Department of Cell Biology and Molecular
Genetics, University of Maryland, College Park, Maryland 20742
Neisserial lipooligosaccharides (LOSs) are a family of complex cell
surface glycolipids. We used mass spectrometry techniques (electrospray
ionization, collision-induced dissociation, and multiple step),
combined with fluorophore-assisted carbohydrate electrophoresis
monosaccharide composition analysis, to determine the structure of the
two low-molecular-mass LOS molecules (LOSI and LOSII) expressed by
Neisseria subflava 44. We determined that LOSI contains one
glucose on both the 
and 
chains. LOSII is structurally related
to LOSI and differs from it by the addition of a hexose (either glucose
or galactose) on the chain. LOSI and LOSII were able to bind
monoclonal antibody (MAb) 25-1-LC1 when analyzed by Western blotting
experiments. We used a set of genetically defined Neisseria
gonorrhoeae mutants that expressed single defined LOS epitopes
and a group of Neisseria meningitidis strains that
expresses chemically defined LOS components to determine the structures
recognized by MAb 25-1-LC1. We found that extensions onto the -chain
glucose of LOSI block the recognition by this MAb, as does further
elongation from the LOSII chain. The LOSI structure was determined
to be the minimum structure that is recognized by MAb 25-1-LC1.
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INTRODUCTION |
Neisseria gonorrhoeae and
Neisseria meningitidis are important human pathogens.
Although there are many more cases of gonorrhea than meningococcal
meningitis, meningitis is a much more serious disease due to its
associated mortality. The importance of lipooligosaccharide (LOS) in
the pathogenesis and immunobiology of these microbes is well
established (1, 2, 9, 16, 19, 23, 41). LOSs are a family
of complex glycolipid molecules found on the outer surfaces of the
outer membranes of gram-negative bacteria (1, 2, 9, 12, 16, 19,
22, 41, 53). They possess many antigenic determinants that are
important in natural and acquired immunity (24, 37, 51,
52). In recent years, biologists have focused their efforts on
the study of LOS as a potential vaccine candidate (5, 19,
20).
Gonococcal and meningococcal LOSs have been examined by chemical
(11, 18, 22), biological (17), and
immunological techniques (28, 42), as well as through
visualization by silver staining sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) gels (26, 43). LOS is an
amphipathic molecule that consists of a hydrophilic carbohydrate moiety
attached to a hydrophobic lipid A moiety through two molecules of the
acidic sugar 3-deoxy-D-manno-2-octulosonic acid
(KDO). The oligosaccharide (OS) is multiantenniary and branches at two
basal heptose (Hep) residues, forming three elongation centers defined
as the , , and 
chains (11, 22, 33, 50) (Fig.
1). The chain elongates from Hep I
and may contain several structures that are mimics of human
carbohydrate epitopes (1, 30). -Chain (extending from
Hep II) expression is modulated by the expression of lgtG
(4), and the chain may be composed of a single glucose
(Glc) or lactose, or Glc with additional sugars (12, 51).
The chain has been found in all strains examined and consists of a
GlcNAc or GlcNAc (acetate) linked to Hep II. Occasionally, this chain
is elongated by the addition of galactose (15). Some
positions of Hep I and Hep II are also available for
phosphoethanolamine (PEA) or phosphate addition (10, 29). Most of the genes that mediate gonococcal and meningococcal LOS biosynthesis have been cloned and characterized (4, 7, 13, 14,
25, 27, 38, 40, 45, 54).
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FIG. 1.
Composite structure of N. gonorrhoeae LOS.
Dotted lines, alternative LOS structures. MAb reactivities are
underlined. Genes involved in LOS biosynthesis are indicated. *,
possible site to which PEA or phosphate can be added. The broken line
indicates the addition of an alternate chain, joined to HepI as a
1-4 linkage.
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Monoclonal antibody (MAb) 1B2 binds to gonococcal LOSs that possess
terminal lactosamine residues (16, 23); these LOSs are
generally considered to be high-molecular-mass LOS molecules. Neisseria subflava 44 cells are able to react in colony
blotting experiments with MAb 1B2. However, N. subflava 44 was found to only express low-molecular-mass LOS components when its
LOS was analyzed by SDS-PAGE (D. C. Stein, unpublished
observations). This suggested that other structural motifs could bind
MAb 1B2. We obtained MAb 25-1-LC1, which reacts with several neisserial LOSs with different immunotypes. This suggested that this MAb binds to
a common core epitope. Our studies indicated that N. subflava 44 LOS can bind this MAb very strongly, indicating that this strain's LOS would be a good candidate for determining the specificity of the MAb. This study was undertaken to define the LOS
structure expressed by N. subflava 44, identify structures recognized by MAb 25-1-LC1, and determine the nature of this
organism's ability to bind MAb 1B2.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, oligonucleotides, and culture
conditions.
All bacterial strains used in this study are listed in
Table 1. All Neisseria strains
were grown in phosphate-buffered gonococcal broth (39)
plus growth supplements (49) and 0.042% sodium bicarbonate or on gonococcal agar base (Difco) plates at 37°C in a
CO2 incubator.
Chemicals and reagents.
All chemicals used for this study
were reagent grade or better and were purchased from Sigma Chemical Co.
(St. Louis, Mo.) unless otherwise specified. Tris-Tricine gels (16.5%)
and running buffer were obtained from Bio-Rad Laboratories (Richmond,
Calif.). The fluorophore-assisted carbohydrate electrophoresis (FACE)
monosaccharide composition analysis kit was from Glyco Inc. (Novato,
Calif.). Acetic acid was from Fisher Scientific Co. (Silver Spring,
Md.). MAbs 25-1-LC1 and 2-1-L8 were made in the laboratory of Wendell Zollinger, Walter Reed Army Institute of Research, Washington, D.C. MAb
B5 was a gift from Margaret A. Gidney, Institute for Biological
Sciences, National Research Council, Ottawa, Canada.
LOS purification and SDS-PAGE analysis.
LOSs were purified
from acetone-powdered organisms by the hot phenol-water method
(48). Proteinase K-treated whole-cell lysates were
prepared from 18- to 20-h cultures by the procedure of Hitchcock and
Brown (21). Approximately 0.1 µg of LOS was subjected to
SDS-PAGE on a 16.5% Tris-Tricine gel in Tris-Tricine running buffer in
accordance with the protocol suggested by manufacturer. The gel was
fixed overnight in 40% ethanol-5% acetic acid, and the LOS was
visualized by silver staining (46).
MS analysis of LOS structure.
LOS was subject to mild acid
hydrolysis, extraction, and methylation (6) to produce
samples for characterization by mass spectrometry (MS). The methods
used in this study were described previously (31).
Preparation of MAb 25-1-LC1.
BALB/c mice were immunized
intraperitoneally at weeks 0 and 3 with a saline suspension (0.1 ml/mouse) of N. meningitidis containing an equal mixture of
strain M981 (L5) and strain 8032 (L nontypeable). The suspension
contained approximately 105 live bacteria per ml. At weeks
5, 7, and 10, the mice were immunized intraperitoneally with a saline
solution of LOS (200 µg/ml) prepared from strains M981 and 8032 (0.1 ml/mouse). Spleens were harvested 3 days after the final immunization,
and lymphocytes were fused with P3X63-Ag 8.653 mouse myeloma cells at a
ratio of 4:1, as previously described (32). Positive
clones were selected by an enzyme-linked immunosorbent assay using
plates coated with M981 LOS or 8032 LOS. One clone, producing MAb
25-1-LC1, was selected for further analysis. Western blot analysis was
used to confirm the binding of the MAb to one of the LOS bands
expressed by M981 LOS. Ascitic fluid was produced by injecting 5 × 106 hybridoma cells into pristine-primed BALB/c mice.
Ascitic fluid was collected, and aliquots were stored at 
20°C.
FACE monosaccharide composition analysis.
Purified LOS (~5
µg) was hydrolyzed in 1% acetic acid for 2 h at 80°C. The
hydrolysate was centrifuged (12,000 × g, 20 min), and
the supernatant containing the OSs was collected. For sugar composition
analysis, the OS was treated in accordance with the procedure provided
by Glyko Inc., with the only difference being that the OS was
hydrolyzed with 4 N HCl for 2 h instead of with 2 N
trifluoroacetic acid for 5 h.
Western blot and colony blot analyses.
After SDS-PAGE, LOSs
were electrotransferred onto an Immobilon-P membrane (Millipore Corp.,
Bedford, Mass.) in a Tris-glycine-methanol buffer (0.025 M Tris, 0.192 M glycine, 20% methanol) at a constant voltage of 100 V for 1 h
in accordance with the protocol provided by Bio-Rad Corp. After being
air dried for 1 h, the membrane was processed by the same
procedure as that used for colony bloting, which was described
previously (39).
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RESULTS |
SDS-PAGE profile of N. subflava 44.
N.
gonorrhoeae strain F62 expresses two predominant LOS components
that can be visualized based on their different electrophoretic mobilities on SDS-PAGE gels. The data presented in Fig.
2A show the typical electrophoretic
pattern obtained with LOS isolated from this strain. The chemical
structures of these two components have been determined
(50), and it is the faster-migrating component that binds
MAb 1B2. Derivatives of F62 that lack the ability to make this LOS
component have been constructed (3), and they produce
truncated LOS molecules (Fig. 2A, lanes 2 and 3). N. subflava produced two small low-molecular-mass LOS components
(Fig. 2A, lane 4).
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FIG. 2.
SDS-PAGE profiles and colony blot analysis of various
Neisseria strains. LOS samples from different
Neisseria strains were analyzed on a Tris-Tricine gel, and
their reactivities with MAb 1B2 were detected with a colony blot of a
duplicate gel. SDS-PAGE gel; (b) colony blot with MAb 1B2. Lanes: 1, N. gonorrhoeae F62; 2, N. gonorrhoeae F62
 lgtA; 3, N. gonorrhoeae F62 lgtA
lgtE; 4, N. subflava 44.
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When the gonococcal cells were analyzed by colony-blotting experiments
with MAb 1B2, only F62 cells gave a detectable signal (Fig. 2B, lanes 1 to 3). While LOS isolated from N. subflava 44 possessed
SDS-PAGE mobilities similar to those of the genetically defined
structural derivatives of F62 (F62 lgtA
lgtE and F62 lgtA), N. subflava
44 cells were able to bind MAb 1B2 in a colony-blotting experiment
(Fig. 2B). When LOS was purified and analyzed by Western blotting of
SDS-PAGE gels, only LOS isolated from F62 was able to bind MAb 1B2
(data not shown). These data suggested that LOS isolated from N. subflava 44, while possessing some structural similarities to that
from F62, as evidenced by its ability to bind MAb 1B2, was structurally different.
Characterization of N. subflava 44 LOS structures by
MS.
In order to determine why N. subflava 44 could bind
MAb 1B2 when analyzed on a colony blot yet failed to bind this MAb when analyzed on a Western blot, we used MS to determine the structure of
the LOS expressed by N. subflava 44. LOS was subjected to
mild-acid hydrolysis, extraction, and methylation to produce samples
for characterization by MS. Since the release and derivatization
chemistry modifies the nascent reducing terminal KDO by adding chemical artifact peaks to the mass spectrum, additional
m/z peaks arise from the multiple charge states
commonly observed in electrospray ionization. In the study of N. subflava 44 OS, the variations were of two types: phosphorylation
of the Hep II moiety and addition of monomers to the nonreducing
termini of the and chains (glycoform distributions). MS
profiles allowed us to identify the natural abundance of parent
structures by the summed intensity of the associated peaks, and this
measured abundance was consistent across multiple isolations and sample
preparations. In the single-charge range of the spectrum (Fig.
3A, m/z 1,400 to
1,800), five principal ions were observed and each was paired with a
satellite ion due to phosphorylation (94 Da; plus PO3Me).
The five principal OS fragments are structurally related and differ
only by the modification or elimination of the KDO. The KDO analogs and
their phosphorylated satellite peaks were identified as follows: KDO
methyl ketoside methylester, m/z 1,698.9/1,792.6;
KDO lactone, m/z 1,652.7/1,746.6; KDO with
acetone eliminated, m/z 1,582.7/1,676.5; KDO
methyl addition product, m/z 1,713.7/1,806.6. A
final pair of ions suggests that the OS has completely lost its
terminal KDO moiety (m/z 1,422.7/1,516.6) (Fig.
3A). While this spread of signal across multiple peaks did diminish
sensitivity, we were able to demonstrate a structural relationship when
collision-induced spectra were compared (data not shown).
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FIG. 3.
MS analysis of N. subflava 44 LOS structure.
(A) MS profile with a structural representation of the reducing-end
KDO. The structural assignment was based on the mass difference between
fragments and MSn analysis of each product. (B)
MS2 characterization of OS lacking a KDO
(m/z 1,422.4). Methylation differentiates
terminal and branched residues, identifying overall topology and
glycosidic sequence. (C) MS3 analysis of the -chain
product (m/z 711.3). Linkage assignment from
resonance activation of smaller-mass products provides enhanced
structural detail. (D) MS2 analysis showing
more-informative fragments of minor abundance that provide localization
of the phosphorylated residue, Hep II.
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The collision spectra of the five principal peaks (Fig. 3A;
m/z 1,422.7, 1,582.7, 1,652.7, 1,698.8, and
1,713.7) and those of the peaks for the associated phosphorylated
satellite ions indicated a common structural motif. That motif was
clearly identified by MS2 analysis of the ion lacking a KDO
moiety, m/z 1,422.7 (Fig. 3B). The loss of two
nonreducing terminal residues (terminal HexNAc [tHexNAc]
and terminal hexose [tHex]), the loss of a
tHex disaccharide (tHex-Hex), and the
elimination of a terminal trisaccharide (tHexNAc-Hex-Hep) from the parent ion allowed us to define the core topology for the and chains of this hexasaccharide, (Fig. 3B). The structure was
further clarified by studying the fragments arising from the rupture
between Hep I and Hep II, separating the -chain fragment from the
-chain fragment (represented in peaks of m/z
711.3 and 734.2, respectively). Selection and resonance activation of
the -chain fragment, m/z 711.3, provided a
terminal relationship (tHex-Hex m/z
241.2/445.1), as well as linkage information for this trisaccharide
(Fig. 3C). The increments of +88 and +132 Da are due to the respective
sequence fragments defined by cross-ring cleavages
(m/z 329.2 and 577.2), indicating that the
linkage between each monomer is (1-4) (36). Selection and
activation of the alternate Hep-Hep rupture fragment,
m/z 734.2, provided detailed characterization of
this trisaccharide, (data not shown), indicating that the chain
contained a single Hex and that the chain contained a single HexNAc.
The replacement of protons on a phosphoryl group
(R-OPO3H2) during methylation provided
increments in the mass of the residue of 94 Da. Collisional activation
of these analogs causes elimination, (126 Da), leaving an unsaturated
OS product. Even though this appears to be the most favored path to
disassembly, other less-abundant fragments were also observed, allowing
us to localize the original phosphate site. Thus, phosphorylation on
the Hep II moiety of the OS lacking KDO (m/z 1,516.6), as well as on
those of the other KDO analogs, was indicated. With KDO as a lactone
(m/z 1,652.7), phosphorylation was indicated by
the 94-Da increment (m/z 1,746.6), and
MS2 (two-step) analysis of this product indicated a single
major elimination fragment (m/z 1,620.5; data not
shown). Amplification of the mass range between
m/z 900 and 1,500 in this spectrum showed a
series of nonreducing terminal losses (tHex,
tHexNAc, and tHex-Hex), which were not
phosphorylated and which, by their differences, indicate residue
location (Fig. 3D). The fragment at m/z 941.5 indicates that phosphorylation occurs in the Hep II portion of the OS,
and the sequential loss of the other terminal moieties places the site
on Hep II. While the exact carbon on Hep II could not be ascertained
from these studies, its identification as C-4 cannot be supported by
these spectra.
While MS studies indicated that there were two major components found
in N. subflava 44 LOS, several larger components were present in LOS purified from this organism, but they were present in
quantities too small to allow for their structural determination (data
not shown). The structure described in Fig.
4 provides the carbon backbone for the
largest molecule and has been named LOSII. Hep I is linked through KDO
to lipid A. The chain consisted of a Hex disaccharide; a Hex
monosaccharide accounted for the chain. Approximately one-third of
this OS was phosphorylated. Further study showed the presence of a
component that possessed a single Hex on the chain (named LOSI).
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FIG. 4.
Structural schematic of OSs produced by N. subflava 44. The carbohydrate backbones of the two predominant OSs
expressed are depicted. Both OSs possess , , and chains
analogous to those seen in N. gonorrhoeae and N. meningitidis.
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FACE monosaccharide composition analysis of N. subflava
44 LOS.
MS analysis allowed us to define the chemical backbone of
the LOS components expressed by N. subflava 44. However,
this type of analysis did not allow us to define the nature of the Hex
and N-acetylhexosamine contained within this LOS. We
identified the composition of the sugars contained in these structures
by FACE monosaccharide composition analysis. N. subflava 44 LOS was hydrolyzed to individual sugars by treatment with HCl. Under
these conditions, KDO is destroyed and N-acetyl groups are
extracted from sugars containing this modification. OS samples were
re-N-acetylated by acetic anhydride in sodium bicarbonate
buffer prior to analysis on FACE gels. Figure
5 shows the results of monosaccharide
composition analysis of LOS isolated from N. subflava 44. Since this methodology is able to detect ~5-ng amounts of sugars, it
is necessary to include in these analyses reagent controls, as some
commercial sources of reagents have detectable levels of glucose in
them. The absence of any detectable signal in lanes 1 (reagent control; 1% acetic acid) and 7 (high-pressure liquid chromatography water) demonstrated that all of the signal obtained was derived from the LOS
sample.
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FIG. 5.
FACE monosaccharide composition analysis of LOSs. Lanes:
1, 1% acetic acid; 2, MONO ladder standard 2 (100 pmol of each
monosaccharide); 3, N. gonorrhoeae F62 lgtD
LOS; 4, N. subflava 44 LOS; 5, N. gonorrhoeae F62
lgtA lgtE LOS; 6, N. gonorrhoeae
F62 lgtA lgtF lgtG+ LOS; 7, blank control (high-pressure liquid chromatography-grade
H2O).
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N. gonorrhoeae F62 lgtD contains glucose,
galactose, and N-acetylglucosamine in a molar ratio of
1:2:2. The data presented in Fig. 5, lane 3, indicated that FACE
analysis was able to detect the presence of all three of these sugars
in LOS isolated from this strain. FACE analysis was also able to
correctly detect the presence of the constituents found in LOSs
isolated from F62 lgtA lgtE and F62
lgtA lgtF lgtG+ (Fig. 5, lanes
5 and 6). The amount of N-acetylglucosamine detected in each
of these LOSs was less than expected. We have determined that the
-chain N-acetylglucosamine is underrepresented under the
hydrolysis conditions employed (data not shown).
FACE analysis indicated that the predominant sugar contained in
N. subflava 44 is glucose (Fig. 5, lane 4). We were able to detect a very small amount galactose when larger quantities of LOS were
examined. Likewise we were able to detect
N-acetylglucosamine (data not shown). Since LOSI possessed
one Hex on both the and chains and one
N-acetylhexosamine in the chain and since LOSII possesses one more Hex on the chain than does LOSI, we have concluded that both Hex's of LOSI are glucose and that LOSII is a
disaccharide containing either Glc or Gal as the terminal Hex on the
chain.
Characterization of structures recognized by MAb 25-1-LC1.
We
have previously determined that MAb 25-1-LC1 reacted quite strongly
with LOS expressed by N. subflava 44. In order to define the
structures found in this LOS that are responsible for its binding, the
SDS-PAGE and MAb binding profiles of LOS isolated from N. subflava were compared to the SDS-PAGE and MAb binding profiles
obtained from various strains of N. gonorrhoeae whose LOSs
has been structurally modified by genetic means (3). The data indicated that LOS with glucose on both the and chains bound MAb 25-1-LC1 (i.e., LOS isolated from N. gonorrhoeae
15253 lgtE and N. gonorrhoeae F62
lgtA lgtE lgtG+ (Fig.
6, lanes 2 and 3). However, LOS isolated
from N. gonorrhoeae F62 lgtA lgtF
lgtG+ failed to bind this MAb (Fig. 6, lane 4). These
data indicate that when the - and -chain glucoses are present,
LOS can bind MAb 25-1-LC1.
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FIG. 6.
SDS-PAGE and Western blot analysis of LOS isolated from
various neisserial species. (A) Silver stain of an SDS-PAGE gel. (B)
Western blot of a duplicate gel obtained with MAb 25-1-LC1. Lanes: 1, N. subflava 44; 2, N. gonorrhoeae 15253 lgtE; 3, N. gonorrhoeae F62 lgtA
lgtE lgtG+; 4, N. gonorrhoeae F62
lgtA lgtF lgtG+; 5, N. gonorrhoeae F62 lgtA lgtG+.
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To further characterize the epitope recognized by MAb 25-1-LC1, we
wished to determine if other additions to and/or modifications of the
chain might affect the ability of this MAb to bind to LOS. While
N. meningitidis 35E (L2) LOS and N. meningitidis
M981 (L5) LOS both possess a single glucose in the chain, they
differ in the structures of their chains. L2 LOS possesses
Gal1-4GlcNAc1-3Gal1-4Glc in the chain, while L5 LOS
possesses Gal1-4GlcNAc1-3Gal1-4Glc1-4Glc in the chain (10, 29). LOS isolated from these strains was examined by SDS-PAGE and Western blot analysis using MAb 25-1-LC1. The
data presented in Fig. 7 show that the
high-molecular-mass LOS isolated from L2 and L5 strains failed to bind
MAb 25-1-LC1. This indicates that further elongation onto the -chain
lactose blocks the epitope recognized by MAb 25-1-LC1. The data in Fig. 7 also show that the smaller LOS expressed by L5, which contains two
glucoses in the chain (29), reacts with MAb 25-1-LC1. This indicated that the presence of the second glucose in the chain, like the presence of galactose, does not interfere with the
ability of MAb 25-1-LC1 to bind this LOS.
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FIG. 7.
SDS-PAGE and Western blot analysis of LOS. (A) Silver
stain; (B) Western blot with MAb 25-1-LC1; (C) colony blot with MAb
25-1-LC1. Lanes: 1, N. meningitidis 35E (L2); 2, N. meningitidis M981 (L5); 3, N. subflava 44 LOS.
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Requirement of the first glucose of the chain for -chain
extensions.
The experiments described above demonstrate that MAb
25-1-LC1 is able to bind neisserial LOS when the chain contains a
glucose or a sucrose. In order to determine if any -chain sugars
were required for MAb 25-1-LC1 binding, we tested LOS isolated from F62
lgtA lgtF lgtG+ for its ability
to bind MAb 25-1-LC1. The data in Fig. 6, lane 4, show that F62
lgtA lgtF lgtG+ LOS failed to
bind MAb 25-1-LC1.
However, when the chain is deleted, it is possible that the chain may not be added because of a biosynthetic requirement for the
presence of at least the glucose of the chain before the first
-chain glycosyltransferase can act. Furthermore, in the absence of
the addition of the -chain glucose, a PEA can be variably added.
Plested et al. (34) described MAb B5, which requires PEA
on the 3 position of Hep II for antibody recognition. If F62
lgtA lgtF lgtG+ had a glucose
instead of a PEA on the chain, LOS isolated from this strain should
now bind this MAb. The data in Fig. 8
show that F62 lgtA lgtF lgtG+
could bind MAb B5. This indicates that in N. gonorrhoeae,
when lgtF was defective, no -chain extension by LgtG
occurred. A similar phenomenon was also found in N. meningitidis (25). This demonstrates that the
-chain glucose, added by LgtF, is needed before LgtG (which is
constitutively expressed in this strain) can begin -chain elongation.
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FIG. 8.
SDS-PAGE and Western blot analysis of LOS. (A) Silver
stain; (B) Western blot with MAb B5. Lanes: 1, N. gonorrhoeae F62 lgtA lgtF
rfaK; 2, N. gonorrhoeae F62 lgtA
lgtF; 3, N. gonorrhoeae F62 lgtA
lgtF lgtG+; 4, N. gonorrhoeae F62
lgtA lgtE.
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DISCUSSION |
Previous studies in our laboratory have identified several
nonpathogenic Neisseria spp. that possess the genetic
potential for expressing gonococcus- and meningococcus-like LOS
structures (3). Our genetic analysis of one of these
strains, N. subflava 44, indicated that it possesses
functional lgtA, lgtB, lgtE, and lgtG genes
(3). Therefore, this strain should express the same lacto-N-neotetraose structure found in the chain of
N. gonorrhoeae F62 and N. meningitidis MC58.
However, SDS-PAGE analysis of its LOS indicated that it produced two
major small isoforms, which we have called LOSI and LOSII. In this
study, MS techniques showed that LOSI possesses one Hex on both the and chains and that LOSII contains an additional Hex on the chain. There is no indication that phosphorylation of LOSI occurs,
while about one-third of the OSs in LOSII are phosphorylated at Hep II.
Banerjee and coworkers (4) demonstrated that, in the
absence of lgtG activity, the chain consists solely of a
PEA added to C-3 of Hep II. The fact that N. subflava LOS
contains a glucose added to C-3 in the chain precludes the presence
of PEA on this residue. Since only one-third of the LOSs expressed by
N. subflava 44 contain PEA yet all of the molecules possess
glucose on C-3 of the chain, this modification is at a different
location and its addition may not be required for the export of LOS to
the surface of the gonococcus. These observations also demonstrate that
PEA addition is another potential source of structural variability in
neisserial LOS.
Using FACE monosaccharide composition analysis, we show that the
predominant Hex in N. subflava 44 LOS was glucose; galactose made up less than 10% of the total sugar. The FACE analysis methods we
employed allow for rapid compositional determinations of an unknown LOS
structure. By combining genetic, structural, and compositional analysis, we conclude that LOSI has one glucose on both the and chains and that LOSII is structurally related to LOSI but differs from
it by the addition of either a glucose or galactose on the chain.
The location of galactose in the LOS cannot be determined by these
analysis. However, since a small amount of high-molecular-mass LOS that
can bind MAb 1B2 is made and since N. subflava possesses an
intact lgt gene cluster needed to make this molecule, it
strongly suggests that the galactose is located at the reducing
terminus of the molecule. This structural heterogeneity would not be
detected by MS or SDS-PAGE because these LOS components would have the
same masses and gel mobilities. This points to the need for better
reagents that can recognize these types of differences in the LOS structures.
Both LOSI and LOSII of N. subflava 44 bound MAb 25-1-LC1,
suggesting that this antibody has a specificity for core elements of
neisserial LOS. To determine the specificity of this MAb, several isogenic strains of N. gonorrhoeae F62 and 15253 that
expressed single genetically defined structures were constructed. These reactivities are summarized in Fig. 9.
LOS from strains 15253 lgtE and F62 lgtA
lgtE lgtG+ possess the same structure as
isoform LOSI and they bind MAb 25-1-LC1 with the same intensity as
N. subflava 44. N. meningitidis M981 (L5) LOS
possesses a single glucose in the chain and elongates the chain
from the first glucose. While the predominant bands expressed by these
strains failed to bind MAb 25-1-LC1, a truncated LOS expressed by
N. meningitidis M981 (L5), which contains two glucoses in
the chain, reacted with MAb 25-1-LC1. These experiments indicated
that (i) the -chain glucose is necessary for binding by MAb
25-1-LC1, (ii) LOSI is the minimum structure recognized by MAb
25-1-LC1, and (iii) further extension of the chain and further
extension of the chain block the epitope bound by MAb 25-1-LC1.
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|
FIG. 9.
LOS structure and MAb binding. Shown is a diagrammatic
representation of some of the LOS components expressed by the strains
and their reactivities with specific MAbs. +, the antibody can bind the
structure; , absence of binding by the structure.
|
|
LOS isolated from N. gonorrhoeae F62 lgtA
lgtF lgtG+ failed to bind MAbs 2C7, 3G9, and
25-1-LC1 but bound MAb B5, which requires the presence of PEA on the 3 position of Hep II for binding. The fact that LOS isolated from F62
lgtA lgtF lgtG+ can bind MAb B5
indicates that, when the chain is truncated and lacks the first
glucose, -chain extension does not occur, even when the strain
possesses a functional lgtG gene. This has allowed us to
further define the order by which sugars are added onto a growing OS.
This sequence can be defined as follows: after the addition of Hep I,
Hep II is added, the -chain GlcNAc is added, the -chain glucose
is added, and then further extension of the chain or chain is possible.
Yamasaki et al. (52) characterized MAb 2C7, which
recognizes a similar gonococcal structure for antibody recognition
(lactose extensions off of both Hep I and Hep II). This antibody binds LOS when the chain is extended by the addition of galactose. In our
studies, MAb 2C7 did not bind to N. subflava LOS, indicating that one can use the differential binding of MAb 2C7 and MAb 25-1-LC1 to differentiate chains that contain glucose or lactose.
This work was supported by a grant from the National Institutes
of Health to D.C.S. (AI 24452) and a grant to V.R. (GM45054).
We thank Paul Rick, Uniformed Services University, for allowing us to
use his FACE system.
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Abstract
Introduction
Present address: Department of Chemistry, University of New
Hampshire. Durham, NH 03824.
Present address: Walter Reed Army Institute of Research,
Department of Bacterial Diseases, Division of Communicable Diseases and
Immunology, Silver Spring, MD 20910.
