3C NMR studies; chemical shifts are quoted with reference to acetone. A 500 MHz Bruker spectrometer was used for "P NMR, and chemical shifts were related t o external 80% H,PO,.

RESULTS
Purification of LAM, LM, and PIMs-A key development in the present demonstration of a progressive structural lineage between PIMs, LM, and LAM was the satisfactory resolution of the three. Initial extraction of M. leprae or M. tuberculosis H37Ra with organic solvents or detergent favored removal of the simpler PIMs, notably PIM2, the dominant member (12, 20). PIM2 and the deacylated product (dPIMz; Gro-P-Ins-Manz) were readily purified ( Fig. 1) and characterized (20); the structure described by Lee and Ballou (20) for the M. tuberculosis dPIM2 was shown to also apply to the product from M. leprae (results not shown). Further extraction of mycobacteria with refluxing ethanol removed LAM, LM, and the residual PIMs. These were purified on columns of Sephacryl S-200 in detergent; the presence of deoxycholate in buffers apparently led to dissociation of aggregates of LAM, Demonstration of the purity of the key mycobacterial products. A, two pg each of purified LAM, LM, and PIM, from M. leprae were applied to polyacrylamide gels as described (10). Gels were stained with a AgN03-periodic acid reagent (9). B, thin layer chromatography of 1) the Gro-P-Ins-Man2 from M. tuberculosis and 2) the Gro-P-Ins-Man6 from M. leprae on silica gel in 1-butanol/ pyridine/acetic acid/water (5:5:1:3). The plate was charred with 10% H2S04. M. W., molecular weight. LM, and the more polar PIMs resulting in a satisfactory resolution of all three (Fig. 1). In both M. leprae and M. tuberculosis, the major PIM which survived the early extractions with organic solvents to be solubilized by refluxing ethanol proved to be PIMs (13). Some of the characteristics of PIM6 from M. tuberculosis, crucial to subsequent work, are shown in Fig. 2. The pure Gro-P-Ins-Man6 showed six clear anomeric singlets between 6 5.4 and 6 5.8, assigned to the six a-Manp units. A broad singlet at 6 4.3 was assigned to the H-2 of Ins (21) (Fig. 2B). I3C-DEPT NMR also showed six wellresolved anomeric C-1s between 6 104-98 (Fig. 2C). The two Cs with small coupling at 6 83 and 6 80 were assigned to C-2 and C-6 of Ins, shifted downfield due to a-Man substitution and displaying coupling due to the adjacent 1-P residue. The eight Cs on the negative axis were assigned to the CH2s as indicated. The signal at 6 69 was assigned to the C of the CH2-0-P unit of Gro; other signals were assigned as indicated. IMs was obtained from PIM6 by sequential deacylation and dephosphorylation. . Accurate quantitation of the relative amounts of these derivatives was not possible under the conditions. Nevertheless, the combined evidence supported the structure proposed for PIM6 in the seminal work of Lee and Ballou (13) (Fig. 2 A ) . Also, this rich source of PIM6, a scarce product in previous work (13) and its key physical properties, were key factors in the subsequent analysis of LAM.
The Presence of the Gro-P-Ins-Manz Unit in LM-The availability of a bacterial endo-a-D-mannosidase, capable of cleaving a-1-6-linked D-Manp residues (16), which are known to dominate the mannan backbone of LAM and LM (lo), presented the opportunity to look specifically for a PIM unit in LAM/LM. Initial digestion of [3H]Ins-dLM with an exoa-D-mannosidase and purification of the product on Bio-Gel in 0.1 N CH3COOH (10) resulted in an exomannosidaseresistant [3H]Ins-containing product (Fig. 3A), analysis of which had previously suggested a linear a-1-6-linked nonaor decamannoside with some single t-a-D-Manp side chains attached to C-2 (10). This product proved susceptible to endoa-(1+6)-mannosidase, yielding a [3H]Ins-containing small molecular weight product after ion-exchange chromatography. Negative ion FAB/MS showed two ("1)-ions at m/z 657 and m/z 819 (Fig. 3B) corresponding in weight to Gro-P-Ins-Manz and Gro-P-Ins-Man3, respectively. These two were readily resolved on DEAE-Sephadex (Fig. 3C). The second peak corresponded to Gro-P-Ins-Manz from the native PIMz of M. tuberculosis in several thin layer chromatography solvents, and the 'H NMR spectra were similar (Fig. 4) marked by evidence of two a-Manp H-1 singlets at 6 5.11 and 6 5.19. Thus, LM apparently contained a Gro-P-Ins-Man2 unit similar to that from PIM,. The first peak (Gro-P-Ins-Mans) showed a similar spectrum with an additional resonance at 6 5.08 indicative of a third a-Manp residue.
Substitutions on the Ins Unit of LAM-The 31P NMR spectra of PIMs and LAM showed single resonances at 6 -0.10 and 6 -0.14, respectively, which, combined with previous evidence (9, lo), demonstrated that the phosphodiester in both occurred in similar environs. To establish the locations of the P and Man residues on the Ins unit in LAM, the expected product (13). The Ins derivative from LAM was shown to be identical to that from Gro-P-Ins-Man6 by coelution from two different capillary GC columns (HP-1 and DB-23) and by an identical EI/MS fingerprint ( m / z 256, 212, 87, 75). Thus, it was concluded that the Ins unit of LAM, like those from PIMz and PIM6, is substituted at C-1, C-2, and C-6. In order to differentiate between the phosphorylated and mannosylated positions, dLAM was dephosphorylated with HF, and the products applied to a column of Bio-Gel P-100, in H20. The retained P-free carbohydrate fractions (also free of the majority of Ara) were pooled, methylated, hydrolyzed, reduced, acetylated, and compared with like products obtained from per-Me-IM6 by GC/MS. The one non-acyclic acetate derived from LAM was identical to that obtained from PIM6 in terms of molecular weight ((M+NH4)+, m/z 388)), retention time on the two GC columns, EI/MS fingerprint ( m / z 200, 191, 75), and thus was identified as 2,6-Ac2-1,3,4,5-Me4-Ins. Accordingly, the substitutions on the Ins unit of LAM were considered to be identical to these in the PIMs. In the more highly mannosylated PIMs, multiple glycosylation occurs at the C-6 position of Ins whiie a single, constant a-Manp residue occupies C-2 (12, 13, 20, 23). Seeking the same arrangement in LAM, the per-0-alkylated fragments of PIM6 were first generated by subjecting per-Me-IM6 to partial acid hydrolysis, reduction with NaB[2H]4, ethylation with C2[2H]61, and GC/MS analysis. The strategy was to apply the known GC/MS features of these fragments to the search for like fragments in LAM. The total ion chromatogram of the products from per-Me-IM6 and the mass spectra of two of the key fragments/compounds, both per-0-alkyl oligomannosyl inositols, are shown in Fig. 5, and the properties of the full range of per-0-alkyl oligomannosyl alditols and per-0-alkyl oligomannosyl inositols are presented in Table I of the substitutents on Ins (either OCH3, OC2[2H]5, or Man) were deduced from the known substitution pattern on Ins of the PIMs rather than from primary mass spectral data, as indicated above. Nevertheless, by relying on GC/MS analysis and available information on the structure of the PIMs, it was possible to identify all of the ensuing Ins-containing dimers, trimers, and tetramers and also the per-0-alkyl oligomannosyl mannitols, and attribute MS features to each of them. The most important fragments/compounds arising from these experiments were 1, 4, 8, 10, 13, and 14 (Table I).
These, if present in LAM, would help demonstrate the presence of a single terminal Man unit at C-2 on Ins and growth of the mannosyl chain at C-6.
Selected ion GC/MS analysis and coinjection GC were applied to the population of per-0-alkyl oligoglycosyl alditols and inositols arising from LAM. Thus, recognition of the diagnostic ions (mlz 252 and 312) for compound 1 (t-Manp-( 1 4 ) -I n s -S c E t ; see Table I for (Table I).

. 1 I E t~-n -M a n p -( l~) n -~-M a n p -( l~) n -~-M a n p -( l~) -I n s -c E
In summary, recognition of these key fragments and others (Table I, footnote d ) demonstrate that only a single a-Manp is on C-2 of the Ins of LAM and the extended mannan emanates from C-6. The evidence was not definite as to the structure of the mannan core beyond the initial Ins-linked mannobiose unit. However, several of the distinctive fragments arising from LAM but absent from PIMs combined with previous evidence (10) strongly suggest both 1 4 -t y p e chain extension in addition to branching at the 2-position within the mannan core.

DISCUSSION
In 1939, Anderson (24) isolated from the human tubercle bacillus a phospholipid fraction, which on hydrolysis yielded glycerophosphoric acid, mannose, and the hexahydric alcohol "inositide." Alkaline saponification of the phospholipid yielded a "phosphorus-containing glycoside," which on de-phosphoryIation produced inositol dimannoside. Some 25 years later, Lee and Ballou (13) arrived at the complete structure of the family of phosphatidylinositol mannosides from M. tuberculosis and M. phlei. They showed unequivocally that the mannoses were attached glycosidically at positions 2 and 6 of the myo-inositol ring (20) and that chain elongation occurred at the latter position (13). The present paper, again 25 years later, demonstrates that the biologically important lipopolysaccharides of mycobacteria, lipoarabinomannan, and lipomannan are based on these same phosphatidylinositol mannosides and are probably the products of yet further glycosylation. The postulated structure of LAM from M. tuberculosis H37Ra is shown in Fig. 6.
Although the present body of evidence clearly proves a structural relationship between the PIMs of mycobacteria and the LAM/LM combination, and rectifies our earlier inability to produce the requisite chemical evidence (IO), a few  Evidence for the presence of the Ins-containing fragments 1,4, 8, 10, and 13 in LAM is discussed in the text.
* Retention time on 12 m HP-1 column programed as described (19). specifics are still lacking. In particular, the key identification of the substituted inositol derivative arising from LAM/LM was based less on independent direct evidence but more through comparison with the substituted inositol obtained from PIM, and thus on earlier evidence (20), a not unreasonable approach given the unambiguity of the earlier work. The susceptibility of LM to endomannosidase was crucial and fortunate in view of the resistance of LAM/LM to other key enzymes such as the PI-specific phospholipase C (10). From these experiments arose convincing evidence of the presence of the Gro-P-Ins-Mann unit in LM and, by implication, in LAM. A variation of the partial depolymerization strategy used in defining the non-reducing ends of LAM (14,26) also provided evidence of a linear a-14-linked mannan backbone with a considerable degree of mannosyl a1-2 branching, altogether reminiscent of the short homomannosyl oligosaccharide described by Maitra and Ballou (25). Nevertheless, the frequency of such a1+2 branch points on the mannan backbone of LAM and the issue of whether they are single t-Manp or mannobiose units has not yet been resolved. Thus, the critical question of a structural relationship between the PIMs and LAM has been addressed. The presence of the two fatty acyl functions, stearyl and tuberculostearyl, previously found exclusively associated with the PIMs (15), has already been shown in LAM (9). Of considerable significance, a lipomannan from Propionibacterium frezdenreichii was also recently shown to contain inositol, glycerol, phosphate, and fatty acids (27), pointing to a covalently linked phosphatidylinositol in an organism phenotypically related to Mycobacterium. Thus, LAM and LM join the PIMs of mycobacteria as members of the biologically important glycosylphosphatidylinositols (11). What bearing this fact may have on the powerful biological activity of LAM is not known but is presumed to be significant considering that many of the biological activities of LAM are dependent on full acylation of the molecule (2, 28). On the other hand, the nature of the non-reducing ends of LAM, which consist of branched arabinans in an avirulent strain of M. tuberculosis (14) and linear al-2-linked oligomannosyl units in a virulent strain, have profound effects on such biological phenomena as the release of tumor necrosis factor.' Thus, rather than the glycosylphosphatidylinositols, a functional paradigm for LAM may be the lipoteichoic acids and lipomannans of Gram-positive bacteria in which the lower regions of the molecule are also key to host-parasite interactions (29). D. Chatterjee, I. Orme, A. Roberts, and P. J. Brennan, unpublished work.