Neisseria gonorrhoeae That Infect Men Have Lipooligosaccharides with Terminal N-Acetyllactosamine Repeats*

Constance M. JohnDagger §, Herman Schneiderparallel , and J. McLeod GriffissDagger **

From the Dagger  Centre for Immunochemistry and the ** Department of Laboratory Medicine, University of California, San Francisco, California 94121 and the  Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D. C. 20307

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Infectious Neisseria gonorrhoeae make relatively large lipooligosaccharides (LOS) that structurally resemble human glycosphingolipids. MS11mkC is an LOS variant of N. gonorrhoeae strain MS11 which was isolated from men at the onset of dysuria (Schneider, H., Griffiss, J. M., Boslego, J. W., Hitchcock, P. J., Zahos, K. M., and Apicella, M. A. (1991) J. Exp. Med. 174, 1601-1605). Delayed extraction matrix-assisted laser desorption and ionization and electrospray ionization mass spectrometry of O-deacylated MS11mkC LOS produced ions consistent with known LOS which have lacto-N-neotetraose (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc; paraglobosyl; monoclonal antibodies (mAbs) 1B2+ and 06B4+) and GalNAcright-arrowlacto-N-neotetraose (gangliosyl; mAb 1-1-M+) oligosaccharides. Ion peaks for a larger LOS which also bound mAb 1B2 indicated the addition of a hexose (+162 Da) to gangliosyl LOS or the addition of a hexose and a N-acetylhexosamine (+365 Da) to paraglobosyl LOS. Analysis of HF-treated and O-deacylated LOS revealed three major components present in a phosphoethanolamine (PEA)0 and a PEA1 series. Digestion of MS11mkC LOS by beta -N-acetylhexosaminidase and beta -galactosidase, alone and sequentially, combined with mAb binding patterns, confirmed the presence of a nonreducing terminal repeating LacNAc ((Galbeta 1right-arrow4GlcNAc)2) on the largest LOS, rather than a parallel oligosaccharide structure.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Lipooligosaccharides (LOS)1 are outer membrane glycolipids that structurally resemble human cell membrane glycosphingolipids (1, 2); their glycose moieties undergo rapid phase variations among oligosaccharides that are structurally identical to those of several glycosphingolipid series (3, 4). In a series of experiments, Schneider et al. (5-7) showed that only LOS variants of Neisseria gonorrhoeae MS11mk that made paraglobosyl and gangliosyl, but not lactosyl LOS, could cause urethritis in healthy male volunteers. An inoculum of only 250 piliated MS11mkC LOS variants caused urethritis in three of seven volunteers who were challenged with them (6), a 43% attack rate that is somewhat higher than the 22% risk of naturally acquired infection (8). Neither the MS11mkA variant, which makes only lactosyl LOS (9), nor sialylated MS11mkC organisms could infect the male urethra (5-7).

MS11mkC organisms, like those gonococci freshly isolated from men with urethritis, make additional LOS molecules that migrate more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than the paraglobosyl and gangliosyl LOS (5, 6). These apparently higher Mr LOS bind the same paraglobosyl and gangliosyl monoclonal antibodies (mAbs) as the lower Mr LOS (1). Their increased mass, therefore, could result from additional substitutions of the lipoidal moieties or basal regions of lower Mr paraglobosyl/gangliosyl LOS (3-5, 10); from duplicative extension of the beta  chain with the same glycose sequences (3, 4, 10, 11), and hence, epitopes, as those of the alpha  chain; or from terminal or internal extensions of alpha  chains (3, 4, 10).

The production of higher Mr LOS by organisms recovered as pyuria develops (5, 7) creates uncertainty as to the LOS phenotype that is most pathogenic and against which preventive measures need be directed. To resolve the structural basis for higher Mr paraglobosyl and gangliosyl LOS, we applied matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) with a time-of-flight (TOF) mass analyzer and electrospray ionization (ESI) MS with a quadrupole mass analyzer, combined with enzymatic and chemical degradations, to the LOS of MS11mkC. Because we had not previously used MALDI-MS techniques to analyze neisserial LOS, we also analyzed the O-deacylated LOS of MS11mkA, which yields a single major hexasaccharide product upon acid hydrolysis and HF treatment, Galbeta 1right-arrow4Glcbeta 1right-arrow4(GlcNAcalpha 1right-arrow2Hepalpha 1right-arrow3)Hepalpha 1right-arrowKdo, as an aid in interpreting spectra of O-deacylated MS11mkC LOS.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Aspergillus niger alpha -galactosidase, anhydrous hydrazine, and LPS from Salmonella typhimurium Ra mutant TV 119 were obtained from Sigma and acetonitrile and methanol from Fisher. A 48% aqueous HF solution was purchased from Mallinckrodt (Muskegon, MI). beta -Galactosidase and beta -N-acetylhexosaminidase, both from jack bean meal, were obtained from Oxford GlycoSystems (Bedford, MA). beta -Glucosidase from sweet almonds was purchased from Boehringer Mannheim. Acrylamide solution, ammonium persulfate, and N, N,N'N'-tetramethylethylenediamine (TEMED) were acquired from Bio-Rad. Resolving and stacking gel buffers were prepared as described by Apicella et al. (12). The enzymatic substrate for immunoblots was obtained from Tropix (Western-Light Chemiluminescent Detection System, Bedford, MA), and blocking and washing solutions were prepared according to recommendations of the manufacturer. MAb 2-1-L8 was provided by Dr. Wendall D. Zollinger (13). The secondary antibody was an anti-mouse-IgG alkaline-phosphatase-conjugated mAb obtained from Sigma.

Bacterial Strains and LOS-- F62 and the A and C LOS variants of MS11mk each were cultured as described previously (5, 14). LOS were extracted and purified from each variant by a modification of the hot phenol-water method (12, 15).

O-Deacylation of LOS-- LOS samples were O-deacylated before MS analyses as reported previously (16, 17). Briefly, LOS (0.1-1.0 mg) was placed in a 1.5-ml polypropylene tube, to which was added 200 µl of anhydrous hydrazine. Samples were kept at 37 °C in a water bath for 20 min and intermittently vortexed and sonicated. The solution was cooled, and 1 ml of chilled acetone (-20 °C) was added dropwise. The precipitated O-deacylated LOS was centrifuged at 12,000 × g for 20 min, and the supernatant was removed. The pellet was washed with cold acetone, centrifuged again, and the supernatant was removed. To the pelleted O-deacylated LOS, 200-500 µl of H2O was added, and the sample was lyophilized.

HF treatment of O-Deacylated LOS-- Phosphoesters were removed by HF treatment because neither the MALDI- nor ESI-MS had sufficient accuracy to distinguish between HexNAc substitutions and HPO3 with PEA ones. The O-deacylated LOS was reacted with 48% aqueous HF to remove phosphoester moieties. From 0.1 to 1 mg of O-deacylated LOS was placed in a 1.5-ml polypropylene tube, and cold 48% aqueous HF was added to make a 5-10 µg/µl solution which then was allowed to react at 4 °C for 16-20 h. Excess HF was removed in a vacuum under a stream of N2 with an in-line NaOH trap (18).

MALDI-MS-- Negative ion MALDI-MS was performed in the linear mode with delayed extraction on a Voyager Elite TOF instrument (PerSeptive Biosystems, Framingham, MA) equipped with a nitrogen laser (337 nm) (19). Analyses were carried out with a 150-ns time delay and a grid voltage of 92-94% of full acceleration voltage, 20 kV, and external calibration. The LOS samples were prepared in 100 mM 2,5-dihydrobenzoic acid. Small amounts (3-10 µl) of sample were desalted with cation exchange beads (Dowex 50X) before crystallization on the MALDI plates. From 50 to 160 scans were averaged and digitally smoothed. Spectra were mass assigned using an external two-point calibration with a standard of O-deacylated LPS prepared from S. typhimurium Ra mutant TV 119; average masses were used for calculating the predicted molecular weight of the LPS. The software of the manufacturer (PerSeptive Biosystems) was used to correct the external calibration of the spectrum of O-deacylated HF-treated MS11mkC LOS, and the peak for O-deacylated HF-treated paraglobosyl LOS at m/z 2509.5 was used as an internal one-point calibrant.

ESI-MS-- Negative ion ESI-MS was performed on a Bio-Q instrument (VG Instruments, Manchester, U. K.) (17). Scans were acquired over a range of m/z 300-3000. Samples were dissolved in H2O/acetonitrile (7/3 v/v) to a final concentration of 0.5 µg/µl, and aliquots of 1-5 µl were injected, using a Rheodyne injector, into a constant stream of 1% acetic acid in H2O/acetonitrile or H2O/methanol (1/1 v/v). A flow rate of 10 µl/min was maintained throughout the analysis. The instrument was calibrated as for MALDI-MS. Spectra were averaged over several scans and digitally smoothed. We compared MALDI and ESI spectra of the MS11mk A and C variant LOS.

Exoglycosidase Digestion of LOS-- According to the manufacturer if the concentration of beta -galactosidase from jack bean meal is kept below 1 unit/ml with a substrate concentration of 15 µM, the enzyme can be used to distinguish galactose-linked beta 1right-arrow3, which is cleaved at a relative rate of 1%, from galactose-linked beta 1right-arrow6 and beta 1right-arrow4, which are cleaved at relative rates of 100% and 75%, respectively (20). beta -Galactosidase from jack bean meal was reconstituted in 50 mM sodium citrate, pH 3.5, to a concentration of 0.8 unit/ml. Approximately 30 µg of LOS was added to 625 µl of enzyme solution to achieve a substrate concentration of approximately 15 µM, and the solution was incubated for approximately 20 h at 37 °C. Digestion was stopped by boiling the solution briefly (2-3 min).

S. typhimurium Ra mutant LPS and MS11mkC LOS were digested with alpha -galactosidase. The reaction in 100 mM sodium citrate buffer, pH 4.0, was for 20 h at 37 °C. The enzyme concentration was 2 units/ml, and from 20 to 100 µg/ml LOS was digested.

Sequential glycosidase digestions of MS11mkC LOS were performed in 100 mM sodium citrate buffer, pH 4.5, first by beta -N-acetylhexosaminidase (10 units/ml) and then by beta -galactosidase (0.5 unit/ml) or beta -glucosidase (5 units/ml). MS11mkC LOS also was digested sequentially with beta -N-acetylhexosaminidase followed by alpha -galactosidase (2 units/ml), but this reaction was performed at pH 4.0. Digestions were carried out for 18-20 h at 37 °C, and the solutions were boiled briefly to inactivate the glycosidases. In one experiment an aliquot of beta -N-acetylhexosaminidase-digested MS11mkC LOS was removed for analysis after inactivation of the enzyme, and beta -galactosidase then was added to a concentration of 0.5 unit/ml, and the digestion continued, as described above.

SDS-PAGE Analysis of LOS-- Native and enzyme-digested LOS were analyzed by SDS-PAGE with silver staining, as described (12). Long (32 cm) 12.9% acrylamide resolving gels with 3% stacking gels were used (12). The resolving gel was prepared using 15 ml of 30% acrylamide to which was added 7 ml of resolving buffer and 12.2 ml of deionized H2O. Just before casting the gel, 300 µl of ammonium persulfate (75 mg/ml) and 10 µl of TEMED were added to the acrylamide, and the solution was mixed well. After casting the gel, 800 µl of H2O was added gently to the top; after 1 h this was removed, and the stacking gel was added. The stacking gel was prepared by adding 1 ml of 30% acrylamide and 1.0 ml of spacer buffer to 7.8 ml of deionized H2O, degassing the solution, and then adding to it, with stirring, 0.1 ml of 10% SDS, 0.1 ml of ammonium persulfate, and 5 µl of TEMED. The stacking gel was added with the comb in place. The gel was run at a constant 15-mA current until the dye front had moved through the stacking buffer, after which the current was increased to 35 mA. The gel was stained with silver using the general procedure of Tsai and Frasch (21), with stopping of the reaction as described by Apicella et al. (12).

Immunoblot with mAb 2-1-L8-- One of duplicate SDS-polyacrylamide gels of native and enzyme-digested MS11mkC LOS was stained with silver, and the other was blotted to nitrocellulose paper for 12 h at 15 V in 20% MeOH with 20 mM tris(hydroxymethyl)aminomethane. The blot was washed in phosphate-buffered saline for 20 min and then incubated in blocking buffer for 2 h. The blot then was washed in phosphate-buffered saline twice for 5 min and incubated overnight at 4 °C with mAb 2-1-L8 diluted in blocking buffer. The blot was washed and incubated for 1 h with secondary antibody in blocking buffer. Bound antibody was detected with a chemiluminescence substrate for alkaline phosphatase and autoradiography.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

MALDI-TOF of MS11mkA LOS-- Negative ion MALDI spectra of O-deacylated and O-deacylated/HF-treated LOS from MS11mkA are presented in Fig. 1, A and B, respectively; their interpretation is based on the known structure of this molecule's glycose moiety and reported structures of neisserial LOS lipoidal moieties. Proposed compositions for deprotonated molecular ions and some observed fragment ions are shown in Table I; there were relatively few molecular ion peaks. Essentially all observed deprotonated ions were singly charged, as were the molecular ion peaks at m/z 2507.2, 2426.8, and 2346.8. 


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Fig. 1.   Negative ion MALDI mass spectra of O-deacylated (panel A) and O-deacylated/HF-treated (panel B) LOS from N. gonorrhoeae variant MS11mkA.

                              
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Table I
Structures and masses of LOS in MALDI mass spectra

The mass of the most abundant molecular ions at m/z 2426.8 was consistent with a (Hex)2HexNAc(Hep)2 PEA(Kdo)2 lipoidal moiety structure and the established structure of the glycose moiety of the MS11mkA LOS (9). The molecular ion peaks at m/z 2507.2 and 2346.8 differed from the peak at m/z 2426.8 by the mass of HPO3 (80 Da). That these represented tri- and monophosphoryl lipoidal moiety variants of the m/z 2426.8 species, with the same glycose moiety, was supported by the presence of a prominent peak at m/z 950.8 that corresponded to deprotonated ions of the diphosphoryl lipoidal moiety, a much less prominent peak for the monophosphoryl lipoidal moiety at m/z 871, and a peak for the triphosphoryl lipoidal moiety at m/z 1030.8 that, albeit present in Fig. 1A, was somewhat more prominent in other spectra of this sample. The peak at m/z 2346.8 corresponded to a double charged peak at m/z 1172.8 in the ESI spectrum of the MS11mkA LOS (data not shown). Its relative abundance, however, was much greater in the MALDI than in the ESI spectrum. The difference in relative abundances suggested that the losses of HPO3 and H3PO4 observed in the MALDI spectrum were partially the result of prompt fragmentation occurring in the source. The small peaks at m/z 2303.3, 2223.3, and 2206.0 differed from the peak at m/z 2426.8 by the loss of PEA (-123 Da), both HPO3 and PEA, or HexNAc (-203 Da for both), and Kdo (-220 Da), respectively.

The peaks observed in the region of m/z 1200-1500 were B-type oligosaccharide fragments ions (19, 22) in which the glycosidic oxygen is retained by the lipoidal moiety. The peaks at m/z 1473.4 and 950.8 corresponded to the glycose and lipoidal fragments, respectively, of the m/z 2426.8 LOS. The peaks at m/z 1429.3, 1252.7, and 1209.8 arose from the losses of CO2 (-44 Da), Kdo (-220 Da), and both Kdo and CO2 (-264 Da), respectively, from the oligosaccharide fragments at m/z 1473.4.

The most prominent peak in the spectrum of the O-deacylated/HF-treated MS11mkA LOS (Fig. 1B) was at m/z 2144.2, 282.6 Da less than the m/z 2426.8 peak in Fig. 1A, and corresponded to the loss of the two lipoidal HPO3 groups and a PEA from the m/z 2426.8 LOS. The peak at m/z 2326.5 was unexpected. It differed from the peak at m/z 2144.2 by 182 Da and most likely resulted from retention of a single laurate during incomplete O-deacylation.

Loss of Kdo (220 Da) from ions at m/z 2144.2 yielded the ion peak at m/z 1924 (Table II). A peak that arose from a B-type glycosidic bond cleavage between the two N-myristoylglucosamines of the lipoidal moiety can be seen at m/z 1756.4; loss of Kdo from these ions yielded those at m/z 1536.5. The peak at m/z 1351.4, a B-type oligosaccharide fragment ion peak, differed from the m/z 1473.4 peak in Fig. 1A by the loss of a PEA moiety (123 Da). The peak at m/z 1306.9 arose from loss of CO2 (-44 Da) from the m/z 1351.4 peak.

                              
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Table II
Structures and masses of MS11mkC LOS from ESI mass spectrometry

MALDI-MS of MS11mkC-- We compared mass spectra of the MS11mk C variant LOS with those of LOS from the A variant. The O-deacylated diphosphoryl variant A LOS molecular ion peak at m/z 2426.8 was replaced in the negative ion MALDI spectrum of the O-deacylated MS11mkC LOS by a series of peaks for molecular ion pairs at m/z 2669.5 and 2792.6, m/z 2872.5 and 2995.6, and m/z 3034.9 and 3158.0 (Fig. 2A). These peaks were not present in the spectrum of the variant A LOS; each pair differed between peaks by a PEA (123 Da). An analogous series of single peaks which lacked phosphoesters (-160 Da or -283 Da) were at m/z 2874.2, 2712.4, and 2509.5 in the MALDI spectrum of the O-deacylated/HF-treated variant C LOS (Fig. 2B). These three molecular ion peaks were consistent with the addition of HexHexNAc (+365 Da) to the MS11mkA m/z 2426.8 molecule (m/z 2792.6) followed by the further additions of HexNAc (+203 Da, m/z 2995.6) and HexHexNAc (m/z 3158.0) to the MS11mkC m/z 2792.6 molecule (Table I).


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Fig. 2.   Negative ion MALDI mass spectra of O-deacylated (panel A), and O-deacylated/HF-treated (panel B) LOS from N. gonorrhoeae variant MS11mkC. A single point internal calibration of the spectrum was performed using the mass of the single charged peak at m/z 2509.5 for O-deacylated paraglobosyl LOS corresponding to the structure shown in Table II with an average calculated Mr of 2510.5. The initial externally calibrated spectrum had assigned this peak at m/z 2508.1.

The peaks at m/z 2630.3 and 2572.0 in the Fig. 2A spectrum were consistent with the loss of a Hex residue (-162 Da) and a Kdo residue (-220 Da), respectively, from the peak at m/z 2792.6. There were several small peaks for B-type oligosaccharide fragments in the m/z 1500-1900 range, including the fragment ion peak at m/z 1840.3 that arose from loss of the lipoidal moiety (-953 Da) from the prominent peak at m/z 2792.6. The peaks at m/z 1796.2 and at m/z 1576.3 arose from the peak at m/z 1840.3 by loss of CO2 (-44 Da) and the loss of CO2 and Kdo (-44 and -220 Da), respectively. The peak at m/z 1716.6 originated from loss of the lipoidal moiety from the (-PEA) peak at m/z 2669.5. Di- and monophosphoryl lipoidal moiety fragment ion peaks were present at m/z 952.1 and m/z 854 (-H2O; -18 Da).

The spectrum of the HF-treated MS11mkC LOS in Fig. 2B contained deprotonated molecular ion peaks at m/z 2874.2, 2712.4, 2509.5, and peaks (not labeled) for the loss of H2O (-18 Da) from the molecular ion peaks. Fragment ion peaks that arose from glycosidic bond cleavage during HF treatment were more prominent in the spectrum of O-deacylated/HF-treated MS11mkC LOS (Fig. 2B) than in the spectrum of similarly treated MS11mkA LOS (Fig. 1B). For example, the prominent peak at m/z 2122.4 and a less prominent one at m/z 2325.7 originated from the molecular ions at m/z 2509.5 and 2712.4 by glycosidic bond cleavage between the N-myristoylglucosamines of their lipoidal moieties (-387 Da). The prominent peak at m/z 1902.5 corresponded to the loss of Kdo (-220 Da) from the respective hemilipoidal m/z 2122.4 molecules, whereas loss of Kdo from the major, m/z 2509.5, molecular ion peak accounted for the m/z 2289.5 ion peak.

The loss of the remainder of the lipoidal moiety from the peak at m/z 2122.4 (Fig. 2B) produced the peak at m/z 1717.6 observed at m/z 1716.6 (calculated m/z 1716.5) in the spectrum of O-deacylated MS11mkC LOS (Fig. 2A). The peak at m/z 1673.4 arose from loss of CO2 (-44 Da) from the peak at m/z 1717.6. The peaks at m/z 1740.3 and m/z 1537.1 are related by loss of a Hex (-162 Da) and a HexHexNAc (-365 Da), respectively, from the m/z 1902.5 peak.

ESI-MS of MS11mkC LOS-- ESI under negative ion conditions yields multiply deprotonated molecular ions (M - nH)n-, where n refers to the number of protons removed and therefore determines the absolute charge state, n = z. Partial lactonization of Kdo residues (-H2O; 18 Da) causes molecular ion peaks to appears as m/z -6 (triple charged) or m/z -9 (double charged) doublets. A negative ion ESI spectrum of O-deacylated MS11mkC LOS is shown in Fig. 3; the proposed compositions for the observed molecular ion species are given in Table II. Peaks corresponding to triple and double charged ions for a particular species have been labeled in Fig. 3 and Table II as A-J, and those marked with an asterisk differ from the next larger labeled peak by a single PEA residue (-123 Da).


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Fig. 3.   Negative ion ESI mass spectrum of O-deacylated LOS from N. gonorrhoeae variant MS11mkC. Except for the lack of PEA residues, the series of peaks (B, D, F, and H) marked by an asterisk are identical to the series of PEA-containing peaks (C, E, G, and I).

The ESI spectrum further confirmed the presence of an Mr 3158.9 LOS species (I ions). The main peaks in the spectrum were consistent with the presence of two basal series, a PEA1 series and a PEA0 series, each with alpha  chains that increased by the alternating addition of HexNAc and Hex residues to the Mr 2428.2 lactosyl LOS molecules (A ions) that MS11mkC LOS have in common with MS11mkA LOS. The Mr 2428.2 LOS was present in the triple charged state at m/z 808.1 and 808.2 in ESI spectra for MS11mkC and MS11mkA LOS, respectively. This molecular species was only a minor component of the MS11mkC LOS, but it predominated in the spectrum of MS11mkA LOS (not shown). The alternating +HexNAc and +Hex additions to the Mr 2428.2 molecules formed the B-J ion series. A HexNAc addition to the Mr 3158.9 moiety was absent, but triple charged +HexHexNAc ions could be identified at m/z 1172.8 (J ions), albeit in low abundance, that were consistent with an LOS molecule with three HexHexNAc additions to the MS11mkA lactosyl LOS. A third ((PEA)2) basal series was present but at very low abundances. The (PEA)0 basal series molecules appeared to prefer the double charged state, whereas the (PEA)1 basal series molecules were more abundant in the triple charged state.

Digestion by Exoglycosidases-- The mass spectra provided information about the composition of the higher mass LOS molecules made by MS11mkC but not about sequence or anomeric configuration. We obtained this information by digesting MS11mkC LOS with various exoglycosidases and analyzing the results with SDS-PAGE. We used S. typhimurium Ra LPS and gonococcal F62 LOS as positive controls and standards with which to interpret the gels. The LOS made by MS11mkC migrate in SDS-PAGE as three primary components: the fastest migrating paragloboyl LOS (LOS1); a middle, gangliosyl, component (LOS2); and a slowest migrating, hence largest, component, LOS3 (Fig. 4). In Fig. 4, MS11mkA LOS, which has a nonreducing terminal beta -galactose (9), appears as a single band (lane A); after digestion with beta -galactosidase, two bands are seen (lane B), likely indicating that cleavage of the nonreducing terminal beta -galactose did not go to completion. In contrast, beta -galactosidase essentially completely hydrolyzed MS11mkC LOS3 and, as expected, LOS1. This confirmed that the additional Hex on the 3034.9/3158.0 ion pair in Fig. 2A, LOS3, was a nonreducing terminal beta -galactose.


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Fig. 4.   Long 12.9% polyacrylamide gel of LOS digested with beta -galactosidase from jack bean meal. Approximately 350 ng of LOS was loaded per well. Shown are MS11mkA (lane A), MS11mkA digested with beta -galactosidase (lane B), F62 (lane C), F62 digested with beta -galactosidase (lane D), MS11mkC digested with beta -galactosidase (lane E), MS11mkC (lane F), F62 (lane G), and F62 digested with beta -galactosidase (lane H). Reactivities with mAbs shown to the right and by the placemarks on the left were determined previously by immunoblots (5).

Subsequent digestion with beta -N-acetylhexosaminidase resulted in complete hydrolysis of LOS2 (data not shown), as expected. The sequential digestion of MS11mkC LOS with beta -N-acetylhexosaminidase followed by beta -galactosidase removed a single beta -galactose from LOS3 and caused it to migrate as if it were LOS2, as shown in Fig. 5. These results confirmed that the additional HexNAc on the 2872.5/2995.6 ion pair in Fig. 2A (LOS2) was a nonreducing terminal beta -N-acetylglucose or beta -N-acetylgalactose and that the terminal beta -galactose of LOS3 was attached to this beta -N-acetylhexosamine and blocked its digestion by the beta -N-acetylhexosaminidase.


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Fig. 5.   Long 12.9% SDS-PAGE of LOS from MS11mkA digested with beta -N-acetylhexosaminidase, and MS11mkC digested first with beta -N-acetylhexosaminidase and then with either beta -galactosidase or beta -glucosidase as described under "Experimental Procedures." The beta -glucosidase enzyme will hydrolyze either beta -glucose or beta -galactose at the nonreducing terminus. Approximately 200 ng of LOS was loaded per well, based on the starting material.

The enzyme beta -glucosidase from sweet almonds will cleave nonreducing terminal glucose, galactose, or fucose in the beta -D configuration, whereas beta -galactosidase and alpha -galactosidase are specific for beta -galactose and alpha -galactose, respectively. The migration of LOS1-LOS3 was essentially the same after either beta -glucosidase or beta -galactosidase digestion following beta -N-acetylhexosaminidase treatment, which would be expected if the substrates for either enzyme were the same beta -galactose moieties (Fig. 5).

The LPS from S. typhimurium Ra expresses a single nonreducing terminal alpha -galactose. Electrophoretic migration of this LPS was faster after digestion with alpha -galactosidase; however, the electrophoretic migrations of the MS11mkC LOS components were not altered by alpha -galactosidase digestion (not shown).

MS11mkC LOS was blotted with mAb 2-1-L8 before and after digestion by beta -N-acetylhexosaminidase and after digestion by beta -N-acetylhexosaminidase followed by beta -galactosidase (Fig. 6). MAb 2-1-L8 binds to LOS expressing nonreducing terminal lactosyl (Galbeta 1right-arrow4Glcright-arrow) groups; it did not bind MS11mkC LOS prior to digestion (Fig. 6, lane A). However, after beta -N-acetylhexosaminidase digestion, a single band was observed (lane B) in the immunoblot which diminished in size after a subsequent digestion with beta -galactosidase (lane C). This result indicates that one MS11mkC LOS component has a nonreducing terminal (HexNAcbeta 1right-arrowGalbeta 1right-arrow4Glcright-arrow) moiety that was digested by beta -N-acetylhexosaminidase to expose a nonreducing terminal (Galbeta 1right-arrow4Glcright-arrow) group that then was bound by mAb 2-1-L8 and provided a substrate for beta -galactosidase. A single nonreducing terminal (HexNAcbeta 1right-arrowGalbeta 1right-arrow4Glcright-arrow) group would not be bound by mAbs 2-1-L8, 1B2, or 1-1-M (Fig. 4) and thus is consistent with the minor, fastest migrating component of the MS11mkC LOS observed in SDS-PAGE (Fig. 4).


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Fig. 6.   Immunoblot of 2-1-L8 binding to SDS-PAGE separated LOS from MS11mkC (lane A), MS11mkC LOS digested with beta -N-acetylhexosaminidase (lane B), MS11mkC LOS digested first with beta -N-acetylhexosaminidase and then with beta -galactosidase (lane C), and F62 LOS (lanes D and E).


    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Both ESI- and MALDI-TOF-MS can be used to detect molecular ions of O-deacylated bacterial LOS, particularly in the negative ion mode, and can assay qualitatively the inherent heterogeneity of complex LOS mixtures. The introduction of a time delay between the ionization and extraction ("delayed extraction") in MALDI-TOF-MS of LOS produces improvement in mass resolution and accuracy as well as a significant reduction in metastable decay processes compared with continuous extraction MALDI. Also with delayed, compared with continuous, extraction prompt fragment ions from both the lipoidal moiety and the oligosaccharide portions of the molecule can be observed as well defined peaks by raising the laser power above the threshold (19). These prompt fragments originate from cleavage of the glycosidic bond between Kdo and the lipoidal moiety, producing Y-type lipoidal moiety ions and B-type oligosaccharide ions (19, 22). ESI-MS has been shown to have excellent dynamic range for the analysis of complex mixtures of O-deacylated LOS (17) but requires approximately 1 order of magnitude more sample (3-10 µg) than is required for delayed extraction MALDI (19). There have been few direct comparisons of the application of the two techniques to O-deacylated LOS, and we chose to use both methods.

O-Deacylation of the LOS by mild hydrazine treatment was performed for two primary reasons (19). One rationale is to increase the water solubility of the LOS, which thereby increases its amenability for ESI-MS and for co-crystallization with standard MALDI matrices. The other reason is to remove acetone-soluble contaminants in the acetone precipitation step.

The LOS made by MS11mkC migrate in SDS-PAGE as three primary components and a fourth minor component; the slowest (LOS3) and fastest (LOS1) migrating primary components bind mAb 1B2, whereas the middle component (LOS2) binds mAb 1-1-M. The fourth and fastest migrating component that binds neither mAb is visible in quite low abundance; exoglycosidase digestions revealed that it lacks the terminal beta -galactose of the paraglobosyl lacto-N-neotetraose. We sought to solve the structure of LOS3.

ESI and MALDI-MS of O-deacylated LOS made by MS11mkC produced molecular and fragment ions that were consistent with the known structure of gonococcal paraglobosyl (LNnT; LOS1) and gangliosyl (GalNAcright-arrowLNnT; LOS2) molecules (2, 23, 24). Gonococcal LOS have been shown to be characterized by production of molecules with a conserved basal region and varying oligosaccharide branches that represent a series of biosynthetic precursors (24-26). Thus, paraglobosyl and/or gangliosyl LOS (LOS1 and LOS2) are most likely biosynthetic precursors of the larger component, LOS3.

Mass spectrometric peaks for the higher Mr component (LOS3) were consistent with either the addition of a single hexose (+162 Da) to the gangliosyl LOS or the addition of both a hexose and an N-acetylhexosamine (+365 Da) to the paraglobosyl LOS. Ions consistent with the addition of a single hexose to the paraglobosyl LOS were not observed, indicating that if the paraglobosyl is a biosynthetic precursor to LOS3, either the Hex or the HexNAc is always added when the other is present.

An addition of Hex to the gangliosyl LOS could be 1) to the terminal beta -GalNAc of the alpha  chain; 2) to the terminal alpha -GlcNAc of the gamma  chain; 3) to form a fourth branch, such as that found on the second heptose (11); or 4) to the molecule internally.

The results from the exoglycosidase digestions of the MS11mkC LOS reveal that beta -galactosidase digestion causes essentially complete hydrolysis of a single terminal beta -galactose from LOS3 and that LOS3 does not express nonreducing terminal beta -N-acetylhexosaminidase, alpha -galactose, or beta -glucose. Thus, if LOS3 is formed by the addition of Hex to the gangliosyl LOS, the Hex has to be on the beta -GalNAc of the alpha  chain because LOS3 is not susceptible to digestion by beta -N-acetylhexosaminidase. Sequential treatment of LOS3 with beta -N-acetylhexosaminidase followed by beta -galactose and then again with beta -N-acetylhexosaminidase revealed loss of a single beta -galactose followed by loss of beta -N-acetylhexosamine (not shown). Because only a single beta -galactose moiety was hydrolyzed by beta -galactosidase, Galbeta 1right-arrowHexNAc could not have been added to the gamma  chain alpha -GlcNAc or elsewhere on the paraglobosyl LOS as this would create two nonreducing terminal beta -galactose moieties. The data do exclude addition to the gangliosyl LOS by possibilities 2, 3, and 4 above but do not rule out the internal addition of a HexHexNAc disaccharide to the paraglobosyl LOS. However, the only possible structure of LOS3 that is consistent with the data if either LOS1 or LOS2 serves as a biosynthetic precursor is the addition of a beta -galactose monosaccharide to the beta -GalNAc of the gangliosyl LOS or the addition of a beta -Galright-arrowHexNAc disaccharide to the paraglobosyl LOS (26).

Theoretically, the oligosaccharides of the 1B2 and 1-1-M epitopes on the MS11mkC LOS (LOS1-3) could be a beta  chain formed by attachment to the second heptose (Hep2). However, recently published genetic and antigenic analyses do not support this possibility. Gonococcal strain MS11mk (27) was isolated previously as its variants A, B, C, and D, based on differences in LOS phenotype (5). The gene (lgtG) encoding the glycosyl transferase that forms the alpha 1-3 link between Hep2 and glucose, the first monosaccharide found on the beta  chain of the gonococcal LOS (11), was recently identified (28) and found to be out of frame in strain MS11mkC, as provided by H. S. to D. C. Stein, but called just MS11 by Banerjee et al. (28). Thus, the gene required for the first step in the biosynthesis of a beta  chain lactose is not transcribed by MS11mkC gonococci. This is in accordance with the prior structural analysis of the LOS of the MS11mkA variant which revealed the presence of an alpha  chain lactosyl group but no beta  chain (9).

In addition, Banerjee et al. reported that mAb 2C7, which binds only gonococcal LOS with a beta  chain lactose (11, 28, 29), does not bind MS11mkC (28). mAb 2-1-L8 binds nonreducing terminal lactosyl moieties on MS11mkA LOS; 2-1-L8 binding is inhibited by the presence of beta  chain oligosaccharides (28) but does not compete with binding of 2C7 to beta  chain lactosyl groups (29). Digestion of MS11mkC LOS with beta -N-acetylhexosaminidase led to the formation of a single LOS with a 2-1-L8 epitope (lactosyl moiety attached to Hep1), confirming the expression of a nonreducing terminal alpha  chain HexNAc-Lac moiety by MS11mkC.

Analysis of O-deacylated/HF-treated (dephosphorylated) LOS showed that the three most abundant LOS molecules were present in (PEA)0 and (PEA)1 basal region series (Table III). This is also evidence that there is not an additional beta  chain on the second heptose because a beta  chain on gonococcal LOS (11) and a single PEA substituent (25) have both been found to be linked to the 3-position of Hep2, and thus the presence of one could preclude the other. Several gonococcal LOS that have an alpha  chain but no beta  chain have been reported (24, 25); however, the only gonococcal LOS reported to have a beta  chain also have an alpha  chain (11, 28, 30). Thus, all of the data support our conclusion that the oligosaccharide chains on LOS1-3 which form the 1B2 and 1-1-M epitopes (5) are not beta  chains attached to Hep2 but rather are attached to Hep1 of the basal region.

                              
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Table III
Proposed structures of major O-deacylated LOS from MS11mkC

mAb 1B2 binds LOS3, so its structure must account for this immunochemistry. 1B2 was raised to a linear N-acetyllactosamine glycosphingolipid, lacto-N-norhexaosylceramide or nLc6Cer, (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc-ceramide) (31) and is specific for terminal LacNAc disaccharides (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow) (2, 11). It does not react with Galbeta 1right-arrow3GlcNAcbeta disaccharides (Lc4Cer) (2). Neither a beta -galactose addition to the gangliosyl LOS alpha  chain nor a Galbeta 1right-arrow4GalNAc addition to the paraglobosyl LNnT would create a terminal LacNAc, so the only remaining possibility is a Galbeta 1right-arrow4GlcNAcbeta right-arrow addition to the nonreducing terminal paraglobosyl LOS molecule (see Table III).

Linkage of the disaccharide Galbeta 1right-arrow4GlcNAcbeta to the nonreducing terminal alpha -GlcNAc of the gamma  chain would require activity of an enzyme that is not known for LOS biosynthesis by either N. gonorrhoeae or Neisseria meningitidis (26), whereas its addition to the paraglobosyl LNnT would require only that lgtA, the beta -glucosaminyltransferase of Neisseria, use the terminal galactose of LacNAc, as well as that of lactose as an acceptor, which seems quite feasible. Also, addition of LacNAc to the gamma  chain GlcNAc would create two nonreducing terminal beta -galactose residues, not one. A (LacNAc)2-Lac, on the other hand, would exactly mimic the oligosaccharide of nLc6Cer and explain all of the mass spectral, electrophoretic, and immunochemical data.

The presence of a (LacNAc)2-Lac structure would imply that further LacNAc additions would be biosynthetically feasible, and these were sought in the mass spectra. In the ESI spectrum a small peak consistent with triple charged (HexNAc-Hex)2-LNnT(PEA)1 ions was observed at m/z 1172.8. The poor accuracy of the experimental Mr for the peak compared with others in the spectrum (see Table II) is likely caused by the low abundance of the ions. The assignment of this peak is supported by the observation of LOS from freshly isolated strains from 36 men of one or more SDS-PAGE LOS bands that bound mAb 3F11 (same specificity as mAb 1B2) (1) but migrated more slowly than paraglobosyl LOS (5). A peak consistent with (Hex-HexNAc)2-LNnT(PEA)0 ions was observed in the spectrum but was so small as to be insignificant.

A few fresh urethral isolates make three LOS molecules that bind mAb 1B2, and most make two. Similarly, most make at least two and sometimes three, higher Mr LOS that bind mAb 1-1-M (5). Additions of (LacNAc)1-2 and GalNAc-LacNAc repeats to paraglobosyl LNnT structures would explain fully the alternate binding of mAbs 1B2 and 1-1-M to higher Mr LOS of urethral isolates (5). The immunochemical data suggest that terminal polylactosamine (pLacNAc) structures are routinely made by urethral isolates of N. gonorrhoeae.

Although minor O-deacylated LOS components of MS11mkC were observed in ESI that were not detected in MALDI-TOF spectra, at least 10 times more sample was required to detect the higher mass species in ESI analysis suggesting that MALDI as a technique may be more sensitive. However, this is partially caused by the high rate of ion transmission characteristic of TOF mass analysis. Our results highlight the potential applicability of sequential glycosidase digestion, SDS-PAGE, and immunoblots coupled with negative ion ESI or delayed extraction MALDI-MS to provide structural information with significantly reduced amounts of LOS.

    ACKNOWLEDGEMENT

We thank John Kim of the Mass Spectrometry Facility of the Oakland Children's Hospital for assistance with the electrospray analysis.

    FOOTNOTES

* This work was supported by NIAID National Institutes of Health Grant AI21620 (to J. M. G.), the Biomedical Research Technology Program of the NIH National Center for Research Resources (A. Burlingame, Director) Grant RR01614 (to the UCSF Mass Spectrometry Facility), and the Research Service of the Department of Veterans Affairs. This is Report 93 from the Centre for Immunochemistry, University of California, San Francisco.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: VA Medical Center (111W1), 4150 Clement St., San Francisco, CA 94121. Fax: 415-221-7542; E-mail: cjohn{at}vacom.ucsf.edu.net.

parallel Present address: School of Public Health Johns Hopkins University, 1165 Ross Bldg., Division of Infectious Disease, 720 Rutland Ave., Baltimore, MD 21205.

The abbreviations used are: LOS, lipooligosaccharide(s); PAGE, polyacrylamide gel electrophoresis; mAb(s), monoclonal antibody(ies); MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; TOF, time of flight; ESI, electrospray ionization; Kdo, 2-keto-3-deoxyoctulosonic acid; TEMED, N, N,N'N'-tetramethylethylenediamine; PEA, phosphoethanolamine; LPS, lipopolysaccharide.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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