THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 hy The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No. 13, Issue of May 5, pp. 7447-7454,1989 Printed in U.S.A.

New-found Phenolic Glycolipids in Mycobacterium bouis BCG PRESENCE OF A DIGLYCOSYLATED GLYCOLIPID*

(Received for publication, June 13, 1988)

Alain Vercellone and Germain Puzo From the Centre de Recherche de Biochimie et Genktique Cellulnires du Centre National de la Recherche Scientifique, 118 route de Narbonne, 31062 Toulouse Cedex, France

A crude phenolic glycolipid extract from Mycobac- terium bovis bacille Calmette-Guerin (BCG) was frac- tionated until homogeneity at the intact level into four phenolic glycolipids called B, B-1, B-2, and B-3 ac- cording to their polarity. The apolar one, which is the most abundant was assigned to the well-known myco- side B. The B-2 and B-3 phenolic glycolipids were purified by direct-phase high performance liquid chro- matography using a 5 pm Spherisorb column but were only recovered in small amounts (3 mg). A linear gra- dient of 0-20% methanol in chloroform was used. The B-1, B-2, and B-3 glycolipids were subjected to suitable modern analytical techniques selected for their poten- tial to elucidate the structure at the intact level. De- sorption chemical ionization-mass spectrometry al- lowed the molecular mass of B-3 to be determined as 1652 Da for the major homolog establishing the molec- ular formula as Thus, the B-3 polar phenolic glycolipid contained two deoxyhexoses, one molecule of phenolphthiocerol esterified by two mole- cules of mycocerosic acid. Using two-dimensional ‘H NMR (correlated chemical shift and nuclear Overhau- ser effect spectroscopy) at the intact level the B-3 oligosaccharide structure was determined as an a-L- Rhap-(1 + 3)-2-O-Me-a-~-Rhap. This is the first re- port of a diglycosylated phenolic glycolipid in a non- pathogenic mycobacteria. The disaccharide unit, the antigenic determinant, appears to be characteristic of M. bovis BCG. This polar glycolipid B-3 and the apolar ones, B-1 and B-2, were reactive in enzyme-linked immunosorbent assay against serum from rabbit hy- perimmunized with M. bovis BCG.

The renewed interest in the detection, purification, and structural elucidation of the phenolic glycolipids of pathogenic mycobacteria has mainly been prompted by recent advances in the field of their immunological properties (1). The Myco- bacterium leprae-specific antigen was characterized by Bren- nan and co-workers (2) as a phenolic glycolipid formed by a unique trisaccharide consisting of 3,6-di-O-Me-P-~-Glcp- (1+4)-2,3-di-O-Me-a-~-Rhap~-(l-+2)-3-O-Me-a-~-Rh~. It

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: Rhap, rhamnopyranose; PheG1, phe- nolic glycolipid; BCG, bacille Calmette-Guerin; Fucp, fucopyranose; Glcp, glucopyranose; TMS, trimethylsilyl; HPLC, high performance liquid chromatography; MS, mass spectrometry; EI, electron impact; DCI, desorption chemical ionization; COSY, chemical shift correla- tion spectroscopy; NOESY, nuclear Overhauser effect and exchange spectroscopy; GC, gas chromatography.

is successfully used as serologic marker for the screening of lepromatous patients (3, 4). It also seems to be involved in the absence of cell-mediated immunity response by inducing suppressor lymphocytes which could inhibit the reactivity of helper T-cell clones in lepromatous patients (1, 5, 6). More- over, it was reported that this glycolipid may protect M. leprue from the antimicrobial activity of activated human macro- phages (7).

Related phenolic glycolipids were observed on M. kansasii which belongs to the non-tuberculous Mycobacteria group. They differ from those of M. leprue by their tetrasaccharidic structure and the monosaccharide composition. The M. kun- sasii phenolic glycolipids share a common core: 2-0-Me-4-0- Ac-c~-~-Fucp-(lj3)-2-O-Me-cu-~-Rhap-(l-+3)-2,4-di-O-Me- a-L-Rhap and differ by the monosaccharidic residue located at the nonreducing end (8, 9). For the major phenolic glyco- lipid (PheG1 K-I), it is a new-found sugar: the 2,6-dideoxy-4- 0-Me-arabino-a-D-hexopyranose and for the minor one (PheG1 K-11) 2,4-di-O-Me-a-D-mannopyranose (10, 11). A related phenolic glycolipid was isolated from M. tuberculosis strain Canetti. Its oligosaccharidic moiety presents the follow- ing trisaccharidic structure: 2,3,4-tri-O-Me-a-~-Fucp- (1+3)-a-~-Rhap-(1+3)-2-0-Me-a-~-Rhap (12). All these gly- colipids were found antigenic and seem to be promising species markers (13, 14).

It is well known that M. bovis BCG contains a monoglyco- sylated 2-O-Me-a-~-Rhap phenolic glycolipid also found in M. tuberculosis strain Canetti (15,16). Besides this glycolipid, we identified new phenolic glycolipids not yet described in M. bo& BCG. These glycolipids could share common epitopes with those of M. fuberculosis leading to false positive enzyme- linked immunosorbent assay tests when the M. tuberculosis phenolic glycolipid is used for the screening of tuberculous patients. Also, by analogy to the immunological properties of the M. leprue phenolic glycolipid we can assume that one of these new phenolic glycolipids could be involved, by the stimulation of the T suppressor cells, in the conflicting results observed in the protection against M. tuberculosis by M. bovis BCG (17). So, the purification and the structural elucidation of these new-found phenolic glycolipids are key steps for a further study of their activity on T-cells.

A more reliable way to determine the structure of minor glycolipids would be to directly analyze a suitably purified intact glycolipid before any degradation. The purification of phenolic glycolipids extracted in small amounts from M. bovis BCG cell wall was a difficult and long task. This was mainly due to the heterogeneity of the M. bovis BCG phenolic gly- colipid mixture resulting from the presence of molecular en- tities which differ by both the lipidic and sugar parts. By using gradient HPLC, the mixture was fractionated into the three minor phenolic glycolipids called B-1, B-2, and B-3. Modern analytical tools were selected for their ability to

7447

7448 New-found Phenolic Glycolipids in M. bovis BCG

elucidate the structure of the B-1, B-2, and B-3 glycolipids in their native forms. DCI-mass spectrometry with ammonia as reagent gas and two-dimensional 'H NMR spectroscopy (ho- monuclear chemical shift correlation, nuclear Overhauser ef- fect) were the key for their structural characterization. This study allowed us to establish the structure of three phenolic glycolipids B-1, B-2, and B-3 described for the first time in M. bouis BCG.

EXPERIMENTAL PROCEDURES

Phenolic Glycolipid Purification-From 1300 g of M. bouis BCG cells, approximately 15 g of lipidic extract were obtained (8). These lipids were applied to a column (4.5 X 50-cm) of Florisil (60-100 mesh) which was irrigated with 600 ml of 25, 75, and 100% CHC13 in petroleum ether followed by a gradient elution solvent of 4 liters of chloroform/methanol from 10:0 to 9:l. Separation was checked by thin layer chromatography (CHC13/CH30H, 9:l). The more polar phenolic glycolipid-containing fractions were rechromatographed by flash chromatography on a silicic acid (Kieselgel G60,230-400 mesh, Merck, Darmstadt, West Germany) column. The eluent consisted of 100-ml fractions of chloroform and 1, 2, 4, 8, and 10% methanol in chloroform. The CHC13 and 1% CH30H-CHC13 eluates contained B while B-1 was present in the 2% CH30H eluate still contaminated by B; B-2 and B-3 appeared in both the 4 and 8% CHIOH eluates. For final purification the 2% CH3OH eluate was chromatographed by reverse-phase HPLC (5-pm Spherisorb ODs2 (7.5 mm, 300 mm) Soci6t6 Francaise Chromato. Colonne, Paris) eluted by 2 ml/min of chloroform/methanol (1:l); B-2 and B-3 containing fractions were injected, in 20-4 aliquots, into a HPLC apparatus equipped with a direct-phase column (5-pm Spherisorb (4.6 mm, 250 mm)) using a gradient elution solvent of methanol in chloroform (from 0 to 20%). The compounds appeared as single peaks using a UV detector ( A = 275 nm). 6 mg of B-l,2.5 mg of B-2 and 3.0 mg of B-3 were obtained as white compounds.

Preparation of Deacylated Phenolic Glycolipids-200 pg of B-3 was dissolved in 100 pl of 10% KOH in benzene/methanol(l:l) in a sealed tube at 110°C for 18 h. After evaporation under N2, 300 p1 of CHC13 and 300 pl of H20 were added, then acidified with diluted HZSO, to pH 4. The washed CHCl3 phase was applied to a Sep-pak silicic cartridge (Waters Associates, Inc., Milfort) which was irrigated first with CHC13 to remove the fatty acids, and with 10% methanol in chloroform to obtain the dB-3.300 pg of B-1 and 200 pg of B-2 were dissolved in 200 pl of anhydrous ether to which 2 mg of AlLiD, were added for 18 h under stirring. After destruction of the hydride by acetone, followed by acidification, the products were partitioned between ether and water. After that, the procedure described above was applied.

anolyzed with 2 M HC1 in dry methanol at 95°C for 1 h. After Glycolipid Methanolysis Procedure-100 pg of glycolipid were meth-

evaporation under Nz, the residue was applied to a Sep-pak C-18 cartridge (Waters Associates, Inc., Milfort, MA) which was eluted successively with methanol to elute the methyl glycosides and with chloroform to remove the lipids.

Acetylation-50 pg of phenolic glycolipid were dissolved in 50 p1 of acetic anhydride-anhydrous pyridine (1:l) in a sealed vial at 60 'C for 20 min. After cooling, the mixture was evaporated under NP.

Analytical Methods-HPLC was conducted on a Gilson apparatus, model 302, model 802 manometric module, and Waters model 450 variable wavelength detector.

Routine gas chromatography was performed on a Girdel series 30 equipped with a OV1 wall-coated open tubular, inner diameter 0.3- mm, 25-m capillary column using nitrogen gas at a flow rate of 2.5 ml/min equipped with a flame ionization detector. A temperature program from 100 to 200°C at a speed of 3 "C/min and an injector temperature of 180 "C were used for TMS methyl glycoside analysis while the fatty acid methyl esters were analyzed with a temperature program from 150 to 290 "C, at a speed of 2 'C/min and an injector temperature of 260°C.

'H NMR was performed on a Bruker AM-3OOWB spectrometer ('H at 300 MHz). One-dimensional spectra were recorded by using 90" pulses, 3600 Hz spectral width, 32 K data points, 1-s recycle time, and 2.62-s acquisition time. Sample was dissolved in C2HC13 at a concentration of 3 mg/0.3 ml, proton spectra were measured at 30 "C, and chemical shifts are referenced to external tetramethylsilane. For each experiment 16-64 scans were accumulated. For the two-dimen- sional 'H NMR spectroscopy spectra were collected using a 256.1024

points time domain matrix over 2074 Hz along the t l and t2 direc- tions. Both t l and t2 were zero-filled to 2048 points. Sine-bell window functions were used along the t l and t2 directions prior to two- dimensional Fourier transformation. Two-dimensional correlation spectroscopy (COSY) was performed using a (90"-t1-90"-acquisi- tion (t2))n (n = 256) sequence. Two-dimensional nuclear Over- hauser effect and exchange spectroscopy (NOESY) sequence was: (9O0-tl-90"-T,-90"t2)n (n = 256). 128 scans were accumulated for each experiment with a mixing time of 0.45 s.

DCI-MS was conducted on a Finnigan MAT 311A mass spectrom- eter. GC-MS was performed on a Nermag model R 10/10 quadrupole mass spectrometer connected to a PDP-8M computer. Infrared was conducted on Perkin-Elmer 177 spectrophotometer.

Immunological Procedures-Rabbit antiserum was obtained by in- jection of 1 ml of a water-in-oil emulsion prepared by mixing 0.5 ml of phosphate-buffered saline, pH 7.2, containing 5 mg of whole M. bo& BCG and 0.5 ml of Freund's incomplete adjuvant. The emulsion was injected intradermally into 3-month-old Fauve de Bourgogne rabbits. 3 weeks later, a booster was made with 3 mg of M. boois BCG cells in water-in-oil emulsion as described above. Bleeding was begun at the end of 15 days.

Enzyme-linked Immumsorbent Assay-Pure glycolipids were dis- solved at a concentration of 25 pg/ml in absolute ethanol/hexane (1:l). Aliquots (50 pi) of the suspension were applied to the wells of a polystyrene microtiter plate (Nunc-Immuno Plate I) and allowed to evaporate at 37 "C overnight. The wells were saturated with 200 p1 of 30% powdered milk and 0.1% Tween 20 in phosphate-buffered saline at 37 "C for 2 h. The plate was washed three times and rabbit immunserum to M. bouis BCG diluted 1:50 was added followed by a 2-h incubation at 37 "C and further washings. Serum dilution was made in phosphate-buffered saline containing 0.4% powdered milk. Then 100 pl of P-galactoside-conjugated donkey anti-rabbit Ig (F(ab') 2 fragment) was added followed by a 2-h incubation at 37 "C. After washing five times, 100 p1 of the substrate solution (orthonitrophenyl- P-D-galactoside (Sigma)), were added. Incubation was for 1 h at 37 "C. Absorbance was read at 405 nm with a Multiskan apparatus (Flow Laboratories, Inc., McLean, VA). The washing solution used through- out the assay consisted of phosphate-buffered saline containing 1% milk.

RESULTS

Purification and Identification of B-1, B-2, and B-3 Phenolic Glycolipids

The CHC13/CH30H-soluble lipid from M. bovis BCG was extracted with ether; the ether-soluble lipids were triturated with acetone. The acetone soluble part (15 g) was fractionated on a florisil column. The CHCI3/CH30H (9:l) eluted glyco- lipids were applied to a flash chromatography silicic acid column. The eluates CHCL and CHCL/CH30H (99:1, v/v) contained, as shown by silicic acid thin layer chromatography analysis, homogeneous mycoside B (1 9). The 2% methanol eluate (200 mg) contained a slightly more polar glycolipid contaminated by the mycoside B. More polar glycolipids (150 mg) are observed in eluates containing 4 and 8% of methanol. The apolar fraction (2% methanol eluate) was subjected to reverse-phase HPLC using a C-18 bonded silica column. The glycolipid called B-1 was easily separated from the mycoside B using an isocratic mobile phase (CHCl&HSOH, 1:l V/V)

at a flow rate of 2 ml/min. The 4 and 8% methanol fractions were subjected to direct-phase HPLC with a 5-pm Spherisorb column. The mobile phase was linear gradients of methanol in chloroform. A representative profile of such a fractionation is shown in Fig. 1 (glycolipids B and B-1 were added as internal standards). The 4 and 8% methanol fractions ap- peared to be composed by at least two other kinds of glycolipid called B-2 (2.5 mg) and B-3 (3 mg) observed at 13.3 and 17.9 min, respectively. Thus, from the crude glycolipid fraction, three quantitatively minor glycolipids, B-1, B-2, and B-3, characterized by their HPLC retention times, were purified until silicic acid thin layer chromatography homogeneity (Fig. 2).

New-found Phenolic Glycolipids in M. bovis BCG 7449

1322

io 2'0 RETENTION TIME (rnin)

FIG. 1. Direct-phase HPLC of phenolic glycolipids B, B-1, B-2, and B-3. A direct-phase analytical column was used. The mobile phase was a linear gradient of 0-20% chloroform/methanol (82, v/v) (Solvent B) in chloroform during the first 9 min, then 20- 100% over a period of 16 min at a flow rate of 1 ml/min.

. . . . .

FIG. 2. Thin layer chromatography of crude acetone soluble (AS) lipid and the HPLC purified B, B-1, B-2, and B-3 phe- nolic glycolipids from M. bovis BCG. The thin layer plate was run in CHC13/CH30H (9:l) and sprayed with 0.2% anthron in H2S04 then heated at 50 "C for 5 min.

Glycolipids B-1, B-2, and B-3 show identical UV absorption spectra characteristic of an aromatic group (intense peak at 220 nm and two lower intensity peaks at 274 and 282 nm) (18). Moreover their 'H NMR spectra show two doublets centered at 6 = 6.95 and 7.14 ppm typical of parasubstituted phenolic groups (19, 20). These data and the two proton quintets centered at 6 = 4.82 ppm assigned to the two methine protons located at the ester linkages in the lipidic chain support the fact that all these glycolipids belong to the phe- nolic glycolipid class.

Structure Elucidation of the B-1, B-2, and B-3 Phenolic Glycolipids

Mass Spectral Analysis-B-1, B-2, and B-3 phenolic glyco- lipids either in their native forms or as peracetylated deriva- tives were analyzed by DCI-mass spectrometry using ammo- nia as reagent gas (21). In all the analyses, mass spectra exhibited, in the molecular mass range, intense signals as- signed to adduct molecular ions (M + NH4)+, allowing accu- rate molecular weight determination. Fig. 3 shows the DCI- mass spectrum of glycolipid B-3. In the high mass range, the five sets of peaks separated by 14 Da observed at m/z 1628, 1642, 1656, 1670, and 1686 were attributed to adduct molec- ular ions. These data suggest that the B-3 glycolipid is com- posed of at least five molecular entities which probably differ by the aliphatic chain length. At low mass range, a first set of fragment ions is observed at m/z 1482, 1496, 1510, 1524, and 1538. They arise from their respective molecular ions by

13b0 1350 1400

1524 I1

1500 1550

1600 1650 1700

mfz

FIG. 3. Partial DCI-NH3 mass spectrum of the B-3 phenolic glycolipid.

m/z

FIG. 4. EI-mass spectrum of the peracetylated B-3 phenolic glycolipid derivative.

the loss of 146 Da assigned to an anhydro-deoxyhexose. A second set of fragment ions observed at m/z 1322,1336,1350, 1364, and 1378 was attributed to the aglycone part linked to the ammonium ion, in agreement with a diacylphenolphthi- ocerol molecule. The 160-Da mass difference between these two sets of fragment ions was assigned to an anhydro-mono- 0-Me-deoxyhexose. So, this data suggests the following par- tial structure for B-3, deoxyhexosyl+O-Me-deoxyhexosyl-, diacylphenolphthiocerol. Furthermore, the B-3 molecular weight heterogeneity arises from the chain length of the aglycone part supporting a unique structure of the oligosac- charide moiety. Such an assignment is in agreement with the pyrolysis EI-mass spectrum of the peracetylated B-3 glyco- lipid shown on Fig. 4. The cleavage reactions at the glycosidic bond yield the expected fragment oxonium ions at m/z 475 and 273 which are shifted to m/z 487 and 282 when the perdeuteroacetylated derivative is analyzed.

The B-1 and B-2 phenolic glycolipid DCI-mass spectra data

7450 New-found Phenolic Glycolipids in M. bovk BCG

are summarized in Table I. From these data and their pyro- lysis EI-mass spectra, one can propose the following partial structure for the major B-1 and B-2 homologs: mono-0-Me- deoxyhexosyl-diacylphenolphthiodolone and deoxyhexosyl +diacylphenolphthiodolone, respectively.

' H NMR Analysis-"H NMR studies on the native and the peracetylated glycolipids were performed by one-dimensional and two-dimensional chemical shift correlation spectroscopy (COSY) and two-dimensional nuclear Overhauser effect spec- troscopy (NOESY) (23, 24). The one-dimensional 'H NMR spectra of the native and peracetylated B-3 glycolipids are shown in Figs. 5 and 6. The B-3 proton anomeric resonance region shows two unresolved doublets at 6 = 5.47 and 6 = 5.17 ppm assigned to H-1 and H-l', respectively. For glycolipids B-1 and B-2, only the signal at 6 = 5.47 ppm was observed. The integration of the anomeric proton signals supported a monosaccharidic structure for B-1 and B-2 and a disaccharidic unit for B-3. The relative deshielding of the H-1 to 5.47 ppm from the normal 5 ppm value for this type of proton, is consistent with the proximity of the aromatic nucleus. So, this H-1 probably characterized the 0-Me-deoxyhexosyl res- idue linked to the diacylphenolphthiocerol part of the B-3

TABLE I Molecular weight and relative abundance determinations of the homologs which constitute the B-1 and B-2 phenolic glycolipids

These data arise from the DCI-MS (NH,) mass spectra. The numbers in brackets correspond to the relative abundance (%).

Adduct molecular Fragment ions Mass dif- ions (M + NHJ' (F + NH,)' ference assignment

Sugar

B-1 1252 (90) 1092 (80) 160 1266 (100) 1106 (95) 160 1280 (50) 1120 (60) 160 Mono-methyl 1294 (70) 1134 (70) 1308 (70)

160 Deoxyhexose 1148 (65) 160

1322 (30) 1162 (35) 160

B-2 1448 (63) 1302 (50) 146 1462 (45) 1316 (40) 146 1464 (18)" 1318 (18)" 146 1476 (100) 1330 (100) 146 Deoxyhexose 1490 (42) 1344 (55) 146 1492 (18)" 1346 (18)" 146 1504 (42) 1358 (30) 146 1518 (36) 1372 (32) 146

The isotopic abundances were subtracted.

- I I 1 I

7 6 5 4 3 2 1 PPm

FIG. 5. B-3 phenolic glycolipid 'H NMR spectrum (3 mg) in CDCL solution.

5.4 5.2 5.0 4.8 4.2 4.0 3.8 ppm

FIG. 6. Partial 'H NMR spectrum of the peracetylated B-3 phenolic glycolipid (3 me) in CDCls solution.

glycolipid. After B-3 peracetylation (Fig. 6) the H-1 and H-1' resonate at 6 = 5.51 and 6 = 4.96 ppm, respectively, and appear as well-resolved doublets. They show identical Jl,2 values of 1.7 Hz suggesting that in both sugar residues the H-1 and H-2 are in eq. positions (25, 26). These J1,2 values are identical to those of the mycoside B H-1, the sugar part of which is 2-O-Me-a-~-Rhap. Between the H-1 and H-1' signals, other non-anomeric proton resonances were observed and assigned to the deshielded gem-acetoxyl sugar protons. Two triplets centered at 6 = 5.16 ppm and 6 = 5.09, J = 9.8 Hz could arise from the H-4 of rhamnopyranoses. Further- more the two quadrets centered at 6 = 5.28 ppm ( J = 10 Hz, J = 3.3 Hz) and at 6 = 5.15 ppm ( J = 3.3 Hz, J = 1.7 Hz) are also in agreement with the H-3 and H-2 resonance of Rhap. Each of these two signals integrates for one proton, suggesting that one of the undeshielded protons H-2 or H-3 of the mono- 0-Me-deoxyhexosyl residue is involved in the glycosidic link- age and the other is the gem-methoxyl proton. The sugar proton assignment was achieved by two-dimensional "H NMR COSY (23, 24). A representative spectrum (Fig. 7) indicates the correlations that led to the identification of the relevant protons allowing type sugar determination. Beginning at 5.51 ppm with the deshielded H-1 we found a cross-peak showing a connectivity to the H-2 resonance at 6 = 3.73 ppm. From this H-2 a connectivity to the H-3 resonance at 6 = 4.16 ppm was found. The H-3 signal multiplicity corresponds to a quadret ( J 3 , 4 = 9.8 Hz, J2,3 = 3.2 Hz) indicating that proton H-2 is in eq. position while the H-3 and H-4 are in ax. positions. Likewise the triplet centered at 6 = 5.16 ppm (J3,4

= J4,5 = 9.8 Hz) was attributed to the H-4 proving that H-3 and H-5 are transdiaxial toward H-4. These observations are in accord with the fact that these resonances are due to H-1, H-2, H-3, and H-4 of a a-rhamnopyranosyl residue. Further- more, the multiplet, centered at 6 = 3.75 ppm assigned to H- 5, is connected to a resonance at 1.17 ppm attributed to the methyl deoxy-group (C-6H3). These data agree with a 2-0-Me or 3-0-Me-aRhap for the sugar residue linked to the aglycone. Likewise the distal monosaccharide residue was identified as a-Rhap (H-1': doublet, 6= 4.96 ppm, JlP2, = 1.8 Hz; H-2': quadret, 6 = 5.15 ppm, JlV2, = 1.8 Hz, JZe3, = 3.35 Hz; H-3': quadret, 6 = 5.28 ppm, J2'3' = 3.3 Hz, J 3 r 4 , = 10.1 Hz; H-4': triplet, 6 = 5.09 ppm, J3r4p = J4,5, = 9.8 Hz; H-5': multiplet, 6 = 4.12 ppm; C-6H3: 6 = 1.25 ppm. From this study the interglycosidic linkage remains undetermined and the a-ano- meric configuration for the rhamnopyranoses must be con- firmed since the J1,2 values are quite similar for the a- and 8- anomers, 1.7 and 1 Hz, respectively. These two points were unambiguously resolved by 'H NMR two-dimensional NOESY analysis of the B-3 peracetylated derivative (Fig. 8).

New-found Phenolic Glycolipids in M. bovis BCG 7451

I I I

5 4 3 2 i ppm

FIG. 7. Two-dimensional 'H-'H shift correlation spectrum at 300 MHz of the peracetylated B-3 phenolic glycolipid de- rivative (3 mg) in CDCls solution.

Two kinds of cross-peak connecting resonances to other res- onances can be observed within 0.3 nm (23, 241, those due to interactions with protons borne by the same sugar residue and those due to interactions with protons on neighboring sugars. So, for methyl-a-Rhap one expects to see 1-2 equa- torial intraring connectivity while for P-anomers one expects 1-3 and 1-5 axial intraresidue connectivities. Starting from the H-1 resonance we found three cross-peaks allowing con- nectivities to the H-2 (6 = 3.73 ppm), the methoxy group (singlet, 6 = 3.53 ppm) and to the ortho aromatic protons (doublet, 6 = 6.95 ppm). These observations support the a- anomeric configuration for the 0-Me-Rhap residue and its linkage by the anomeric carbon to the phenolic aglycone. Moreover, the proximity within 0.3 nm of H-1 and the methoxyl group suggests its location on the C-2 of the sugar ring. Likewise the distal Rhap H-1' is connected to H-3 (6 = 4.16 ppm) and to H-2' (6 = 5.15 ppm). We can conclude that interglycosidic coupling occurs between the C-1' and the C-3 of the residues a-Rhap and 2-O-Me-a-Rhap, respectively. This assignment was also confirmed by GC-MS using the TMS method (28). The B-3 glycolipid was permethylated (29), methanolyzed, and the methyl glycosides converted into TMS derivatives were analyzed by GC-MS. From their reten- tion times and fragmentation pathway, the two products were assigned to permethyl a-Rhap and methyl 2,4-di-O-Me-3-0- TMS-a-Rhap (peaks m/z 146,88) supporting a 1+3 intergly- cosidic linkage. Finally, from DCI-mass spectrometry and extensive 'H NMR analysis the following B-3 disaccharide structure is proposed

Rhap - (la-3)-2-O-Me-Rhap 1 a-aglycone.

-I

7 6 5 4 3 PPm

FIG. 8. Two-dimensional correlated nuclear Overhauser ef- fect spectroscopy at 300 MHz of the peracetylated B-3 phe- nolic glycolipid derivative (3 mg) in CDCls solution.

Similar 'H NMR analysis applied to the phenolic glycolipids B-1 and B-2 led us to propose for their carbohydrate moieties the structures 2-0-Me-a-Rhap and a-Rhap, respectively.

The D or L series of the a-Rhap and 2-O-Me-a-Rhap, constituents of the carbohydrate part of B-1, B-2, and B-3, was determined according to the procedure described by Ger- wig et al. (27). Glycolipids B-1 and B-2 were solvolyzed in the presence of (-)-2-butanol to yield butyl glycoside diastereo- isomers which were separated from the aglycon by silica sep- pak cartridge chromatography. Thus, they were further deriv- atized into TMS and analyzed by capillary GC. By cochro- matography with authentic L standards all the Rhap and 2-0-Me-Rhap were identified as belonging to the L series.

Aglycone Structures The B-3 'H NMR spectrum (Fig. 5 ) shows a singlet at 6 =

3.32 ppm assigned to a methoxyl group borne by the aliphatic chain which characterized a phenolphthiocerol structure. This singlet is also observed in the B-2 'H NMR spectrum, but its integration gives only one proton suggesting, in agreement with the DCI-MS observations (Table I), that the B-2 aglycon from the major homolog corresponds to phenolphthiodolone. It is missing in the B-1 'H NMR spectrum, characterizing the presence of phenolphthiodolone.

The B-1, B-2, and B-3 aglycone parts obtained by metha- nolysis were alkalinolyzed to yield the phenol glycol cores and fatty acids. After methylation by diazomethane, the methyl fatty esters were separated from the phenol glycol cores by a silicic Sep-pak cartridge, and analyzed by GC-MS. B-3 and B-2 fatty esters are structurally similar and mainly composed of 2,4,6-trimethyl-trieicosanoate (m/ z 88, 101, 129, 171, 281,

7452 New-found Phenolic Glycolipids in M. bovis BCG

339, and 410) and two other CZ7 and CZ9 homologs. The B-1 fatty ester composition differs by the presence of C-16, C-18, and also by CZ4 and c26 chains ( m l z 74, 87, 367, and 410) which are unmethylated at C-2 and C-4. Moreover, the com- bination in B-1 of short (C16 and Cls) and long chains (CZ4 and c26) explains the lower B-1 molecular weight compared with B-2. The B-1, B-2, and B-3 phenol glycol cores were analyzed by mass spectrometry using complementary ioniza- tion modes E1 and DCI-NH3. The B-3 phenol glycol DCI mass spectrum (Fig. 9) shows four peaks in the high mass range assigned to (M + NH,)' ions allowing the determination of their molecular weights (534, 548, 562, and 590) which are in agreement with a phenolphthiocerol structure. Moreover, the fragment ions observed at mjz 204 and those present in the EI-mass spectrum ( m l z 73, 107, 316, and 376) (20) allow the proposition of the following structure for the major hom- olog.

""" + """" 107 346

I I

(- HI I

; I j I I

,' \ CHA(CH~)~~-CHJXH~+CH-(CH~)~-CH+"H-CH, I I I I

OH I I OH I

CHJI I ( - H , + N H : ~ L " -_ "_ [ +

204 73

STRUCTURE I

\

/ B-3: phenolphthiocerol, R = CH"OCH3, M , = 548, m/z 204, 73

\

/ B-1: phenolphthiodolone, R = C=O.

A similar phenolphthiocerol structure was found for the minor B-2 phenol glycol core, while for B-1, as previously suggested, DCI-MS analysis confirms a phenolphthiodolone structure. From these data the B-1 and B-2 structures are presented in Table I1 while Fig. 10 illustrates the B-3 glycolipid structure.

Immunoreactivity of B-1, B-2, and B-3 Phenolic Glycolipids Preliminary serological investigations show that B-1, B-2,

and B-3 are reactive against polyclonal hyperimmune rabbit

antiserum raised against M. bouis BCG (Fig. 11). The lower activity of the polar glycolipids B-2 and B-3 in relation to B can be correlated with their lower abundance (relative abun- dance 0.3% compared to the B (100%)) at the cell-wall surface. However, this assumption is not in agreement with the similar reactivity observed between the antigens B and B-1 (Fig. 11) (B-1, relative abundance: 0.6%). This fact can be explained by a cross-reaction of the epitopes B-1 toward the antibodies against the glycolipid B since their carbohydrate parts are identical (2-O-Me-a-~-Rhap).

DISCUSSION

The purification of intact biological molecules is a key step in determining their structure by modern instrumental analy- sis. The present study clearly demonstrates the complexity of the phenolic glycolipid fraction extracted from M. bouis BCG previously described as containing only glycolipid B. To frac- tionate this mixture, a simple and nondestructive method mainly based on reverse-phase and gradient direct-phase HPLC was successfully developed. Besides the well-known mycoside B, three phenolic glycolipids called B-1, B-2, and B-3 were purified in their native form until homogeneity and identified according to their relative HPLC retention times. The purification method described here might be applicable to the possible fractionation of a crude phenolic glycolipid mixture from M. marinum (30) and M. tuberculosis strain Canetti. In previous studies we demonstrated that the use of Fab-mass spectrometry and Fab-tandem mass spectrometry was the key for the establishment of the true structure of the

0

FIG. 10. Proposed structure of the polar B-3 phenolic gly- colipid found in M. bouis BCG.

0.50?

i Rh I

204

m/z

FIG. 9. DCI-NH3 mass spectrum of the B-3 phenoglycol core.

B B-1 8-2 8-3 FIG. 11. Enzyme-linked immunosorbent assay reactivity of

the B, B-1, B-2, and B-3 phenolic glycolipids against non- immunized rabbit serum (open columns) and polyclonal rabbit immunoserum against M. bouis BCG (shaded columns). The plate was coated with each glycolipid at 1.25 pglwell. A serum dilution of 150 was used.

TABLE I1 Proposed structures for the B-1, B-2, and B-3 M. bovis BCG phenolic glycolipids

Phenolic glycolipids Sugar residues Phenol glycols Fatty acids

B-1 8-0-Me-a-L-Rhap phenolphthiodolone C16, Cls, and C-2, C-4 un-

B-2 u-L-Rhap phenolphthiodolone (major) mycocerosic c26, czn c m B-3 u-L-Rhap (1-3)-2-O-Me-a-~-Rhap phenolphthiocerol mycocerosic c z 6 , C Z ~ , CB

branched Czl and c26

New-found Phenolic Glycolipids in M. bovis BCG 7453

major phenolic glycolipid PheGl K-I isolated from M. kansasii (8). These analytical techniques allowed us to reveal and to locate a new found sugar in nature: the 2,6-dideoxy-4-0-Me- a-D-arabino-hexopyranose at the nonreducing end of the PheGl K-I tetrasaccharide (9, 11). The tendency of these amphiphatic molecules to aggregate into micelles in polar matrices (eg. glycerol, thioglycerol) required the development of more suitable matrices as (for instance the monobutyl triethylene glycol (31)) for the success of their analysis by Fab-MS. However, Fab sensitivity was low, and increasing it required investigations into the solution chemistry behavior of these molecules. Moreover, the PheGl K-I tetrasaccharide sequence was established by partial acid hydrolysis followed by 'H NMR spectroscopy, and the interglycosidic linkage determination was based on the TMS procedure which is more suitable than the alditol acetate method (28). All these chemical reactions are sample-consuming and furthermore they can introduce artifacts leading to incomplete structure elucidation since some sugar residues contained by the gly- colipid oligosaccharide moieties are acidolabile. This obser- vation is illustrated by the discovery of a particular sugar, cited above, on the M. kansasii phenolic glycolipid (10). Thus, to increase sensitivity and in the aim of achieving structural elucidation at the intact level, new developments based on suitable modern analytical tools are proposed first, DCI-mass spectrometry (32), second, two-dimensional 'H NMR spec- troscopy (homonuclear chemical shift 'H-'H correlation spec- troscopy and nuclear Overhauser effect and exchange spec- troscopy). DCI-ionization mode using ammonia as reagent gas was found to be a powerful technique for intact phenolic glycolipid analysis. Molecular weights were unambiguously determined. Furthermore, the partial sugar structures and their sequences were established from the rearranged frag- ment ions resulting from the glycosidic cleavage. Finally the DCI/NH3 ionization mode is more sensitive, by a factor of 10, than Fab-MS to analyze these amphiphatic molecules. How- ever, for highly glycosylated glycolipids Fab-MS analysis re- mains a prodigious ionization technique (33). Two-dimen- sional 'H NMR analysis appeared, in the present study, as a decisive technique for the structural analysis of phenolic glycolipids in their native form. The sugar type contained by the different phenolic glycolipids analyzed was characterized by two-dimensional 'H NMR COSY while the interglycosidic bonds and anomeric configurations were unambiguously as- signed by two-dimensional 'H NMR NOESY. In previous works the mannose and rhamnose anomeric configurations were dubiously attributed either by the Jl,z coupling constant or by the chemical shift of the H-1, H-5, and H-6 sugar protons under their native or peracetylated forms. An unam- biguous anomeric assignment results from the JISC-I,H.l cou- pling constant value determined on the 13C NMR undecou- pling spectra (25). However, this technique required an amount of sample which is often too large with regard to the mycobacteria cell-wall content. Thus, 'H NMR NOESY analysis is a key technique for the anomeric characterization of rhamnose on a small scale (1 mg). The proposed B-1, B-2, and B-3 structures differ from that of the B mycoside (2-0- Me-a-L-Rhap l 4 i a c y l phenolphthiocerol) either by the lip- idic or by the sugar parts. Glycolipid B-1 is distinguishable from mycoside B by the phenolphthiodolone aglycone struc- ture B-2 mainly by the sugar residue structure (a-L-Rhap) and B-3 by the disaccharidic structure of its oligosaccharide moiety. More interesting is the structural comparison of the M. bouis BCG phenolic glycolipid and those from M. tuber- culosis strain Canetti. From this study, we can conclude that glycolipids B, B-2, and probably B-1 are shared by M. bouis

BCG and M. tuberculosis. To date, the polar diglycosylated phenolic glycolipid (B-3) has not been described in M. tuber- culosis. But, the triglycosylated phenolic glycolipid found in M. tuberculosis strain Canetti (PheG1 T-I) differs from B-3 only by the distal permethylated Fucp monosaccharide resi- due. The M. leprae and M. kansasii phenolic glycolipid anti- genic determinants were characterized as the distal disaccha- ride unit. Thus, cross-reactivity between B-3 and the PheGl T-I immunoserum can be expected, leading to false positive enzyme-linked immunosorbent assay testing when PheGl T- I is used as marker for the screening of tuberculous patients recently vaccinated by M. bouis BCG.

Quantitatively minor phenolic glycolipids are also observed in M. leprae and M. kaansasii cell-walls. Their structures differ from that of major ones by the sugar type and by the methoxyl group location on the sugar ring, but present the same number of glycosidic units. In M. bouis BCG monoglycosylated (B, B- 1, B-2) and diglycosylated B-3 phenolic glycolipid antigens are observed. Thus, as for the M. auium complex, different subspecies could be identified according to their phenolic glycolipid antigen composition. Furthermore, if certain of these glycolipids are recognized by T-cell suppressors, an explanation might be offered for the apparently conflicting results observed in the protection by M. bouis BCG vaccina- tion against M. tuberculosis.

Acknowledgments-We wish to express our gratitude to Dr. Georghiu for preparing the M. bouis BCG culture and also Dr. G. Marchal for useful discussions. We thank Maryse Bon and Philippe Serval for recording NMR spectra.

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REFERENCES Gaylord, H., and Brennan, P. J. (1987) Annu. Rev. Microbiol. 4 1 ,

Hunter, S. W., Fujiwara, T., and Brennan, P. J. (1982) J. Biol.

Menzel, S., Harboe, M., Bergsvik, H., and Brennan, P. J. (1987)

Fujiwara, T., Hunter, S. W., Cho, S.-N., Aspinall, G. O., and

Mehra, V., Brennan, P. J., Rada, E., Convit, J., and Bloom, B. R.

Prasad, K. H., Mishra, R. S., and Nath, I. (1987) J. Ezp. Med.

Neill, M. A,, and Klebanoff, S. J. (1988) J. Exp. Med. 167 , 30-

Fournib, J.-J., Riviere, M., and Puzo, G. (1987) J. Biol. Chern.

Riviere, M., Fournib, J.-J., and Puzo, G. (1987) J. Biol. Chem.

Fournib, J.-J., Riviere, M., Papa, F., and Puzo, G. (1987) J. Biol.

645-675

Chem. 257,15072-15078

Znt. J . Leprosy 5 5 , 617-624

Brennan, P. J. (1984) Infect. Immun. 43,245-252

(1984) Nature 3 0 8 , 194-196

165,239-244

42

262 , 3174-3179

2 6 2 , 14879-14884

Chem. 262,3180-3184 Fournie, J.-J., Riviere, M., and Puzo, G. (1987) Eur. J. Biochem.

168,181-183 Daffb, M., Lacave, C., Lanbelle, M.-A., and Lanbelle, G. (1987)

Papa, F., Riviere, M., Fournib, J. J., Puzo, G., and David, H.

Torgal-Garcia, J., David, H., and Papa, F. (1988) Annu. Znst .

MacLennan, A. P., Randall, H. M., and Smith, D. W. (1961)

Daffi., M., LanBelle, M. A., Lacave, C., and Lankelle, G. (1988)

Mustafa, A. S., Kvalheim, G., Degre, M., and Godal, T. (1986)

Gastamhide-Odier, M., and Sarda, P. (1970) Pneurnologie 142 ,

Hunter, S. W., and Brennan, P. J. (1981) J. Bacteriol. 147,728-

Tardelli, E., Draper, P., and Payne, S. N. (1884) Carbohydr. Res.

Hansen, G., and Munson, B. (1978) Anal. Chem. 5 0 , 1130-1134

Eur. J. Biochem. 167 , 155-160

(1987) J. Clin. Microbiol. 2 5 , 2270-2273

Pasteur/Microbwl. 139 , 289-294

Biochem. J. 80,309-318

Biochem. Biophys. Acta 958,443-449

Infect. Zmrnun. 5 3 , 491-497

241-255

735

131,346-352

7454 New-found Phenolic Glycolipids in M. bovis BCG

22. Reinhold, V. N., and Carr, S. A. (1982) Anal. Chem. 54,499-503 23. Yu, R. K., Koerner, T. A. W., Scarsdale, J. N., and Prestegard,

24. Scarsdale, J. N., Yu, R. K., and Prestegard, J. H. (1986) J. Am.

25. Kasai, R., Okihara, M., Asakawa, J., Mizutani, K., and Tanaka,

26. Haverkamp, J., De Bie, M. J. A., and Vliegenthart, J. F. G. (1975)

27. Genvig, G. J., Kamerling, J. P., and Vliegenthart, J. F. G. (1978)

J. H. (1986) Chem. Phys. 42,27-48

Chem. SOC. 108,6778-6784

0. (1979) Tetrahedron 35 , 1427-1432

Carbohydr. Res. 39,201-211

Carbohydr. Res. 62,349-357

28. Rivibre, M., Fournie, J. J., Monsarrat, B., and Puzo, G. (1988) J.

29. Ciucanu, I., and Kereck, F. (1984) Carbohydr. Res. 131 , 209-217 30. Sarda, P., and Gastambide-Odier, M. (1967) Chem. Phys. Lipids

31. Rivibre, M., FourniB, J. J., Vercellone, A., and Puzo, G. (1988)

32. Westmore, J. B., and Alauddin, M. M. (1986) Mass Spectrom.

33. Egge, H., and Peter-Katalinie, J. (1987) Mass Spectrom. Rev. J.

Chromatogr. 446, 87-95

1,434-444

Biomed. Environ. Mass Spectrom. 1 6 , 275-278.

Rev. J . 5,381-465

6,331-393