From the 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
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ABSTRACT |
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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 (Gal 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 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,
Gal Materials--
Aspergillus niger 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
( 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
S. typhimurium Ra mutant LPS and MS11mkC LOS were digested
with
Sequential glycosidase digestions of MS11mkC LOS were performed in 100 mM sodium citrate buffer, pH 4.5, first by
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.
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.
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 (
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 (
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
( 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 (
The peaks at m/z 2630.3 and 2572.0 in the Fig.
2A spectrum were consistent with the loss of a Hex residue
(
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
(
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 ( ESI-MS of MS11mkC LOS--
ESI under negative ion conditions
yields multiply deprotonated molecular ions (M
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 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
Subsequent digestion with
The enzyme
The LPS from S. typhimurium Ra expresses a single
nonreducing terminal
MS11mkC LOS was blotted with mAb 2-1-L8 before and after digestion by
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 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 (GalNAc 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
The results from the exoglycosidase digestions of the MS11mkC LOS
reveal that Theoretically, the oligosaccharides of the 1B2 and 1-1-M epitopes on
the MS11mkC LOS (LOS1-3) could be a In addition, Banerjee et al. reported that mAb 2C7, which
binds only gonococcal LOS with a 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 1
4GlcNAc
1
3Gal
1
4Glc; paraglobosyl; monoclonal
antibodies (mAbs) 1B2+ and 06B4+) and
GalNAc
lacto-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
-N-acetylhexosaminidase and
-galactosidase, alone and
sequentially, combined with mAb binding patterns, confirmed the
presence of a nonreducing terminal repeating LacNAc
((Gal
1
4GlcNAc)2) on the largest LOS, rather than a parallel oligosaccharide structure.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
chain with the same glycose sequences (3, 4, 10, 11), and
hence, epitopes, as those of the
chain; or from terminal or
internal extensions of
chains (3, 4, 10).
1
4Glc
1
4(GlcNAc
1
2Hep
1
3)Hep
1
Kdo, as an aid in interpreting spectra of O-deacylated
MS11mkC LOS.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-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).
-Galactosidase and
-N-acetylhexosaminidase, both from jack bean meal, were
obtained from Oxford GlycoSystems (Bedford, MA).
-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.
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.
-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
1
3, which is cleaved at a relative rate of 1%,
from galactose-linked
1
6 and
1
4, which are cleaved at relative rates of 100% and 75%, respectively (20).
-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).
-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.
-N-acetylhexosaminidase (10 units/ml) and then by
-galactosidase (0.5 unit/ml) or
-glucosidase (5 units/ml).
MS11mkC LOS also was digested sequentially with
-N-acetylhexosaminidase followed by
-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
-N-acetylhexosaminidase-digested MS11mkC LOS was removed
for analysis after inactivation of the enzyme, and
-galactosidase then was added to a concentration of 0.5 unit/ml, and the digestion continued, as described above.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (21K):
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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.
Structures and masses of LOS in MALDI mass spectra
123 Da), both HPO3 and PEA, or HexNAc (
203 Da
for both), and Kdo (
220 Da), respectively.
44 Da), Kdo (
220
Da), and both Kdo and CO2 (
264 Da), respectively, from the oligosaccharide fragments at m/z 1473.4.
44 Da) from the m/z 1351.4 peak.
Structures and masses of MS11mkC LOS from ESI mass spectrometry
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).
View larger version (25K):
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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.
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).
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.
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.
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).
View larger version (31K):
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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).
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.
-galactose
(9), appears as a single band (lane A); after digestion with
-galactosidase, two bands are seen (lane B), likely
indicating that cleavage of the nonreducing terminal
-galactose did
not go to completion. In contrast,
-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
-galactose.
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Fig. 4.
Long 12.9% polyacrylamide gel of LOS
digested with -galactosidase from jack bean meal. Approximately
350 ng of LOS was loaded per well. Shown are MS11mkA (lane
A), MS11mkA digested with
-galactosidase (lane B),
F62 (lane C), F62 digested with
-galactosidase
(lane D), MS11mkC digested with
-galactosidase
(lane E), MS11mkC (lane F), F62 (lane
G), and F62 digested with
-galactosidase (lane H).
Reactivities with mAbs shown to the right and by the
placemarks on the left were determined previously by
immunoblots (5).
-N-acetylhexosaminidase
resulted in complete hydrolysis of LOS2 (data not shown),
as expected. The sequential digestion of MS11mkC LOS with
-N-acetylhexosaminidase followed by
-galactosidase
removed a single
-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
-N-acetylglucose or
-N-acetylgalactose and
that the terminal
-galactose of LOS3 was attached to
this
-N-acetylhexosamine and blocked its digestion by the
-N-acetylhexosaminidase.
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Fig. 5.
Long 12.9% SDS-PAGE of LOS from MS11mkA
digested with -N-acetylhexosaminidase, and MS11mkC
digested first with
-N-acetylhexosaminidase and then
with either
-galactosidase or
-glucosidase as described under
"Experimental Procedures." The
-glucosidase
enzyme will hydrolyze either
-glucose or
-galactose at the
nonreducing terminus. Approximately 200 ng of LOS was loaded per well,
based on the starting material.
-glucosidase from sweet almonds will cleave nonreducing
terminal glucose, galactose, or fucose in the
-D
configuration, whereas
-galactosidase and
-galactosidase are
specific for
-galactose and
-galactose, respectively. The
migration of LOS1-LOS3 was essentially the
same after either
-glucosidase or
-galactosidase digestion
following
-N-acetylhexosaminidase treatment, which would
be expected if the substrates for either enzyme were the same
-galactose moieties (Fig. 5).
-galactose. Electrophoretic migration of this
LPS was faster after digestion with
-galactosidase; however, the electrophoretic migrations of the MS11mkC LOS components were not
altered by
-galactosidase digestion (not shown).
-N-acetylhexosaminidase and after digestion by
-N-acetylhexosaminidase followed by
-galactosidase
(Fig. 6). MAb 2-1-L8 binds to LOS expressing nonreducing terminal lactosyl (Gal
1
4Glc
) groups; it
did not bind MS11mkC LOS prior to digestion (Fig. 6, lane
A). However, after
-N-acetylhexosaminidase
digestion, a single band was observed (lane B) in the
immunoblot which diminished in size after a subsequent digestion with
-galactosidase (lane C). This result indicates that one
MS11mkC LOS component has a nonreducing terminal
(HexNAc
1
Gal
1
4Glc
) moiety that was digested by
-N-acetylhexosaminidase to expose a nonreducing terminal
(Gal
1
4Glc
) group that then was bound by mAb 2-1-L8 and
provided a substrate for
-galactosidase. A single nonreducing
terminal (HexNAc
1
Gal
1
4Glc
) 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 -N-acetylhexosaminidase (lane B),
MS11mkC LOS digested first with
-N-acetylhexosaminidase
and then with
-galactosidase (lane C), and F62 LOS
(lanes D and E).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-galactose of the paraglobosyl
lacto-N-neotetraose. We sought to solve the structure of
LOS3.
LNnT; 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.
-GalNAc of the
chain; 2) to the terminal
-GlcNAc of the
chain; 3) to form a fourth branch, such as that found on the second
heptose (11); or 4) to the molecule internally.
-galactosidase digestion causes essentially complete
hydrolysis of a single terminal
-galactose from LOS3 and
that LOS3 does not express nonreducing terminal
-N-acetylhexosaminidase,
-galactose, or
-glucose.
Thus, if LOS3 is formed by the addition of Hex to the
gangliosyl LOS, the Hex has to be on the
-GalNAc of the
chain
because LOS3 is not susceptible to digestion by
-N-acetylhexosaminidase. Sequential treatment of
LOS3 with
-N-acetylhexosaminidase followed by
-galactose and then again with
-N-acetylhexosaminidase revealed loss of a single
-galactose followed by loss of
-N-acetylhexosamine (not shown). Because only a single
-galactose moiety was hydrolyzed by
-galactosidase,
Gal
1
HexNAc could not have been added to the
chain
-GlcNAc
or elsewhere on the paraglobosyl LOS as this would create two
nonreducing terminal
-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
-galactose monosaccharide to the
-GalNAc of
the gangliosyl LOS or the addition of a
-Gal
HexNAc disaccharide to the paraglobosyl LOS (26).
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
1-3 link between Hep2 and
glucose, the first monosaccharide found on the
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
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
chain lactosyl group but no
chain
(9).
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
chain oligosaccharides (28) but does not compete with binding of
2C7 to
chain lactosyl groups (29). Digestion of MS11mkC LOS
with
-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
chain HexNAc-Lac moiety by MS11mkC.
chain on the second heptose because a
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
chain but no
chain have been reported (24, 25);
however, the only gonococcal LOS reported to have a
chain also have
an
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
chains attached to
Hep2 but rather are attached to Hep1 of the
basal region.
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,
(Gal1
4GlcNAc
1
3Gal
1
4GlcNAc
1
3Gal
1
4Glc-ceramide) (31) and is specific for terminal LacNAc disaccharides
(Gal
1
4GlcNAc
1
) (2, 11). It does not react with
Gal
1
3GlcNAc
disaccharides (Lc4Cer) (2). Neither a
-galactose addition to the gangliosyl LOS
chain nor a
Gal
1
4GalNAc addition to the paraglobosyl LNnT would create a
terminal LacNAc, so the only remaining possibility is a
Gal
1
4GlcNAc
addition to the nonreducing terminal
paraglobosyl LOS molecule (see Table III).
Linkage of the disaccharide Gal1
4GlcNAc
to the nonreducing
terminal
-GlcNAc of the
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
-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
chain GlcNAc would create two nonreducing terminal
-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.
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ACKNOWLEDGEMENT |
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We thank John Kim of the Mass Spectrometry Facility of the Oakland Children's Hospital for assistance with the electrospray analysis.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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