Salmonella minnesota R595

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1 THE JOURNAL OF BOLOGCAL CHEMSTRY Val. 260, No. 9, ssue of May 10, pp Printed in 6.S.A. Monophosphoryl Lipid A Obtained from Lipopolysaccharides of Salmonella minnesota R595 PURFCATON OF THE DMETHYL DERVATVE BY HGH PERFORMANCE LQUD CHROMATOGRAPHY AND COMPLETE STRUCTURAL DETERMNATON* (Received for publication, September 7, 1984) Nilofer QureshiS, Paolo Mascagnil, Edgar Ribiil, and Kuni Takayama$** From the SMycobacteriology Research Laboratory, William 8. Middleton Memorial Veterans Hospital, Madison, Wisconsin 53705, the School of Pharmacy, University of London, London, United Kingdom, TRibi mmunochem Research, nc., Hamilton, Montana 59840, and 1) The mtitute for Enzyme Research, Uniuersity of Wisconsin, Madison, Wisconsin The monophosphoryl lipid A (MLA) obtained from the lipopolysaccharides of ~ ~ m o nminne~ota e ~ ~ a R695 was fractionated on a silicic acid column to yield the heptaacyl, hexaacyl, and pentaacyl MLA. Each of these MLAs was methylated with diazomethane to yield the dimethyl derivative and purified to homogeneity by reverse-phase high performance liquid chromatography. The molecular ions obtained by positive ion fast atom bombardment mass spectrometry of purified dimethyl heptaacyl MLA allowed us to establish the molecular formula and M, of C112H211N2023P and , respectively. Cleavage at the glycosidic linkage yielded an oxonium ion of mass 1115, which showed that the distal sugar unit contained one phosphate (dimethyl), two hydroxymyristates, one laurate, and one myristate, while the reducing sugar unit contained two hydroxymyristates and one palmitate. By utilizing twodimensional NMR spectroscopy, we were able to assign all of the protons of dimethyl heptaacyl MLA. This assignment included the B protons of the three acyloxyacyl groups. A substantial downfield shift of the protons at the 3- and 3 -carbons was observed, which indicated that these two positions are occupied by ester groups. Fast atom bombardment mass spectral analysis of the hexaacyl and pentaacyl MLAs showed that these structures were identical to the previously designated TLC-3 and TLC-5 fractions, respectively, from Salmonella typhimurium. From this study, the complete structures of the MLA series found in the LPS of S. minnesota can now be described. The complete structure of lipid A obtained from the lipopolysaccharides (LPS ) of Salmonella typhimurium G30/C21 * This work was supported in part by the Medical Service of the Veterans Administration. A preliminary report of this work was presented at the Annual Meeting of the American Society of Biological Chemists in St. Louis, June 3-7, The costs of publication of this article were defrayed in part by the payment of page charges. This articb must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence should be addressed. The abbreviations used are: LPS, lipo~lysacch~ides; MLA, monophospho~~ lipid A; HPLC, high performance liquid chromatography; FAB-MS, fast atom bombar~ent-mass s~ctromet~, NMR, nuclear magnetic resonance; Me2S0, dimethyl sulfoxide; TLC, thin layer chromatography; OH&, hydroxymyristoyl group; C,,, lauroyl group; G,, myristoyl group; Cl4OC4, myristoxymyristoyl group. with the highest degree of acylation (desi~ated MLA TLC- 3) has been determined (1-3). This lipid was then related to the structural series of MLA containing lower amounts of fatty acids (TLC-5, -7, and -9) as well as to the corresponding diphosphoryl lipid A series (2). These results suggested that microheterogeneity of lipid A exists, based on the number of fatty acids. For the first time, the lipid A from a single bacterial source was completely characterized. The structure of lipid A from Escherichia coli has been studied by many investigators (4-11), and the work is presently almost complete. After the recent studies by Takayama et al. (9, 10) and moto et al. (ll), only the nature of the fatty acids occupying each of the four positions on the disaccharide molecule still needs to be established for the lipid A from this source. A suggestion has been made by Rosner et al. (6) that the lipid A from Salmonella and E. coli are probably identical. Similarly, the lipid A of LPS of Salmonella minnesota has been extensively studied but not precisely characterized (12-18). Uniquely, one prominent form of lipid A from this source appears to contain palmitic acid (13, 17). We have now isolated this particular lipid A (heptaacyl MLA) from the LPS of S. minnesota R595, purified the dimethyl derivative by HPLC to homogeneity, and determined its complete structure by utilizing positive ion FAB- MS and proton NMR spectroscopy. The structure of this form of lipid A was then related to the structures of the two lower forms. Special methods were developed to purify this complex mixture of the structural series of MLA, and these will be described in detail. EXPERMENTAL PROCEDURES Materiab-HPLC-grade chloroform, methanol, acetonitrile, and isopropanol were purchased from Burdick and Jackson Laboratories nc., Muskegon, M. Biosil HA (minus 325 mesh) was purchased from Bio-Rad; Silica Gel H thin layer plates (250 pm) were purchased from Analabs, nc., North Haven, CT. Growth of Bacteria and Preparation of LPS-Cells of S. minmsota R595 were grown in a New Brunswick 28-liter fermentor at 37 C in a LB broth medium (9) supplemented with 1.25 g of Na2HP0,, 0.63 g of KHsPO4, and 5.0 g of dextrose/liter of culture medium. Radiolabeled cells of S. minnesota R595 were grown in 100-ml cultures containing 1.0 mci of [- Clacetate (60 pci/pmol) (Amersham Gorp.). These cells were the source of f C]LPS, which contained the label in the fatty acids. LPS were prepared by the method of Galanos et al. (19) with modification (2). Preparafwn of The LPS (1.8 g) were suspended in 70 ml of 0.1 N HCl and hydrolyzed at 100 C for 15 min. The MLA (1.2 g) was recovered 8s the free acid, as described previously (1). This was applied to a 3.7 X 34-cm silicic acid column packed as a slurry in chloroform. The column was washed with several bed volumes of 5271

2 5272 Structure of Lipid A from S. minnesota chloroform, and lipid A was eluted with a linear gradient of 0 to 16% methanol in chloroform (2 liters). Ten-milliliter fractions were collected. Fraction numbers (72 mg), (147 mg), and (105 mg) contained heptaacyl, hexaacyl, andpentaacyl MLA, respectively. The pooled samples were first applied on the Sephadex LH-20 column to separate out traces of diphosphoryl lipid A as described previously (1) and then passed successively through a 0.8 x 3-cm Chelex 100 column to remove any remaining divalent cations and a 0.8 X 3-cm Dowex50 (H') column to yield the free acids. Chloroform-methanol (4:l) was used for elution. The samples were immediately methylated with a slight excess of freshly prepared diazomethane for 2 min at ambient temperature and dried with a stream of nitrogen. The recovery of the sample was quantitative. HPLC Fractionation-HPLC was performed with two Waters 660 solvent programmers (Waters Associates nc., Milford, MA), a Waters U6K universal liquid chromatograph injector, a variable wavelength detector (model LC-85B, Perkin-Elmer Corp., Analytical nstru- ments, Nonvalk, CN), and a radial compression module (model RCM- 100, Waters Associates nc.). A Radial Pak cartridge (8 mm X 10 cm) (C,,-bonded 10-p silica, Waters Associates nc.) was used at a flow rate of 3 ml/min. For the fractionation of dimethyl MLA, a linear gradient of 20-80% isopropanol in acetonitrile was used over a period of 60 min. The wavelength of the detector was set at 210 nm. Fatty Acid Analyses-Heptaacyl and hexaacyl MLA containing "C-labeled fatty acids were acid hydrolyzed and analyzed by HPLC as described previously (1). Mass Spectral Analysis-Positive ion FAB-MS was performed on an MS-50 mass spectrometer (AE/Kratos, Manchester, England) utilizing a neutral beam of xenon atoms with a translational energy of 8 kev and a discharge current of 40 PA as previously described (2). Proton NMR Analysis-Spectra were recorded on an XL-300 MHz Varian spectrometer. HPLC-purified dimethyl heptaacyl-mla (7 mg) was dissolved in 0.5 ml of benzene-d6/me2s0-d6 (9:1, v/v). Two hundred fifty-six increments of 2048 data points each were collected for the 'H-'H shift correlation experiment. The operating temperature was 33 & 0.5 "C. RESULTS TLC Analysis of Fractionation of MU on a Silicic Acid Column-The fractionation of MLA (as the free acid) on a silicic acid column was followed by analytical TLC. The column fractions were pooled to yield partially purified heptaacyl, hexaacyl, and pentaacyl MLA as indicated by column fractions (lane B), (lane D), and (lane F), respectively, on TLC (Fig. 1): The tetraacyl and lower series of MLA, which appeared in lanes G and H of the chromatogram, were not analyzed. Fatty Acid Analysis of Purified Heptaacyl MLA-TLC-purified heptaacyl MLA with 14C label in the fatty acids was analyzed for total fatty acid content by reverse-phase HPLC of the methyl ester (data not presented). t showed the presence of hydroxymyristic, lauric, myristic, palmitic, and hexadecenoic acids. The hydroxymyristic to normal fatty acids ratio was calculated to be about 4:3. There was some unsaturated fatty acid in the form of Clkl, which apparently accumulated at the expense of lauric acid when cells were grown at 30 "C (18). The fatty acid content of 14C-labeled hexaacyl MLA was similarly analyzed, and the results showed a hydroxymyristate to normal fatty acid ratio of about 4:2 (data not presented). Purification of the Dimethyl Derivative of MLA by Reversephase HPLC-Each of the silicic acid column-purified MLAs was methylated with diazomethane and subjected to prepar- * Portions of this paper (including Figs. 1-4,6-8, and Table 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD Request Document No. 84M-2308, cite the authors, and include a check or money order for $4.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. ative HPLC using a reverse-phase C18-bonded silica cartridge. The results of the HPLC analysis before (A) and after (B) such a fractionation are shown in Figs The dimethyl heptaacyl MLA resolved into one major peak at 51 min, one minor shoulder fraction on the trailing edge of the major peak, and several small shoulders (Fig. 2A). The dimethyl hexaacyl MLA resolved into one major peak at about 41 min and one minor peak on the trailing edge of the major peak (Fig. 3A). There were at least seven other very small peaks present. The dimethyl pentaacyl MLA resolved into one major peak at 31 min and about 10 minor peaks which appeared to include the dimethyl hexaacyl MLA (Fig. 4A). Although TLC of the underivatized samples indicated a high degree of homogeneity (Fig. l), HPLC analysis revealed the purity of the dimethyl heptaacyl, hexaacyl, and pentaacyl MLA to be about 67, 50, and 40%, respectively. From this, only a small amount of impurities generated by varying degrees of derivatization (methylation) would be expected. The nature of these minor peaks in the three fractions is presently under investigation. The purity of the dimethyl heptaacyl, hexaacyl, and pentaacyl MLA was thus established (Figs. 2B, 3B, and 4B), and these HPLC-purified samples were analyzed by positive ion FAB- MS. The purified dimethyl heptaacyl MLA was further analyzed by two-dimensional NMR spectroscopy. Positive on FAB-MS-HPLC-purified heptaacyl, hexaacyl, and pentaacyl MLAs were analyzed by positive ion FAB- MS. The partial spectra of heptaacyl MLA are shown in Fig. 5. t should be noted that because of the weak signal we had to scan a narrow range of 1940 to 2040 mass units in order to obtain a good spectrum of dimethyl heptaacyl MLA in the molecular ion region (Fig. 5A). t contained adduct ion MK', MNa+, and MNH: peaks at m/z 2022, 2006, and 2001, respectively. An MH' peak was observed at m/z The loss of the hydroxyl group at the anomeric carbon yielded an (M- OH)' fragment of mass The loss of a hydroxymyristoyl group gave adduct ion peaks MNa+-OHC14 at mlz 1779 and MH+-OHC1, at m/z 1757, as well as an (M-OH)'-OHC,, peak at m/z 1739 (data not presented). Cleavage at the glycosidic linkage yielded an oxonium ion of mass The loss of a lauroyl group yielded a 1115-C2 peak at m/z 932, and further loss of water yielded a 1115-C12-H20 peak at m/z 914. From these results, the dimethyl heptaacyl MLA has a molecular weight consistent with the formula C112H211N2023P and M, = t contains four hydroxymyristoyl, one lauroyl, one myristoyl, and one palmitoyl residues. The spectrum of dimethyl hexaacyl MLA is shown in Fig. 6. t contained an adduct ion MNH: peak at m/z 1763, and MH+ peak at m/z 1746, and an ("OH)+ fragment of mass An MH+-CH3-H20 fragment appeared at m/z The loss of a hydroxymyristoyl group yielded an MNHZ-OHCM peak at mlz 1536, an MH+-OHC14 peak at mlz 1519, and an (M-OH)+-OHC1, peak at mlz A 1713-OHC14 fragment appeared at mlz Loss of a myristoxymyristoyl group yielded an MH+-Cl40C4 peak at m/z 1309 and an ("OH)+- C,,OC,, peak at mlz Cleavage at the glycosidic linkage yielded oxonium ion of mass LOSS of a lauroyl group yielded a 1115-Cl2 peak at mlz 932, and further loss of water yielded a 1115-C12-HzO peak at mlz 914. The dimethyl hexaacyl MLA has a molecular formula C,H1a1N2022P and M, = The spectrum of dimethyl pentaacyl MLA is shown in Fig. 7. t contained adduct ion MNa' and MNH: peaks at mlz 1558 and 1553, respectively, and an MH' peak at mlz The loss of the hydroxyl group at the anomeric carbon yielded an ("OH)+ fragment of mass An MH+-CH3-H20 fragment appeared at mlz The loss of the hydroxymyristoyl

3 Structure of Lipid A from S. minnesota 5273 > - V r z w? LL rn h FG. 5. Partial positive ion FAB mass spectra of dimethyl heptaacyl MLA. A, raw data on the molecular ion region acquired by scanning at accelerated voltage at 8 kev with 3000 resolutions; B, spectrum of lower mass region. group gave ion peaks MH+-OHC4 at m/z 1309 and ("OH)+at m/z A 1503-OHC14 fragment appeared at m/ z Loss of a lauroyl group yielded a 13o9-Cl2 peak at m/ z 1127, which lost a methyl group to give a 1127-CH3 peak at m/z Cleavage at the glycosidic linkage yielded oxonium ion of mass 904. The loss of a lauroyl group yielded 9O4-Cl2 at m/z 721, and further loss of water yielded 721-H20 at m/z 703 (data not shown). From these results, the dimethyl pentaacyl MLA has a molecular formula Ca~H155N2021P and M, = Fatty Acid Distribution at the Distal and Reducing Units of Dimethyl Lipid A-Fast atom bombardment of dimethyl hexaacyl MLA and dimethyl pentaacyl MLA yielded oxonium ion fragments corresponding to distal units with masses 1115 and 904, respectively (Table ). Based on these results, the 6- distal unit of both heptaacyl and hexaacyl MLA derivatives must contain two hydroxymyristoyl, one lauroyl, and one myristoyl groups. The reducing unit of dimethyl heptaacyl MLA must then contain two hydroxymyristoyl and palmitoyl residues, whereas a similar unit of hexaacyl MLA derivative contains two hydroxymyristoyl residues. The dimethyl pentaacyl MLA differs from the dimethyl hexaacyl MLA in the TABLE Distribution of fatty acids in the lipid As of LPS from S. minnesota R595 based on the generation of oxonium ion on positive ion FAB- MS of purified dimethyl derivative Oxonium ion M, of dimethyl MLA (free acid) derivative (mlz)" Fatty acid content in the unitb Distal Reducing Heptaacyl HC14,Clz,Clr 20HClr,C16 Hexaacyl HC1(,C12,Cl4 20HC14 Pentaacyl HC14,Cz 20HCl4 "Due to fragmentation at the glycosidic bond to yield an intact distal unit. * OHClr, hydroxymyristate; C12, laurate; C14, myristate; C16, palmitate. absence of a myristoyl residue at the distal end. Proton NMR Spectroscopy-By utilizing the solvent of benzene-dg/me2so-ds (9:1, v/v) and the operating temperature of 33 "C on the HPLC-purified dimethyl heptaacyl MLA, a well resolved proton NMR spectrum was obtained as shown in Fig. 8 (upper panel). The 'H assignments were obtained by two-dimensional NMR spectroscopy. A proton-proton shift correlation (20) of the dimethyl heptaacyl MLA afforded all

4 5274 Structure of Lipid A from S. minnesota the "connectivities" needed for the assignment of the two glucosamine ring protons and of the a,p protons of the fatty acids esterified at the various carbons of the disaccharide molecule. Fig. 8 (lower panel) shows an intensity contour map of a COSY (correlated spectroscopy) of the dimethyl heptaacyl MLA in benzene-d6/mezso-ds solution. The two enlargements in Fig. 9 indicate the correlations which led to the identification of the reducing unit (panel A) and of the distal unit (panel B) (see structure in Fig. 10 for location of protons). The assignment of the amide protons of distal unit (NH') and reducing unit (NH) to the doublets at and , respectively, was based on the suppression of the signals by deuterium exchange. Once the two NHs were identified, the cross-peaks that lie off the diagonal in the contour plot allowed the identification of the remaining protons of the two glucosamine rings. For instance, in Fig. 9A, H-2 at shows a correlation to H-1 at and H-3 at The latter was found to be coupled to H-4 at , which in turn is correlated to H-5 at H-61, H-62, and H-40H could then be assigned knowing the position of H-5 and H-4. n a similar manner, the assignment of the second ring protons and of the a and P protons of the fatty acid esters could be completed. Chemical shifts (6) and coupled constants (J) are shown in Table 11. Of particular interest is the presence in the spectrum of protons other than those found on the sugar ring appearing at the downfield region at 5.57, 5.56, and as shown in Fig. 9B. As in the case of similar molecules we have analyzed recently (3, O), we attributed these three downfield signals to the protons of the 0-substituted p-carbon of the acyloxyacyl groups. The two magnetically nonequivalent methyl groups on the phosphate moiety gave four intense lines at (Fig. 8, top). From the data presented here the following conclusions can be drawn. (i) The anomeric proton at is of the (Y type (J1.2 = 2.5) and it belongs to the reducing unit of the disaccharide. Thus a further splitting to yield a three-line signal suggests scalar coupling with the anomeric OH. (ii) The J1,2 = 8.2 of the H-1' proton of indicates that the anomeric configuration of the distal unit is p. (iii) The sites of esterification of the fatty acids are at the 3 and 3' carbons of the disaccharide. This is consistent with the observed downfield shift to 5.71 and , respectively, of the protons at these positions from the normal range that is characteristic of H-3, H-4, and H-6 protons of sugars. (iv) The phosphate group is at the 4'-carbon, since the H-4 OH and H-6' OH protons could be assigned the signals at 5.35 and , respectively. (v) The dimethyl heptaacyl MLA contains four P-hydroxycarboxylate moieties. Acylation occurs at the 0- hydroxy group of three of these fatty acids. group of the fourth moiety was found at coupled to the neighboring /3-proton at Analysis of Products Formed by Excess Methylation of Heptaacyl MU-When the silicic acid column-purified heptaacyl MLAwas incubated in the presence of a large excess of diazomethane (alkaline conditions) overnight at 4 "C, hydroxymyristate was liberated, and additional methyl groups (up to a total of four) were added. This was shown when the HPLC- H -8,OH W-7 H-3' B % ' 2.0- E i FG. 9. 'H-'H connectivities illustrated for the reducing unit (A) and for the distal unit (B) of dimethyl heptaacyl MLA.

5 Structure of Lipid A from S. minnesota 5275 FG. 10. Complete structure of the dimethyl derivative of heptaacyl MLA obtained from the LPS of S. rninnesota R595. The structure of the heptaacyl MLA would not have the two methyl residues on the phosphate group. The structure of the corresponding heptaacyl diphosphoryl lipid A would have a phosphate group at the 1-position. As part of the LPS backbone structure, the 6"position of the diphosphoryl lipid A would be occupied by the 2-keto-3-deoxyoctonate units. CH3 '0 c=o purified methylated product was examined by both positive ion FAB-MS and proton NMR spectroscopy (data not presented). The FAB-MS of this compound showed MH' ions at m/z 1772,1786 (+CH,), and 1800 (+2CH2). Adduct ion MNa' peaks were observed at m/z 1794,1808 (+CHd, and 1822 (+2CH2). ("OH)+ ions appeared at m/z Cleavage of the glycosidic linkage yielded an oxonium ion of mass Based on these results, the new product had M, = 1771, 1785 (+CH,), and 1799 (+2CH2). Proton NMR spectroscopy of the sample in benzene-me2s0 (9:1, v/v) revealed H-3 proton at , whereas the H-3' proton appeared in the envelope region of about 56. This indicated that the hydroxyl group at the 3-carbon was unsubstituted. These results thus showed that when the heptaacyl MLA was treated with excess diazomethane, the hydroxymyristic acid was liberated from the 3- position of the disaccharide. Triethylamine Hydrolysis of the Purified MU-Treatment of the heptaacyl MLA with 3% (v/v) triethylamine (ph 9.4) at 100 "C caused a rapid liberation of the hydroxymyristic acid and a slow release of myristoxymyristic acid (data not presented). No other fatty acid was detected. This was identical to the triethylamine hydrolysis of MLA TLC-3 fraction obtained from S. typhimurium as previously reported (1, 2). These results showed that the alkali-labile ester-linked fatty acids in the heptaacyl MLA are hydroxymyristic and myristoxymyristic acids. With this information, we now have sufficient data to assign the exact location of all 7 fatty acids on this glucosamine disaccharide. This is shown in Fig. 10. DSCUSSON The structure of lipid A obtained from another strain of Gram-negative bacteria, namely S. minnesota R595, can now be described precisely. We have found that an MLA series exists in our lipid A preparation (heptaacyl, hexaacyl, and pentaacyl MLA), which we were able to purify to homogeneity by HPLC as their dimethyl derivatives and characterize by utilizing FAB-MS.3 Proton NMR spectroscopy was also used As in the structural series of MLA found in the lipid A preparation of LPS obtained from S. typhimuriurn (1, Z), the MLAs in the series from S. rninnesota are thought to be structurally related. to further characterize the dimethyl heptaacyl MLA. Early studies by Gmeiner et al. (12) showed that lipid A from S. minnesota contains a glucosamine disaccharide with a Pl4 linkage. Our present work confirms the /3 assignment at the anomeric carbon. Their work also indicated the presence of a 4"phosphate group, which we confirmed by NMR spectroscopy. Batley et al. (16) reported that the reducingend sugar of lipid A from this source must have an a configuration, and we have arrived at the same conclusion, again based on NMR spectroscopy. Wollenweber et al. (17) showed that their unfractionated lipid A preparation obtained from LPS of S. minnesota contains lauroxymyristoyl and palmitoxymyristoyl groups in amide linkages. They did not determine which monosaccharide unit contained the specific acyloxyacyl group. Since they must have dealt with a crude mixture containing, among other things, various forms of lipid A (1, 2), it would be difficult to accurately determine how many fatty acids are present per molecule or where each fatty acid is located in a specific lipid A molecule. Despite these apparent difficulties, Wollenweber et al. (17) suggested that there are 7 fatty acids/molecule in the lipid A mixture obtained from the LPS of S. minnesota. We have now developed a silicic acid column method with high load capacity using chloroform-methanol as the solvent (without using water) to achieve effective separation of a structural series of MLA (as the free acid) according to the degree of acylation. Each of these partially purified MLAs was then methylated to yield the dimethyl ester of MLA (phosphotriester). This derivative was now suitable for preparative-scale HPLC fractionation using the C18-bonded silica cartridge by the reverse-phase mode without resorting to adding a paired ion (tetrabutylammonium ion) that was found to be detrimental to the recovery of the undegraded sample (1). By this procedure, highly purified dimethyl derivatives of the heptaacyl, hexaacyl, and pentaacyl MLA were obtained. We were able to apply loads of up to 6 mg of MLA and achieve good separation. The recovery of sample was quantitative. Positive ion FAB-MS of such a purified derivative gave relatively strong signals for the molecular ions as well as for the

6 5276 Structure of Lipid A from S. minnesota fragments including the diagnostic oxonium ion. Two-dimensional NMR spectroscopy of the dimethyl heptaacyl MLA gave sharp spectra and well defined contour maps in benzene- Me,SO solvent at 33 "C, which allowed us to assign all of the protons of this glycolipid. From these results, we were able to determine the nature and the exact location of the ester groups on the disaccharide. t also allowed us to confirm the position of the phosphate group at the 4"position (12) and the anomeric configuration of both distal and reducing sugars. Based on FAB-MS, the molecular weight of the dimethyl heptaacyl MLA was established to be daltons. Since the oxonium ion had a mass of 1115, the distal unit of this MLA contained one glucosamine unit, one phosphate group, and four fatty acyl groups (two hydroxymyristate, one laurate, and one myristate). t follows that the reducing unit must then contain one glucosamine unit and three fatty acyl groups (two hydroxymyristates and one palmitate). Proton NMR spectroscopy showed that the ester groups on the sugar were at positions 3 and 3', while the hydroxyl group of the 4- and 6'-positions were unsubstituted. t was previously established by Wollenweber et al. (17) that the two nitrogen positions carried the amide-linked fatty acyloxyacyls, which were identified to be palmitoxymyristoyl and lauroxymyristoyl groups. Since proton NMR spectroscopy of the dimethyl heptaacyl MLA showed the presence of three acyloxyacyl groups, the third acyloxyacyl must be the myristoxymyristoyl residue. We have shown that the myristoxymyristic acid along with hydroxymyristic acid were specifically released under mild alkaline conditions, which indicated ester linkages. We established by FAB-MS and NMR spectroscopy of the mild alkali treated dimethyl heptaacyl MLA that the hydroxymyristic acid was liberated from the 3-position of the glycolipid. These results clearly showed that the following fatty acyl groups were attached to the indicated positions of the disaccharide of heptaacyl MLA a palmitoxymyristoyl group on the nitrogen of the reducing sugar (2-position), a hydroxymyristoyl group at the 3-position, a lauroxymyristoyl group on the nitrogen of the distal sugar (2'-position), and a myristoxymyristoyl group at the 3"position. The complete structure of the heptaacyl MLA as the dimethyl derivative is shown in Fig. 10. Heptaacyl MLA is unique because of the presence of the palmitic acid. Although hexaacyl MLA is the major component of the lipid A from s. minnesota, the heptaacyl form is clearly a prominent component. The reducing unit of the heptaacyl form can be related structurally to the lipid Y, which accumulates along with lipid X in the temperaturesensitive mutant of E. coli MN7 at nonpermissive temperature (9, 10). This might suggest that lipid Y serves as an early precursor in the biosynthesis of the heptaacyl lipid A, just as lipid X is probably an early precursor of lipid A of E. coli (9, 21, 22). However, as previously discussed, it is presently difficult to ascribe a metabolic role for lipid Y in E. coli, since the lipid A from this source does not contain appreciable amounts of palmitic acid (10). This may indicate that an insertion-removal mechanism of a transient palmitoyl group is involved in the biosynthesis of the LPS and that the removal process in S. minnesota is sluggish (10). t would be interesting to determine the influence of an additional palmitate group on the numerous biological activ- ities of lipid A. This is appropriate, since we have found that one of the multiple structural requirements for toxicity of lipid A is the presence of a normal fatty acid (23). Ribi et al. (24) showed that their fraction 1 from S. minnesota (heptaacyl MLA) was nontoxic (chick embryo lethality test) and had tumor regression activity (syngeneic guinea pig system). These results were similar to those for the hexaacyl MLA (designated MLA TLC-3) (1, 24). Comparative studies on other biological activities of the heptaacyl and hexaacyl MLA are in progress. Acknowledgments-We thank Dr. Karen Hyver and David Heller for their excellent and generous assistance in the mass spectral studies. We also thank Todd Sievert for growing and harvesting cells of S. minnesota R595. Mass spectral determinations were carried out at the Middle Atlantic Mass Spectrometry Laboratory, a National Science Foundation Shared nstrumentation Facility, The Johns Hopkins University School of Medicine, Baltimore, MD REFERENCES 1. Qureshi, N., Takayama, K., and Ribi, E. (1982) J. Biol. Chem. 257, Qureshi, N., Takayama, K., Heller, D., and Fenselau, C. (1983) J. Biol. Chem. 258, Takayama, K., Qureshi, N., and Mascagni, P. (1983) J. Biol. Chem. 258, Rosner, M. R., Tang, J., Barzilay,., and Khorana, H. G. (1979) J. Biol. Chem. 254, Rosner, M. R., Khorana, H. G., and Satterthwait, A. C. (1979) J. Biol. Chem. 254, Rosner, M. R., Verret, R. C., and Khorana, G. H. (1979) J. Biol. Chem. 254, Van Alphen, L., Lugtenberg, B., Rietschel, E. Th., and Mombers, C. (1979) Eur. J. Biochem. 101, Strain, S. M., Fesik, S. W., and Armitage,. M. (1983) J. Biol. 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7 Structure of Lipid A from S. minnesota 5277 Front ' 1.m Origin. i - * 2

8 5278 Structure of Lipid A from S. minnesota Chemical shift8 (61 and coupling constants (J. in Hz) of protons of dimethyl Table 1 heptaecyl WLA* e m' L i * i! S )