Access the full text.
Sign up today, get DeepDyve free for 14 days.
Loading next page...
/lp/unpaywall/acylation-state-of-the-phosphatidylinositol-hexamannosides-from-i7Ghb8q6J2
References
References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.
- Publisher
- Unpaywall
- ISSN
- 0021-9258
- DOI
- 10.1074/jbc.m303446200
- Publisher site
- See Article on Publisher Site
Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 32, Issue of August 8, pp. 29880 –29889, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Acylation State of the Phosphatidylinositol Hexamannosides from Mycobacterium bovis Bacillus Calmette Gue ´ rin and Mycobacterium tuberculosis H37Rv and Its Implication in Toll-like Receptor Response* Received for publication, April 3, 2003, and in revised form, May 20, 2003 Published, JBC Papers in Press, May 29, 2003, DOI 10.1074/jbc.M303446200 Martine Gilleron‡§, Vale ´ rie F. J. Quesniaux , and Germain Puzo‡ From the ‡Institut de Pharmacologie et de Biologie Structurale du CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex and FRE 2358, Experimental and Molecular Genetics, 3B rue de la Ferrolerie, 45071 Orleans, Cedex 2, France wall. Among these compounds are the lipoarabinomannans The dimannoside (PIM ) and hexamannoside (PIM ) 2 6 phosphatidyl-myo-inositol mannosides are the two most (LAM) and the lipomannans (LM). The importance of these abundant classes of PIM found in Mycobacterium bovis lipoglycans in the immunopathogenesis of tuberculosis is now bacillus Calmette Gue ´ rin, Mycobacterium tuberculosis established, and a rising number of studies in the literature are H37Rv, and Mycobacterium smegmatis 607. Recently, devoted to the delineation of their biological activities (2, 3). these long known molecules received a renewed interest PIM, LM, and LAM derive from the same biosynthetic pathway due to the fact that PIM constitute the anchor motif of an 2 as demonstrated using biochemical (1, 2, 4, 5) and genetic important constituent of the mycobacterial cell wall, the approaches (6 – 8). PIM appear to be the common anchor of LM lipoarabinomannans (LAM), and that both LAM (phos- and LAM, as LM correspond to polymannosylated PIM and phoinositol-capped LAM) and PIM are agonists of Toll- then give rise to LAM by further glycosylation with arabinosyl like receptor 2 (TLR2), a pattern recognition receptor units. This anchor plays a fundamental role in the biological involved in innate immunity. Due to the biological impor- functions of LAM. Indeed, it is now clearly established that tance of these molecules, the chemical structure of PIM most of the LAM immunoregulatory effects are abolished by was revisited. The structure of PIM was recently pub- alkaline hydrolysis, highlighting the importance of the lipidic lished (Gilleron, M., Ronet, C., Mempel, M., Monsarrat, B., part of the anchor (2). Gachelin, G., and Puzo, G. (2001) J. Biol. Chem. 276, 34896 – Mycobacterium bovis BCG 1173P2 (the Pasteur strain) (1), 34904). Here we report the purification and molecular Mycobacterium smegmatis ATCC-607 (1), and Mycobacterium characterization of PIM in their native form. For the first tuberculosis H37Rv ATCC-27294 were found to mainly contain time, four acyl forms of this molecule have been purified, two PIM families, the dimmanosylated (PIM ) and the hexam- using hydrophobic interaction chromatography. Mono- to 2 annosylated (PIM ) ones. PIM , PIM , PIM , and PIM were tetra-acylated molecules were identified in M. bovis bacil- 6 1 3 4 5 observed in very small amounts, suggesting that they are bio- lus Calmette Gue ´ rin, M. tuberculosis H37Rv, and M. smeg- matis 607 using a sophisticated combination of analytical synthetic intermediates. tools, including matrix-assisted laser desorption/ioniza- PIM are known from the 1940s and have been structurally tion-time of flight-mass spectrometry and two-dimen- investigated by Ballou and co-workers in the 1960s (9). By 1965, sional homo- and heteronuclear NMR spectroscopy. studies of deacylated PIM from M. tuberculosis and These experiments revealed that the major acyl forms are Mycobacterium phlei revealed the structure of the saccharidic similar to the ones described for PIM . Finally, we show 2 part. PIM were the highest PIM which were fully character- that PIM , like PIM , activate primary macrophages to 6 2 ized from M. phlei (10) and were shown to contain a penta- secrete TNF- through TLR2, irrespective of their acyla- mannoside of sequence Manp132Manp132Manp13 tion pattern, and that they signal through the adaptor 6Manp136Manp13 attached to position 6 of the myo-inositol, MyD88. whereas a Manp unit is linked to the position 2 of the myo- inositol. Recently, the complete structure of native PIM has been achieved (1, 2). These last studies focused on the characterization A variety of phosphatidyl-myo-inositol mannosides (PIM) - of their lipidic part and unambiguously established the existence based compounds are known to be part of the mycobacterial cell of a tetra-acylated form that was thus far suggested (4). Several biological functions have been recently attributed to * This work was supported by the European Community through the PIM. PIM were shown to recruit natural killer T cells, which Fifth Framework Program TB vaccine program. The costs of publication have a primary role in the local granulomatous response (1, of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”inac- 11). Moreover, a role for surface-exposed PIM as M. tuberculo- cordance with 18 U.S.C. Section 1734 solely to indicate this fact. sis adhesins that mediate attachment to non-phagocytic cells § To whom correspondence should be addressed. Tel.: 33 5 61 17 55 has also been established (12, 13). Analysis of infected macro- 57; Fax: 33 5 61 17 59 94, E-mail: [email protected]. phages revealed that PIM, among other mycobacterial lipids, The abbreviations used are: PIM, phosphatidyl-myo-inositol man- nosides; BCG, bacillus Calmette Gue ´ rin; BLP, bacterial lipopeptide; are actively trafficked out of the mycobacterial phagosome (14). C , palmitate; C , stearate; C , tuberculostearate (10-methyloctadec- 16 18 19 This could be of particular importance relating to the potential anoate); COSY, correlation spectroscopy; ESI-MS, electrospray ioniza- tion-mass spectrometry; Gro, glycerol; HABA, 2-(4-hydroxyphenylazo)- benzoic acid; HMQC, heteronuclear multiple quantum correlation spec- MyD88, myeloid differentiation factor; p, pyranosyl; PI, phosphatidyl- troscopy; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; LAM, myo-inositol; QMA, quaternary methyl ammonium; ROESY, rotating lipoarabinomannans; LM, lipomannans; ManLAM, LAM with manno- frame nuclear Overhauser effect spectroscopy; t, terminal; TLR, Toll- syl extensions; MALDI-Tof-MS, matrix-assisted laser desorption/ like receptor; TMS, trimethylsilyl; TNF-, tumor necrosis factor-; IL, ionization-time of flight-mass spectrometry; myo-Ins, myo-inositol; interleukin; LPS, lipopolysaccharide. This is an Open Access article under the CC BY license. 29880 This paper is available on line at http://www.jbc.org PIM Acyl Forms and TLR Response 29881 role played by these constituents in extending the influence of the bacterium over its surroundings. An unfractionated prep- aration of PIM, as well as phosphoinositol capped LAM (PI- LAM), was recently shown to activate cells via Toll-like recep- tor-2 (TLR-2) (15). Activation of TLR-dependent signaling pathways leads to the activation of genes that participate in innate immune responses, such as expression of cytokines, coactivation molecules, and nitric oxide (16, 17). Finally, PIM as well as ManLAM from Mycobacterium leprae and M. tuber- culosis are presented by antigen-presenting cells in the context of CD1b (18). The high affinity interaction of CD1b molecules with the PIM acyl side chains was then established (19). The FIG.1. Negative MALDI mass spectrum of the PIM -enriched phosphatidylinositol moiety plays a central role in the process fraction C from M. bovis BCG, containing 1, 2, 3, and 4 fatty acyl appendages. of PIM and ManLAM binding to CD1b proteins. Here we investigated the structure of the most polar PIM isolated from M. bovis BCG, PIM . The earlier NMR studies Acetolysis Procedure—200 g of PIM were treated with 200 lof conducted on PIM and PIM by Severn et al. (20) focused on 2 6 anhydrous acetic acid-d /acetic anhydride-d , 1:1 (v/v), at 110 °C for 4 6 the deacylated molecules, thus excluding the study of the lip- 12 h. The reaction mixture was dried under stream of nitrogen and was submitted to acetylation. The mixture was dissolved in acetic anhy- idic moieties. In this study, we investigated the chemical struc- dride/anhydrous pyridine, 1:1 (v/v), at 80 °C for 2 h. The reaction mix- ture of native PIM , focusing on the characterization of the ture was dried under stream of nitrogen. 20 l of chloroform/methanol, different “acyl forms,” using sophisticated analytical tools such 9:1 (v/v), was added and analyzed in MALDI-Tof-MS in positive and as MALDI-MS and two-dimensional NMR. Then we demon- negative modes. strated the capacity of the PIM and PIM acyl forms to stim- 2 6 Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry ulate macrophages to produce cytokines, and we investigated (MALDI-Tof-MS)—Analysis by MALDI-Tof-MS was carried out on a Voyager DE-STR (PerSeptive Biosystems, Framingham, MA) using the the implication of the different TLR in this process. reflectron mode. Ionization was effected by irradiation with pulsed UV EXPERIMENTAL PROCEDURES light (337 nm) from an N laser. PIM were analyzed by the instrument operating at 20 kV in the negative ion mode using an extraction delay PIM Extraction—The PIM-containing lipidic extract was obtained time set at 200 ns. Typically, spectra from 100 to 250 laser shots were through purification of the phenolic glycolipids from M. bovis BCG summed to obtain the final spectrum. All of the samples were prepared 1173P2 (the Pasteur strain) (21) and was briefly summarized in Ref. 1. for MALDI analysis using the on-probe sample cleanup procedure with An acetone-insoluble phospholipids-containing lipid extract was pre- cation-exchange resin (22). The HABA matrix (from Sigma) was used at pared (1) and applied to a QMA-Spherosil M (BioSepra SA, Villeneuve- a concentration of 10 mg/ml in ethanol/water (1:1, v/v). Typically, 0.5 la-Garenne, France) column that was first irrigated with chloroform, l of PIM sample (10 g) in a CHCl /CH OH/H O solution and 0.5 lof chloroform/methanol (1:1, v/v), methanol in order to elute neutral com- 3 3 2 the matrix solution, containing 5–10 cation exchange beads, were pounds. Phospholipids were eluted using ammonium acetate containing deposited on the target, mixed with a micropipet, and dried under a organic solvents. Indeed, 0.1 M ammonium acetate in chloroform/meth- gentle stream of warm air. The measurements were externally cali- anol (1:2, v/v) (fraction A) allowed elution of 750 mg of phospholipids brated at two points with PIM. (enriched in phosphatidyl-myo-inositol di-mannosides (PIM )),and 0.2 M NMR Analysis—NMR spectra were recorded with an Avance ammonium acetate in chloroform/methanol (1:2, v/v) (fraction B) was DMX500 spectrometer (Bruker GmbH, Karlsruhe, Germany) equipped subdivided into two fractions. The first volumes allowed elution of 440 with an Origin 200 SGI using Xwinnmr 2.6. Samples were dissolved in mg of phospholipids (cardiolipids essentially), and the next ones al- CDCl /CD OD/D O, 60:35:8 (v/v/v), and analyzed in 200 5-mm lowed elution of 160 mg of phospholipids (mixture of phosphatidyl-myo- 3 3 2 535-PP NMR tubes at 308 K. Proton chemical shifts are expressed in inositol dimannosides (PIM ) and hexamannosides (PIM )), and finally, 2 6 ppm downfield from the signal of the chloroform ( /TMS 7.27 and 0.2 M ammonium acetate in methanol (fraction C) allowed elution of 55 H /TMS 77.7). The one-dimensional phosphorus ( P) spectra were mg of phospholipids (enriched in PIM ). Repeated lyophilizations were C measured at 202 MHz with phosphoric acid (85%) as external standard necessary to eliminate ammonium acetate salts. PIM from M. smegma- (p 0.0). All the details concerning NMR sequences used and experi- tis (ATCC 607) and M. tuberculosis H37Rv (ATCC 27294) were also mental procedures were detailed in previous study on PIM (1). analyzed. 2 Primary Macrophage Cultures—TLR2- and/or TLR4-deficient mice Purification of the PIM Acyl Forms—Fraction C (20 mg) was loaded obtained by inter-cross from TLR4-deficient mice (23) and TLR2-defi- in 0.1 M ammonium acetate solution containing 15% (v/v) propanol-1 to cient mice (24), TLR6-deficient mice (25) and MyD88-deficient mice octyl-Sepharose CL-4B (Amersham Biosciences) column (20 1.5 cm) (26), and their control littermates were bred under specific-pathogen- pre-equilibrated with the same buffer. The column was first eluted with free conditions in the Transgenose Institute animal breeding facility 50 ml of equilibration buffer and then with a linear propanol-1 gradient (Orle ´ ans, France). Murine bone marrow cells were isolated from femurs from 15 to 65% (v/v) (250 ml each) in 0.1 M ammonium acetate solution and cultivated (10 /ml) for 7 days in Dulbecco’s minimal essential at a flow rate of 5 ml/h. The fractions were collected every 30 min. 20 l medium supplemented with 20% horse serum and 30% L929 cell-con- of each fraction was dried and submitted to acidic hydrolysis (100 lof ditioned medium (as source of M-CSF, as described in Ref. 27). Three trifluoroacetic acid, 2 M,2hat110 °C). The hydrolysates were dried, days after washing and re-culturing in fresh medium, the cell prepara- reconstituted in water, and then analyzed by high pH anion exchange tion contained a homogenous population of macrophages. The bone chromatography for mannose content giving the presented chromato- marrow-derived macrophages were plated in 96-well microculture gram (Fig. 2A). Fractions were pooled according to the purification plates (at 10 cells/well) and stimulated with LPS (Escherichia coli, profile, and repeated lyophilizations were performed to eliminate am- serotype O111:B4, Sigma, at 100 ng/ml), bacterial lipopeptide (Pam - monium acetate salts. An acetone precipitation step was done on each 3 Cys-Ser-Lys , EMC microcollections; at 0.5 g/ml), or PIM preparations fraction in order to eliminate contaminants issued from the propanol-1. 4 at the indicated concentration. Lyophilized PIM preparations were Finally, 1.2 (fraction I), 1 (fraction II), 7.5 (fraction III), and 3 mg solubilized in Me SO and added in the cultures at a non-cytotoxic final (fraction IV) were obtained. 2 concentration of 1–1.5% Me SO (cell viability monitored by 3-(4,5-di- Purification was checked by TLC on aluminum-backed plates of silica 2 methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). gel (Alugram Sil G, Macherey-Nagel, Duren, Germany), using chloro- Alternatively the macrophages were infected with M. bovis BCG form/methanol/water, 60:35:8 (v/v/v), as migration solvent. A sulfuric (Pasteur strain 1173P2; kind gift from G. Marchal, Pasteur Institute, anthron spray and a Dittmer-Lester spray were used to detect carbo- Paris, France; at a multiplicity of infection of 2 bacteria per cell). After hydrates containing lipids and phosphorus-containing lipids, 18 h of stimulation, the supernatants were harvested and analyzed for respectively. cytokine content using commercially available enzyme-linked immu- nosorbent assay reagents for TNF- and IL-12p40 (DuosetR&D M. Gilleron, V. F. J. Quesniaux, and G. Puzo, unpublished data. Systems, Abingdon, UK). 29882 PIM Acyl Forms and TLR Response FIG.2. A, octyl-Sepharose chromatography of the PIM -enriched fraction C from M. bovis BCG. The column was eluted with a propanol-1 linear gradient (15– 65% (v/v)) in 0.1 M ammonium acetate. Fractions were analyzed by high pH anion exchange chromatography for their mannose content. B–F, negative MALDI mass spectra of the fractions I–IV of the octyl-Sepharose chromatography. Fatty acyl compositions were based on the most abundant fatty acyl chains found by gas chromatography-mass spectrometry analysis, i.e. palmitate (C ), tuberculostearate (C ), and 16 19 for a lesser extent stearate (C ) and heptadecanoate (C ). B and C, fraction I corresponded to mono-acylated forms, acylated by 1C (m/z 1543.6) 18 17 16 or 1C (m/z 1585.7) (a, fraction Ia corresponded to fraction 52 of the octyl-Sepharose, and b, fraction Ib corresponded to fraction 62 of the octyl-Sepharose). D, fraction II corresponded to di-acylated forms, acylated by 2C (m/z 1781.8) and C ,C (m/z 1823.9) and in a minor part by 16 16 19 C ,C (m/z 1795.8). E, fraction III corresponded to tri-acylated forms, acylated predominantly by 2C ,1C (m/z 2062.1), and in a minor part by 16 17 16 19 C ,C ,C (m/z 2090.1). F, fraction IV corresponded to tetra-acylated forms, acylated predominantly by 3C ,1C (m/z 2300.3) and 2C ,2C (m/z 16 18 19 16 19 16 19 2342.4). The ions at m/z 2314.3 corresponded to PIM esterified by 2C ,2C . Minor ions at m/z 2272.3, 2286.3, 2328.4, and 2356.4 were attributed 6 16 18 to PIM esterified by 3C ,1C ,3C ,1C ,2C ,1C ,1C , and 1C ,2C ,C or 1C ,1C ,2C , respectively. Species esterified by unsaturated 6 16 17 16 18 16 18 19 16 18 19 16 17 19 fatty acids were also present as each peak consisted of a 2 analog. RESULTS and stearic (C ) acids and in minor amounts heptadecanoic acids (C ), the fatty acid composition of each acyl form was Purification of the PIM Acyl Forms from M. bovis BCG—An determined. We then confirmed mono-acylated forms (1Ac) enriched fraction of phosphatidyl-myo-inositol hexamanno- with C (m/z 1543.6) or C (m/z 1585.7), di-acylated forms sides (PIM ) was purified from M. bovis BCG as described 16 19 (2Ac) with 2C (m/z 1781.8) or 1C ,1C (m/z 1823.9), tri- previously (fraction C (1)). Briefly, PIM are known to be found 16 16 19 acylated forms (3Ac) mainly with 2C ,1C (m/z 2062.1), and in the acetone-insoluble fraction of mycobacterial lipidic ex- 16 19 tetra-acylated forms (4Ac) mainly constituted by 3C ,1C tract (28). The contaminating neutral compounds were elimi- 16 19 (m/z 2300.3) or 2C ,2C (m/z 2342.4). M. tuberculosis H37Rv nated by QMA anion exchange chromatography, irrigated with 16 19 was found to produce as major PIM families PIM and PIM in neutral eluents. The phospholipids were then eluted with am- 2 6 the same acyl forms as the ones described for M. bovis BCG (not monium acetate-containing organic solvents resulting in three shown). fractions, A–C. These fractions were analyzed by ESI-MS in To proceed in the separation of the acyl forms, 20 mg of negative mode (1) and revealed that fraction A mainly includes fraction C were applied on an octyl-Sepharose column, using PIM containing a total of three and four fatty acids in addition to phosphatidyl-myo-inositol; fraction B contains PIM and propanol-1 as eluent (Fig. 2A). The different acyl forms were eluted at concentrations of propanol-1 ranging from 25 to 50% PIM containing a total of three and four fatty acids, and fraction C mainly contains the different acyl forms of PIM . and separated into 4 sub-fractions according to the profile elution determined by the mannose content. Each sub-frac- Fig. 1 presents the negative MALDI spectrum of M. bovis BCG fraction C that is dominated by peaks assigned to deprotonated tion (I to IV) was collected and analyzed by negative MALDI-Tof-MS. molecular ions (M H) revealing the different acyl forms of PIM present in this fraction. From the predominant fatty MALDI-Tof-MS Characterization of the Octyl-Sepharose Col- acids deduced from gas chromatography-mass spectrometry umn Fractions—The mass spectra of the fraction I (Fig. 2, B–C) analysis (not shown), i.e. palmitic (C ), tuberculostearic (C ), revealed that it contained mono-acylated forms of the mole- 16 19 PIM Acyl Forms and TLR Response 29883 1 13 FIG.3. H- C HMQC spectrum of the tetra-acylated forms. Fraction IV of the octyl-Sepharose chromatography of the PIM was dissolved in CDCl /CD OD/D O, 60:35:8 (v/v/v), and the NMR experiment was realized at 308 K. Mannose units are labeled from I to VI; numbers with Ins 3 3 2 correspond to the proton number of the myo-Ins unit, and numbers with letter G correspond to the proton number of the glycerol unit. cules (1Ac). Indeed, the analysis of fraction Ia revealed depro- PIM , corresponding to mono-, di-, tri-, and tetra-acyl forms. tonated molecular ions at m/z 1543.6 characterizing mono- The structural analysis is further detailed below for the most acylated forms with C (Fig. 2B), whereas mono-acylated complex entities, tri- and tetra-acyl forms. forms with C appeared more abundant in fraction Ib (m/z Glycosidic Analysis of Native Tetra-acyl Forms—The se- 1585.7) (Fig. 2C). The mass spectrum corresponding to fraction quence of glycosyl residues in PIM was established using a II showed three peaks with an intensity above 10% and were range of high resolution NMR techniques applied to the tetra- assigned to (M H) ions of di-acylated forms: two major acylated molecules. The H NMR spectrum of the native mol- peaks at m/z 1781.8 and 1823.9 and one minor one at m/z ecules in CDCl /CD OD/D O, 60:35:8 (v/v/v), at 500 MHz 3 3 2 1795.8 (Fig. 2D). The two major peaks were characterized as showed a complex anomeric proton region (between 4.4 and 5.1 (M H) ions of di-acylated forms containing 2C and ppm) (Fig. 3). Anomeric signals were investigated thanks to the 1 13 1C ,1C , respectively, and the minor peak corresponded to H- C HMQC spectrum (Fig. 3). Indeed, from the protons 16 19 (M H) ions of the molecules acylated by 1C ,1C . The resonating between 4.4 and 5.1 ppm, two of them correlated 16 17 mass spectrum of fraction III (Fig. 2E) revealed two peaks of with carbons out of the anomeric zone: proton at 5.02 corre- intensity superior to 10% assigned to tri-acylated forms of the lated with a carbon at 70.6 ppm and proton at 4.53 correlated molecules. The major peak at m/z 2062.1 was attributed to with a carbon at 71.7 ppm. From previous studies (1, 2), they (M H) ions corresponding to tri-acylated forms containing were respectively assigned to H2 of Gro and H3 of myo-Ins. 2C ,1C , whereas the minor one at m/z 2090.1 contains These protons are deshielded due to the presence of gem acyl 16 19 1C ,1C , and 1C . The mass spectrum of fraction IV ap- group. Moreover, the coupling constants measured on the pro- 16 18 19 peared more complex, constituted by a series of peaks (Fig. 2F) ton at 4.53 (J 2.4 Hz and J 10.6 Hz) confirmed its 2,3 3,4 between m/z 2230.2 and 2356.4 and assigned to (M H) ions attribution to myo-Ins H3. The other proton resonances corre- of different tetra-acylated forms. Indeed, the most abundant lating with carbons resonating around 100 ppm accounted for (M H) ions at m/z 2300.3 and 2342.4 characterized tetra- the six mannose units labeled from I to VI in decreasing order acylated forms containing 3C ,1C and 2C ,2C , whereas of their chemical shifts. Indeed, integration of the anomeric 16 19 16 19 the ions at m/z 2314.3 corresponded to tetra-acylated forms signals (H1) proved that the signal resonating at 4.88 ppm containing 2C ,2C . Minor ions at m/z 2272.3, 2286.3, 2328.4, corresponded to two protons (II and III ). The -anomeric 16 18 1 1 and 2356.4 were attributed to the molecules esterified by configuration of the mannoses was deduced from the values of 3C ,1C ,3C ,1C ,2C ,1C ,1C , and 1C ,2C ,1C or the one bound coupling constant ( J ) above 170 Hz, meas- 16 17 16 18 16 18 19 16 18 19 C1,H1 1 13 1C and 1C ,2C , respectively. In addition, species esterified ured on non-decoupled H- C HMQC spectrum (not shown). 16 17 19 by unsaturated fatty acids were also present as each peak The H2 protons of each -Manp unit was determined using consisted of a 2 analog. the COSY spectrum (Fig. 4A) although H2 of system I was Therefore, we have developed a powerful preparative method partly hidden by the H3/H3 of glycerol (Gro). The entire spin of fractionation, leading to the purification of four purified system of Gro can be analyzed from the COSY spectrum (Fig. 29884 PIM Acyl Forms and TLR Response 1 1 FIG.4. Expanded region ( F 4.45–5.1 and F 3.0 – 4.4) of the H- H COSY (A), HOHAHA (B), and ( F 4.45–5.1 and F 3.0 – 4.4) of 2 1 2 1 1 1 the H- H ROESY (C) spectra of the tetra-acylated forms at 308 K. The product was dissolved in CDCl /CD OD/D O, 60:35:8 (v/v/v). Mannose 3 3 2 units are labeled from I to VI; numbers with Ins correspond to the proton number of the myo-Ins unit, and numbers with letter G correspond to the proton number of the glycerol unit. C, intra-residual contacts are expressed only with a number, whereas inter-residual contacts are expressed with the roman number of the concerned unit followed by the number corresponding to the proton number of the unit. 4A) and those of the myo-Ins from the HOHAHA spectrum (Fig. the C2 of units I ( 78.5) and IV ( 79.1) and of C6 of units II ( 4B). The six mannose spin systems could be completely as- 65.8) and VI ( 66.3) revealed the sites of glycosylation of the 1 1 1 13 signed (Table I) using H- H HOHAHA (Fig. 4B) and H- C concerned units. The absence of deshielding concerning the HMQC (Fig. 3). The deshielded values of the chemical shifts of carbons of systems III and V indicated that these units III and PIM Acyl Forms and TLR Response 29885 TABLE I 1 13 H and C NMR chemical shifts of the tetra-acylated forms measured at 308 K in CDCl /CD OD/D O, 60:35:8, v/v/v 3 3 2 1 2 3 4 5 6 Conclusion I 5.02 3.83 3.64 3.39 3.48 3.47/3.64 2-O-Linked 101.0 78.5 70.4 67.3 73.4 61.5 II 4.88 3.92 3.60 3.49 3.65 3.48/3.70 6-O-Linked 101.8 70.1 70.9 67.0 70.5 65.8 III 4.88 3.84 3.58 3.47 3.78 4.02/4.15 Terminal, 6-O-Acylated 101.8 70.1 70.8 67.1 70.7 63.9 IV 4.86 3.70 3.68 3.42 3.42 3.51/3.60 2-O-Linked 98.3 79.1 70.5 67.3 73.0 61.4 V 4.78 3.78 3.53 3.37 3.50 3.47/3.65 Terminal 102.3 70.4 71.0 67.4 71.0 61.5 VI 4.63 3.72 3.59 3.42 3.55 3.54/3.65 6-O-Linked 99.7 70.3 70.8 67.3 70.9 66.3 myo-Ins 3.97 4.06 4.53 3.55 3.17 3.60 3-O-Acylated 76.8 76.5 71.7 70.9 73.1 78.7 Gro 4.19/3.95 5.02 3.79/3.76 1,2-Di-O-Acylated 62.9 70.6 63.6 V were terminal. The chemical shift of the C6 of unit III ( 63.9) Manp, could be assigned by analysis of the two-dimensional 1 13 (Table I) was intermediate between those of the C6 of units I, H- C HMQC spectrum. Indeed, the chemical shifts of H-6/ IV, and V (around 61.5 ppm) that were not glycosylated in C6 H-6 of -Manp unit III (4.02/4.15 ppm) proved that this posi- and those of the C6 of units II and VI (around 66.0) that were tion is also acylated. The slight deshielding of C6 resonance glycosylated in C6. Moreover, H6/H6 protons of unit III were (from 61.5 to 63.9 ppm) confirmed this assignment. deshielded (4.02:4.15) (Table I). Thus, these data demonstrated Therefore, the analytical approach applied to this acyl form that this unit is acylated in position 6. of PIM (containing four fatty acids in total) revealed that the The glycosidic sequence was next deduced from the inter- four positions of acylation were the same as the ones described residual nuclear Overhauser effect contacts observed in the in case of the corresponding acyl form of PIM (1): the positions 1 1 H- H ROESY spectrum (Fig. 4C). This sequence was used C1 and C2 of the Gro, position C3 of the myo-Ins unit, and here to observe short through space connectivities between the position C6 of the Manp unit linked to C2 of the myo-Ins. anomeric proton of each mannose and the proton of the adja- Acyl Distribution of Native Tetra-acyl Forms—The nature of cent glycosidically linked residue. The anomeric proton of spin the fatty acids esterifying the different sites was investigated system I ( 5.02) correlated with H2 of system IV, indicating by mass spectrometry using MALDI-Tof-MS analysis of the that -Manp unit I is linked to the C2 position of -Manp unit acetolysis reaction products of the native tetra-acyl forms of the IV. In the same way, the occurrence of cross-peaks relating H1 molecules (not shown). Acetolysis cleaves the phosphoglycerol (IV)/H6 (VI), H1 (V)/H2 (I), and H1 (VI)/H6 (II) established the linkage without altering the fatty acid esters, leading to two partial linear sequence V-(132)-I-(132)-IV-(136)-VI-(136)- entities: the hexamannosyl-inositol phosphate moiety (Man - II. Anomeric protons of -Manp units II and III were then Ins-P) and the acyl-glycerol residue, as already described in separated showing both correlations with protons belonging to Ref. 1. The hexamannosyl-inositol phosphate moiety was ob- the Ins unit. served in negative mode as [M H] ions, whereas the acyl- The H1 of system III showed a nuclear Overhauser effect glycerol part was analyzed in positive mode as [M Na] ions. contact with proton 2 of myo-Ins, revealing that this unit is As described previously, the acetolysis reaction produces two linked in position 2 of the myo-Ins. The H1 of system II showed populations of Man -Ins-P moieties that differ by the presence two cross-peaks with protons 1 and 6 of myo-Ins. As explained or absence of an acetyl group on the phosphate (1). But as this 1 31 below, two-dimensional H- P HMQC analysis allowed us to acetate present on the phosphate is very labile (mixed anhy- define one of the positions of substitution of the phosphorus as dride), it is partially hydrolyzed when the sample is mixed with position 1 of the myo-Ins. Thus, system II was deduced to be the matrix (HABA diluted in EtOH/H O). To discriminate be- linked in position 6 of the myo-Ins. tween acetate groups and changes of C19 by C16, the reaction Therefore, the glycosidic analysis of the tetra-acylated form was made with perdeuterated acetic acid and acetic anhydride. of the molecules using a combination of scalar and dipolar The positive MALDI-Tof-MS spectrum of the perdeutero- homonuclear and heteronuclear NMR sequences demonstrated acetolyzed of the tetra-acylated molecules showed an intense the structure t--Manp-(132)--Manp-(132)--Manp-(136)- peak at m/z 678.6 assigned to sodium adduct of the di-acylated -Manp-(136)--Manp-(136)-myo-Ins-(241)-t--Manp,asde- C /C Gro (not shown). The negative mass spectrum mainly 16 19 picted in Fig. 5. showed two peaks at m/z 2653.7 and 2695.8 corresponding to Anchor Structure of Native Tetra-acyl Forms—The NMR the Man -Ins-P moiety acylated with 2C and C ,C , respec- 6 16 16 19 strategy developed to study PIM containing a total of 4 fatty tively. Less intense peaks were also observed corresponding to 1 31 acids (1) was used here. From the H- P HMQC experiment, the Man -Ins-P moiety acylated with other combinations of the prochiral H-3 and H-3 Gro protons and the myo-Ins H-1 fatty acids. Taken together, these results indicate that the were easily assigned (not shown). The remaining myo-Ins and glycerol is always acylated by C ,C , and we can postulate 16 19 1 31 Gro protons were then observed on the two-dimensional H- P that the mannose is acylated by a C , whereas the nature of HMQC-HOHAHA spectrum (Fig. 6B) and were assigned from the fatty acid present on the inositol is variable, being predom- their multiplicity and chemical shifts (29) and with the help of inantly C or C . 16 19 1 1 1 13 the H- H HOHAHA spectrum (Fig. 6, C–D). The different Analysis of Native Tri-acyl Forms— H- C HMQC spectrum chemical shifts typified the presence or absence of an acyl of the tri-acylated molecules exhibited the same cross-peaks as appendage. The chemical shifts of the H-1 (4.19/3.95 ppm) and for the tetra-acylated one, except for the inositol spin system H-2 (5.02 ppm) revealed a di-acyl-Gro (29). The downfield shift defined as H1/C1 3.93/70.64, H2/C2 4.04/78.68, H3/C3 of the myo-Ins H-3 resonating at 4.53 ppm then signed the C3 3.27/70.63, H4/C4 3.36/67.63, H5/C5 3.10/73.68, and acyl appendage. A fourth position of acylation, the C6 of the H6/C6 3.60/79.03, indicating that there is no fatty acyl append- 29886 PIM Acyl Forms and TLR Response FIG.5. Structural model of PIM acylated by four fatty acids in total, here 3C /1C . 6 16 19 showed a similar intense peak at m/z 678.6 assigned to the sodium adduct of the di-acylated C /C Gro as the one ob- 16 19 served in the case of the tetra-acylated molecules (not shown). The negative mass spectrum mainly showed one peak at m/z 2460.5 corresponding to the Man -Ins-P moiety acylated with one 1C . Thus, the results indicate that the tri-acylated mol- ecules predominantly exist with 2C and 1C , in agreement 16 19 with the negative MALDI-Tof analysis of the native molecules (Fig. 2E), the glycerol being di-acylated by C /C and the 16 19 mannose bearing a C . Macrophage Stimulation by PIM and PIM —Unfraction- 2 6 ated PIM have been shown to stimulate the murine RAW 264.7 monocytic cell line to produce TNF- (15), and we asked whether purified PIM and PIM were equally pro-inflamma- 2 6 tory. First, unfractionated PIM preparation was tested and shown to stimulate TNF- and IL-12 p40 secretion by primary murine macrophages (not shown). Then two different well de- fined acyl forms of PIM were assayed as follows: a fraction (F6) containing lyso-PI and lyso-PIM (both containing C ) and a 2 16 fraction (F7) containing more acylated forms of PIM (tri-acy- lated and tetra-acylated molecules). Both PIM fractions in- duced TNF- secretion irrespective of their acyl forms (Fig. 7). The concentration of TNF- secreted was clearly sub-maximal (1 ng/ml) as compared with those achieved by stimulation with strong stimuli such as LPS or BLP or after infection with live BCG (15 ng/ml; Fig. 7A). We next asked whether PIM exhibited a similar function. The unfractionated PIM prepa- ration (F1) also stimulated TNF- secretion by macrophages (Fig. 7B). To determine whether the number of fatty acids played a role in the stimulation of TNF- secretion, the purified FIG.6. NMR analysis of the phosphate substituents of the tet- acyl forms of PIM (F2 to F5) were tested for their capacity to 1 1 6 ra-acylated forms. Expanded region (A)( H, 3.00 –5.20) of the H stimulate macrophages to produce TNF-. No clear enrichment one-dimensional spectrum. Expanded region (B)( H, 3.00 –5.20, and 31 1 31 in stimulatory activity was observed with the purified PIM P, 2.00 –7.00) of the H- P 55 ms HMQC-HOHAHA spectrum. Ex- panded zone (C)( H, 3.00 –5.20 and 3.00 –3.30) of the two-dimensional acylated forms (mono- to tetra-acylated molecules) as compared 1 1 H- H HOHAHA spectrum showing the myo-Ins spin systems. Ex- with the unfractionated PIM (Fig. 7B). Only marginal levels of panded zone (D)( H, 3.00 –5.20 and 4.90 –5.20) of the two-dimensional 1 1 IL-12p40 were detected after incubation of macrophages with H- H HOHAHA spectrum showing the Gro spin systems. The product PIM or PIM (data not shown). was dissolved in CDCl /CD OD/D O, 60:35:8 (v/v/v), and all NMR ex- 2 6 3 3 2 periments were realized at 308 K. Thus, both PIM and PIM stimulated TNF- secretion by 2 6 primary macrophages, and this activity seemed independent age on C3. The three fatty acids were found on both positions of from the number and the nature of the PIM and PIM acyl 2 6 Gro and on the -Manp unit on the C2 of myo-Ins. The positive moieties. MALDI-Tof-MS spectrum of deutero-acetolyzed molecules TLR Dependence of PIM Activity—The unfractionated PIM PIM Acyl Forms and TLR Response 29887 FIG.7. Induction of TNF- by primary macrophages stimulated with BCG or PIM - and PIM -purified fractions. Murine bone 2 6 marrow-derived macrophages were infected for 24 h with BCG (at a multiplicity of infection of 2; A) or incubated with PIM and PIM fractions 6 2 (F1 to F7) (all PIM fractions at 30 g/ml; B). F1 corresponds to the PIM family, i.e. a fraction containing all the acyl forms. F2, F3, F4, and F5 correspond to mono- (Ac1), di- (Ac2), tri- (Ac3), and tetra-acylated (Ac4) forms, respectively; F6, a fraction containing lyso-PI and lyso-PIM (both containing C ); F7, a fraction containing more acylated forms of PIM (tri-acylated and tetra-acylated molecules). TNF- was measured in the 16 2 supernatants. Results are mean S.D. from n 2 mice and are from one representative experiment out of two independent experiments. preparation was shown to be a TLR2 agonist, based on reporter for allowing us in a first step to separate PIM from PIM using 2 6 assay with Chinese hamster ovary cell lines transfected with a QMA column and in a second step to separate acyl forms the tlr2 gene (15). Here bone marrow-derived macrophages using an octyl-Sepharose column, similar to that used to purify prepared from mice rendered deficient for TLR2 and/or TLR4, LAM acyl forms, as described by Leopold and Fischer (33) and for TLR6, or for MyD88, the adaptor common to the different applied in our laboratory by Nigou et al. (34). The purification TLR, were used to investigate the TLR dependence of the PIM of the PIM family from M. bovis BCG as well as M. smegmatis and PIM responses. The macrophages were stimulated with 607 and M. tuberculosis H37Rv revealed that very small quan- the fraction (F6) containing lyso-PI and lyso-PIM (both con- tities of intermediate forms (PIM , PIM , PIM , and PIM ) 1 3 4 5 taining C ), and the fraction (F7) containing more acylated 16 were found and that the more polar PIM corresponded to PIM forms of PIM (tri-acylated and tetra-acylated molecules) or 2 and not PIM , in contrast to early conclusions (35) and still with the PIM fraction (F1) and TNF- (Fig. 8) and IL-12 p40 6 reported in the literature (5, 7, 36). The purification to homo- (not shown) secretions were assessed. The production of TNF- geneity of the different PIM using isocratic silicic flash chro- by primary macrophages in response to the PIM and PIM 2 6 matography was unsuccessful, except for the PIM containing fractions is dependent on TLR2, as no TNF- could be detected four fatty acyl appendages in total (2). Indeed, as shown on Fig. in the supernatant of macrophages deficient for TLR2, al- 9, the more polar PIM co-migrate with the different acyl forms though cells deficient for TLR4 were efficiently stimulated by of PIM . these fractions (Fig. 8, B and C). As expected, no cytokine The structural strategy used to characterize PIM acyl forms production was detected in the supernatants of macrophages was similar to the one successfully used to define PIM acyl isolated from the double knock-out mice (TLR2/ and forms (1) and combines the potency of complementary analyt- TLR4/) (Fig. 8, B and C). Thus, PIM are capable of induc- ical techniques, NMR and mass spectrometry. NMR was used ing TNF- secretion via a TLR2-dependent pathway. TLR2 has to characterize the acylation positions, whereas mass spec- been shown to heterodimerize with TLR1 or TLR6 (30), and we trometry gave information concerning the number of fatty ac- next looked at the implication of TLR6 in the PIM-TLR2 inter- ids and the nature of the fatty acids present at each position. action. TLR6 deficiency did not impair the ability of the macro- MALDI or ESI modes were chosen rather than fast atom bom- phages to respond to PIM or PIM (Fig. 8, D–F). MyD88 is 2 6 bardment, because the molecules could be analyzed without involved in most of the TLR-mediated signals (31), including derivatization preventing the loss of any labile substituents. In TLR2 as shown here for the TLR2 agonist BLP and for the best this study, MALDI-MS was chosen rather than ESI-MS as a of the response to the TLR4 agonist LPS (Fig. 8D), and we same deposit could be analyzed in positive or negative mode. showed that macrophages deficient for MyD88 did not respond Indeed, concerning the analysis of the acetolysis products, the to the PIM or PIM stimulation (Fig. 8, E and F). Thus, both 2 6 hexamannosyl-inositol phosphate moiety (Man -Ins-P) was ob- PIM and PIM stimulate TNF- secretion by macrophages 2 6 served in negative mode, whereas the acyl-glycerol residue was through TLR2 and not TLR4. TLR6 is not essential in this analyzed in positive mode. The complete NMR study required response which involves signaling through the adaptor MyD88. some milligrams of purified product. This was detailed for the DISCUSSION more complex acyl forms, the tri-acylated and the tetra-acy- lated ones. The present study constitutes the first complete structural The results obtained are summarized in Table II. The mono- analysis of the native PIM from M. bovis BCG allowing the acylated forms are identified for lyso-PIM with C or C in definition of the structure of the tetra-acylated form of the 6 16 19 position 1 of the glycerol. The di-acylated forms appear as two molecule as depicted in Fig. 5. The saccharidic part is in com- populations almost equally represented as one with 2C and plete agreement with that proposed in the 1960s (for a review, 16 one with 1C and 1C , both fatty acids being on the glycerol see Ref. 32) and determined for the deacylated PIM by Severn 16 19 and structurally corresponding to “true” PIM . In contrast, et al. (20). Beyond this, the analysis presented here allows us to concerning tri-acylated forms, a major acyl form was observed, discriminate the different structures hiding beneath the term containing 2C and 1C , the glycerol being di-acylated by “PIM .” Indeed, the “PIM family” corresponds to a mixture of 16 19 6 6 10 or 12 acyl forms. Purification of these different species C ,C and the mannose bearing a C . They should then 16 19 16 correspond to mono-acylated-PIM (or Ac PIM ). Interestingly, proved to be crucial for the complete structural study of the 6 1 6 native molecules. We developed a rapid and powerful method the acyl form containing 3C was not observed, indicating that 16 29888 PIM Acyl Forms and TLR Response FIG.8. TLR dependence of macrophage stimulation by PIM . Bone marrow-derived macrophages from mice deficient in TLR2 and/or TLR4 (A–C) or deficient for TLR6 or MyD88 (D–F) were incubated with the TLR2 agonist BLP (0.5 g/ml; A and D), the TLR4 agonist LPS (100 ng/ml; A and D), with F6 and F7 (20 g/ml; B and E), or with F1 (20 g/ml; C and F) for 24 h. F1, F6, and F7 are defined in the legend of Fig. 7. TNF- was measured in the supernatants by enzyme-linked immunosorbent assay. Results are mean S.D. from n 2 mice per genotype and are from one representative experiment out of two independent experiments. Indeed, the linear mannan backbone found in LM and LAM is constituted by a linear -(136)-linked Manp, whereas PIM exhibit at its extremity -(132) links. PIM appears to be an end product and may have a specific role. Mammalian TLR proteins are pattern recognition receptors for a wide array of bacterial and viral products (17). Gram- negative bacterial lipopolysaccharide (LPS) activates cells through TLR4, whereas the mycobacterial cell wall lipoglycans, AraLAM, activate cells through TLR2. Recently, Jones et al. (15) identified a secreted TLR2 agonist activity in short term culture filtrates of M. tuberculosis, which they called STF, for “soluble tuberculosis factor.” To determine the identity of the TLR2 agonist present in soluble tuberculosis factor, they used preparative SDS-PAGE. The TLR2 agonist activity was pres- ent in one fraction, with an apparent molecular size of 6 kDa, raising the possibility that the TLR2 agonist was PIM .By using TLC, the authors reported the presence of two major FIG.9. TLC analysis of the major PIM purified from M. bovis species, PIM and PIM . In that case, the precise structure of 1 2 BCG. Lanes 1– 4, purified acyl forms of PIM ; lane 5, M. bovis BCG total the TLR2 agonist was not determined. We confirm here that lipidic extract; lanes 6 –9, purified acyl forms of PIM . TLC was devel- structurally defined acyl forms of PIM stimulated TNF- se- oped with CHCl /CH OH/H O, 60:35:8 (v/v/v), and sprayed with orcinol. 3 3 2 cretion by primary macrophages in a TLR2-dependent fashion. We then demonstrated that the major polar PIM from M. bovis the tri-acylated forms arose from the di-acylated forms contain- BCG and M. tuberculosis, PIM , also stimulated TNF- secre- tion and that this secretion was mediated by TLR2 but not ing C ,C as fatty acyl appendages. The tetra-acylated forms 16 19 were present as essentially two populations, 3C ,1C or TLR4. TLR2 has been shown to heterodimerize with TLR1 or 16 19 TLR6 (30). We show here that the TLR2-mediated stimulation 2C ,2C . The acylation positions were here clearly elucidated 16 19 as being both positions of Gro, the C3 of the myo-Ins unit and of macrophages by PIM and PIM was independent of TLR6 2 6 but that both PIM and PIM signaled via the adaptor molecule the C6 of the Manp linked to the C2 of the myo-Ins unit. Taken 2 6 together, the same conclusions as for the acyl forms of PIM MyD88. could be made. Indeed, the results indicate that the glycerol is It has been demonstrated in the case of LPS that saturated preferentially acylated by C ,C . However, the nature of the fatty acids acylated in lipid A moiety are essential for its 16 19 fatty acid present on the myo-inositol appears highly variable, biological activities. Saturated fatty acids, but not unsaturated being essentially C or C . fatty acids, induce NF-B activation and expression of inflam- 16 19 As mentioned previously (1), the biosynthetic linkage be- matory markers in macrophages (37). In addition, it has been tween PIM , LM, and LAM appears now to be more and more proposed that the shape of lipid A, influenced by the length and evident, as the same anchor structures were found for all these number of acyl chains, asymmetry of acyl groups, and distri- lipoglycans. However, even if the acyl forms found for PIM bution of negative charges, determine the interaction of LPS were exactly the same as the ones found for PIM , LM, and with different TLR (38). This would explain how E. coli LPS, LAM, PIM are not part of the LM/LAM biosynthetic pathway. with a strong conical shape lipid A, interact with TLR4, 6 PIM Acyl Forms and TLR Response 29889 TABLE II Major PIM acyl forms evidenced in M. bovis BCG The relative abundance of the different species for each acyl form was determined from the integration of the corresponding monoisotopic signals in the negative MALDI spectrum of the PIM -enriched fraction C from M. bovis BCG (Fig. 1). Gro Acylation degree m/z Fatty acids nature Manp 6 myo-Ins 3 % Structure 1 1543.6 C C 70 Lyso-PIM 16 16 6 1585.7 C C 30 19 19 2 1781.8 C ,C C C 45 PIM 16 16 16 16 6 1823.9 C ,C C C 55 16 19 16 19 3 2062.1 2C ,C C C C 100 Ac -PIM 16 19 16 19 16 1 6 4 2300.3 3C ,C C C C C 45 Ac -PIM 16 19 16 19 16 16 2 6 2342.4 2C ,2C C C C C 55 16 19 16 19 16 19 10. Hill, D. L., and Ballou, C. E. (1966) J. Biol. Chem. 241, 895–902 whereas Porphyromonas gingivalis or Rhodobacter sphaeroides 11. Apostolou, I., Takahama, Y., Belmant, C., Kawano, T., Huerre, M., Marchal, LPS, with a more cylindrical shape lipid A, are TLR2 agonists G., Cui, J., Taniguchi, M., Nakauchi, H., Fournie, J. J., Kourilsky, P., and Gachelin, G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5141–5146 (38). Here we asked whether different PIM and PIM acyl 2 6 12. Cywes, C., Hoppe, H. C., Daffe, M., and Ehlers, M. R. (1997) Infect. Immun. 65, forms, bearing 1– 4 acyl residues, were equally potent in stim- 4258 – 4266 ulating macrophages to produce TNF-. We show that the 13. Hoppe, H. C., de Wet, B. J., Cywes, C., Daffe, M., and Ehlers, M. R. (1997) Infect. Immun. 65, 3896 –3905 TNF- stimulating activity of PIM is independent from the 14. Beatty, W. L., Rhoades, E. R., Ullrich, H. J., Chatterjee, D., Heuser, J. E., and number and the nature of the acyl moieties present on the Russell, D. G. (2000) Traffic 1, 235–247 molecules. This seemed to be also the case for PIM as both 15. Jones, B. W., Means, T. K., Heldwein, K. A., Keen, M. A., Hill, P. J., Belisle, J. T., and Fenton, M. J. (2001) J. Leukoc. Biol. 69, 1036 –1044 mono- (F6) and tri- and tetra-acylated (F7) forms studied here 16. Heldwein, K. A., and Fenton, M. J. (2002) Microbes Infect. 4, 937–944 had similar activity. 17. Takeda, K., Kaisho, T., and Akira, S. (2003) Annu. Rev. Immunol. 21, 335–376 In infected macrophages, PIM were shown to traffic out of 18. Sieling, P. A., Chatterjee, D., Porcelli, S. A., Prigozy, T. I., Mazzaccaro, R. J., Soriano, T., Bloom, B. R., Brenner, M. B., Kronenberg, M., Brennan, P. J., the mycobacterial phagosome among other mycobacterial lip- and Modlin, R. L. (1995) Science 269, 227–230 ids, such as LAM, and are released to the medium and by- 19. Ernst, W. A., Maher, J., Cho, S., Niazi, K. R., Chatterjee, D., Moody, D. B., Besra, G. S., Watanabe, Y., Jensen, P. E., Porcelli, S. A., Kronenberg, M., stander uninfected cells (14). We show here that both major and Modlin, R. L. (1998) Immunity 8, 331–340 PIM species from M. bovis BCG, M. smegmatis 607, and M. 20. Severn, W. B., Furneaux, R. H., Falshaw, R., and Atkinson, P. H. (1998) tuberculosis H37Rv, PIM and PIM , are agonists of TLR2, Carbohydr. Res. 308, 397– 408 2 6 21. Vercellone, A., and Puzo, G. (1989) J. Biol. Chem. 264, 7447–7454 irrespective of their acylation state. ManLAM from slow-grow- 22. Ludwiczak, P., Brando, T., Monsarrat, B., and Puzo, G. (2001) Anal. Chem. 73, ing mycobacteria such as M. bovis or M. tuberculosis do not 2323–2330 show such pro-inflammatory effects or TLR2-dependent activ- 23. Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K., and Akira, S. (1999) J. Immunol. 162, 3749 –3752 ity (39). TLR2 may be present intracellularly, recruited to 24. Michelsen, K. S., Aicher, A., Mohaupt, M., Hartung, T., Dimmeler, S., macrophage phagosomes, where they sample the content of the Kirschning, C. J., and Schumann, R. R. (2001) J. Biol. Chem. 276, vacuole, and contribute to elaborate an inflammatory response 25680 –25686 25. Takeuchi, O., Kawai, T., Muhlradt, P. F., Morr, M., Radolf, J. D., Zychlinsky, appropriate for defense against a specific pathogen (40). The A., Takeda, K., and Akira, S. (2001) Int. Immunol. 13, 933–940 trafficking and export of PIM and the more polar PIM from 2 6 26. Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999) Immunity 11, 115–122 M. bovis or M. tuberculosis could thus contribute to maintain- 27. Muller, M., Eugster, H. P., Le Hir, M., Shakhov, A., Di Padova, F., Maurer, C., ing the smoldering activation state of the infected macrophage Quesniaux, V. F., and Ryffel, B. (1996) Mol Med. 2, 247–255 and of the neighboring cells in the tuberculous granuloma 28. Gilleron, M., Vercauteren, J., and Puzo, G. (1993) J. Biol. Chem. 268, 3168 –3179 through TLR2 activation, and contribute to the innate immu- 29. Wang, Y., and Hollingsworth, R. I. (1995) Anal. Biochem. 225, 242–251 nity necessary to contain latent infection. 30. Ozinsky, A., Underhill, D. M., Fontenot, J. D., Hajjar, A. M., Smith, K. D., Wilson, C. B., Schroeder, L., and Aderem, A. (2000) Proc. Natl. Acad. Sci. Acknowledgments—We gratefully acknowledge Dr. J. Nigou (CNRS, U. S. A. 97, 13766 –13771 Toulouse, France) and Dr. B. Ryffel (CNRS, Orle ´ ans, France) for helpful 31. Takeuchi, O., and Akira, S. (2002) Curr. Top. Microbiol. Immunol. 270, 155–167 discussions and carefully reading the manuscript. 32. Goren, M. B. (1984) in The Mycobacteria, a Sourcebook (Kubica, G. P., and REFERENCES Wayne, L. G. eds) Vol. 15, Part A, pp. 379 – 415, Marcel Dekker, Inc., New York 1. Gilleron, M., Ronet, C., Mempel, M., Monsarrat, B., Gachelin, G., and Puzo, G. 33. Leopold, K., and Fischer, W. (1993) Anal. Biochem. 208, 57– 64 (2001) J. Biol. Chem. 276, 34896 –34904 34. Nigou, J., Zelle-Rieser, C., Gilleron, M., Thurnher, M., and Puzo, G. (2001) 2. Gilleron, M., Nigou, J., Cahuzac, B., and Puzo, G. (1999) J. Mol. Biol. 285, J. Immunol. 166, 7477–7485 2147–2160 35. Ballou, C. E., Vilkas, E., and Lederer, E. (1963) J. Biol. Chem. 238, 69 –76 3. Nigou, J., Gilleron, M., and Puzo, G. (2003) Biochimie (Paris) 85, 153–166 36. Guerardel, Y., Maes, E., Elass, E., Leroy, Y., Timmerman, P., Besra, G. S., 4. Khoo, K. H., Dell, A., Morris, H. R., Brennan, P. J., and Chatterjee, D. (1995) Locht, C., Strecker, G., and Kremer, L. (2002) J. Biol. Chem. 277, Glycobiology 5, 117–127 30635–30648 5. Besra, G. S., Morehouse, C. B., Rittner, C. M., Waechter, C. J., and Brennan, 37. Lee, J. Y., Sohn, K. H., Rhee, S. H., and Hwang, D. (2001) J. Biol. Chem. 276, P. J. (1997) J. Biol. Chem. 272, 18460 –18466 16683–16689 6. Schaeffer, M. L., Khoo, K. H., Besra, G. S., Chatterjee, D., Brennan, P. J., 38. Netea, M. G., van Deuren, M., Kullberg, B. J., Cavaillon, J. M., and Van der Belisle, J. T., and Inamine, J. M. (1999) J. Biol. Chem. 274, 31625–31631 Meer, J. W. (2002) J. Immunol. 23, 135–139 7. Kremer, L., Gurcha, S. S., Bifani, P., Hitchen, P. G., Baulard, A., Morris, H. R., Dell, A., Brennan, P. J., and Besra, G. S. (2002) Biochem. J. 363, 437– 447 39. Means, T. K., Wang, S., Lien, E., Yoshimura, A., Golenbock, D. T., and Fenton, M. J. (1999) J. Immunol. 163, 3920 –3927 8. Kordulakova, J., Gilleron, M., Mikusova, K., Puzo, G., Brennan, P. J., Gicquel, B., and Jackson, M. (2002) J. Biol. Chem. 277, 31335–31344 40. Underhill, D. M., Ozinsky, A., Hajjar, A. M., Stevens, A., Wilson, C. B., 9. Lee, Y. C., and Ballou, C. E. (1965) Biochemistry 4, 1395–1404 Bassetti, M., and Aderem, A. (1999) Nature 401, 811– 815
Journal
Journal of Biological Chemistry – Unpaywall
Published: Aug 1, 2003