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J Biol Chem, Vol. 274, Issue 51, 36550-36558, December 17, 1999
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From the Department of Biochemistry and
Biotechnology, Tottori University, Tottori 680-8553, the
¶ Institute of Physical and Chemical Research
(Riken), Wako 351-0198, Japan, and the
Maxwell Finland Laboratory for Infectious Diseases, Evans
Memorial Department of Clinical Research and Department of Medicine,
Boston University, Boston, Massachusetts 02118
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Lipo-oligosaccharides (LOS) produced by
Neisseria gonorrhoeae are important antigenic and
immunogenic components of the outer membrane complex. Previously, we
showed that murine monoclonal antibody (mAb) 2C7 did not cross-react
with human glycosphingolipids but identified the LOS epitope that is
widely expressed in vivo and in vitro (Gulati,
S., McQuillen, D. P., Mandrell, R. E., Jani, D. B., and
Rice, P. A. (1996) J. Infect. Dis. 174, 1223-1237). In the present study, we analyzed the structure of
gonococcal strain WG LOS containing the 2C7 epitope and investigated
the structural requirements for expression of the epitope. We
determined that the WG LOS components are Hep[1]-elongated forms of
15253 LOS that have a lactose on both Hep[1] and Hep[2] (Yamasaki,
R., Kerwood, D. E., Schneider, H., Quinn, K. P., Griffiss,
J. M., and Mandrell, R. E. (1994) J. Biol.
Chem. 269, 30345-30351). In addition, we found that expression
of the 2C7 epitope within the LOS is blocked when the Hep[2]-lactose
is elongated. Based on the structural data of these LOS and the results
obtained from immunochemical analyses, we conclude the following: 1)
mAb 2C7 requires both the 15253 OS minimum structure and the
N-linked fatty acids in the lipoidal moiety for expression
of the epitope; 2) mAb 2C7 binds to the LOS that elongates the lactose
on Hep[1] of the 15253 OS, but not the one on Hep[2]; and 3) the
2C7 epitope is expressed on gonococcal LOS despite the presence of
human carbohydrate epitopes such as a lactosamine or its
N-acetylgalactosaminylated (globo) form. Our study shows
that the conserved epitope defined by mAb 2C7 could potentially be used
as a safe site for the development of a vaccine candidate.
Neisseria gonorrhoeae causes one of the major sexually
transmitted human diseases. The potential severity of complications of
gonococcal infection has provided impetus to develop a preventive anti-gonococcal vaccine. In response to natural gonococcal infection, the antibody response is primarily directed against pili, outer membrane proteins, and lipo-oligosaccharide
(LOS)1 (1, 2). Prior vaccine
attempts using gonococcal pilus, porin, and opacity protein have failed
to produce a broadly protective immune response (3-5). Therefore,
several investigators have been concentrating efforts to utilize LOS as
potential vaccines.
Recent immunochemical and structural studies have shown that the
oligosaccharide (OS) structure governs complex antigenic variations
among gonococcal LOS (6-14). The OS moiety consists of a conserved
core and a structurally variable region. Gonococci synthesize this
variable region by adding a glycose moiety sequentially on the
conserved core trisaccharide,
GlcNAc-Hep[2]-Hep[1]2 to
express two different types of OS elongation; OS elongates from
Hep[1] only or from both Hep[1] and Hep (2). Different OS
structures thus produced result in heterogeneous antigenic expression
of LOS whose molecular masses vary in the range of 3-7 kDa (6).
In addition to complex LOS expression, studies by us and others (8, 11)
have shown that gonococci may mimic human glycolipids or may change
serum sensitivity by modifying their LOS (15-17). The 4.5-kDa LOS
produced by strain F62 contains a lactoneotetraose moiety that is
linked to the Hep[1] residue of the core trisaccharide as described
above (11). This tetraose is the carbohydrate moiety also found in a
human glycosphingolipid, paragloboside (8, 11). Furthermore, in
vitro experiments showed that gonococci utilize exogenous cytidine
monophospho-N-acetylneuraminic acid and sialylate their LOS
(17). As a result of sialylation, gonococci become resistant to
complement-mediated killing (15, 16). Thus, gonococci may evade human
immune defenses by mimicking human carbohydrate epitopes or altering
their serum sensitivities.
Despite the frequency of antigenic heterogeneity of gonococcal LOS
often accompanied by cross-reaction with human glycosphingolipids or
gangliosides, our previous studies (18) had indicated the possibility
to overcome the potential problems inherent in the variability of
gonococcal LOS in order to develop LOS-based vaccines. Our recent
studies showed that murine mAbs, 3G9 and 2C7, do not cross-react with
human glycosphingolipids or ganglioside (18). Of the epitopes defined
by these mAbs, the 2C7 epitope is widely expressed in vivo
and in vitro (7, 18). In addition, mAb 2C7 is bactericidal
and opsonic against gonococci converted to serum resistance by
sialylation or strains that are otherwise intrinsically serum-resistant
(18). Further study with mAb 2C7 showed that its anti-idiotypic
antibody elicited a bactericidal and opsonic immune response in
experimental immunization (19). Collectively, these data indicated that
mAb 2C7-defined epitope could potentially be utilized as a safe vaccine target.
Both previous and very recent work have provided some insights on the
mAb 2C7-defined epitope. First, mAb 2C7 did not bind to the lipid A
moiety of the LOS but bound to its tyraminated OS, which suggested that
the mAb recognizes the OS moiety of the LOS (18). Second, inert binding
of the mAb to some LOS samples has shown that LOS lacking OS on the
Hep[2] may not have the 2C7 epitope (18, 20). Third, immunoblot
analysis using truncated gonococcal LOS has suggested the importance of
a lactose on Hep[2] for expression of 2C7 epitope (21). However,
those previous studies have not yet precisely confirmed whether mAb 2C7
recognizes gonococcal LOS of a specific OS elongation pattern. Neither
the 2C7 epitope nor the structural requirements necessary for its expression have been defined.
Among LOS recognized by the mAb 2C7 (7, 18, 21), LOS produced by an
isolate from disseminated gonococcal infection (DGI), 15253, is the
only one whose OS structure has been determined (13). Definition of LOS
structure other than 15253 LOS is essential for us to determine not
only the specific OS elongation pattern that mAb 2C7 recognizes but
also the common epitope expressed among various gonococcal LOS.
Therefore, in the present study, we studied the structures of LOS
components produced by another DGI isolate, WG (22). In addition to the
structural analysis, we investigated structural requirements for the
binding of mAb 2C7 to several additional gonococcal LOS thereby
characterizing the 2C7 epitope. We determined that the major WG LOS
contains a Gal LOS and mAbs--
We used the following gonococcal LOS that have
been prepared previously: F62 (11), MS11mk (variant A) (12), WG (22), 15253 (13), 24-1 (18). 15253 lgtE mutant LOS was provided by Dr. Emil
C. Gotschlich (The Rockefeller University, New York), and JW31R LOS was
a gift from Dr. Herman Schneider (The John Hopkins University,
Baltimore). Murine IgM mAbs, 3F11 and 1-1-M (6, 7, 10, 11, 23), were
provided by Dr. Michael A. Apicella (University of Iowa, Iowa City) and
Dr. Robert E. Mandrell (USDA/ARS, Albany), respectively. The production
of mAb 2C7 (7, 18) has been described previously.
LOS Modification and PAGE Blot Analysis--
Partial deacylation
with NaOH and dephosphorylation of LOS were done as described
previously (10, 12, 24). LOS samples were also de-O-acylated
with anhydrous hydrazine by modifying the method by Haishima et
al. (25); LOS were treated in anhydrous hydrazine at room
temperature (~25 °C) for 2 h. The resulting LOS were further
treated with 4 M KOH for 4 h at 120 °C for complete deacylation (25, 26). Enzymatic treatments of WG, 15253, and JW31R LOS
were done as described previously (10).
PAGE Blot and ELISA--
Intact LOS and chemically and
enzymatically modified LOS were separated with a Bio-Rad mini-PROTEAN 2 Cell (Bio-Rad), and blots were immunostained with subsequent treatments
of mAbs, goat anti-mouse IgG, or IgM Abs (alkaline phosphatase
conjugate) and Western BlueTM Stabilized Substrate (Promega
Co. Madison) (10, 12, 24). TLC immunostaining of LOS was done as
described previously (10) by using anti-mouse IgG (alkaline phosphatase
conjugate) and the substrate as described above. ELISA was done as
follows: wells (Maxi Soap, Nunc, Roskilde, Denmark) coated with LOS
samples (up to 200 ng) were reacted sequentially with a primary
antibody, a corresponding secondary antibody (alkaline phosphatase
conjugate), and then with a substrate solution
(p-nitrophosphate). Absorbancies at 405 nm were measured
with a Multiskan MS (Labsystems, Helsinki, Finland). After determining
the concentration of mAb 2C7 for ~50% binding to the intact 15253 LOS, the binding of the mAb to the LOS (100 ng) was measured in the
presence of inhibitors (25-800 ng). The secondary antibodies
used in this study were purchased from Sigma.
Oligosaccharide (OS) and Carbohydrate Analyses--
Preparation
of OS, composition, and methylation analyses were done as described in
previous studies (10-13, 27) unless otherwise stated. A major OS
component (dephosphorylated) was obtained after gel chromatography (a
Bio-Gel P-6 column, <400 mesh, 1.6 × 90 cm, 100 mM
ammonium acetate) and subsequent desalting with Bio-Gel P-2
chromatography. For gas chromatography-MS analysis, a JEOL AX505HA
spectrometer was used. The following partially methylated alditol
acetates were identified in the major WG OS component: 2,3,4,6-tetra-O-Me-Gal; 2,3,6-tri-O-Me-Glc;
2,4,6-tri-O-Me-Gal; 3,6-di-O-Me-GlcN(Me)Ac;
3,4,6-tri-O-Me-GlcN(Me)Ac; 2,6,7-tri-O-Me-Hep; and 4,6,7-tri-O-Me-Hep. The partially methylated alditol
acetates gave identical fragmentation patterns to those as reported
previously (11-13, 27).
Mass Spectral Analyses--
Negative ESI (electrospray
ionization) mass spectra of de-O-acylated WG LOS were
obtained with a triple stage quadruple mass spectrometer (Finnigan MAT
TSQ 700) equipped with the ESI ion source (28). Samples were dissolved
in water/acetonitrile = 1:1 (v/v) including 0.1% ammonia
solution. The ESI mass spectrometric conditions are as follows:
potential difference in the negative ion mode, 3 kV; capillary
temperature, 200 °C; sample flow rate, 5 ml/min; sheath and
auxiliary gas, nitrogen; sheath gas pressure, 60 pounds/square inch;
collision gas, argon; collision energy, 30 eV; collision gas pressure,
0.4 Pa. The data were processed with a DEC Station 5000/120 computer.
The FAB (fast atom bombardment)-mass spectra of oligosaccharides (OS)
were obtained with the Finnigan MAT TSQ 700 mass spectrometer equipped
with the FAB ion source (29, 30). Samples were dissolved in water. The
FAB mass spectra were recorded under the following conditions: primary
beam, xenon; accelerating voltage of the primary ion, 8 kV; collision
gas, argon; collision energy, 30 eV; collision gas pressure, 0.4 Pa. The data were processed with a DEC Station 2100 computer.
NMR Analysis--
All NMR experiments were run on a JEOL 600 MHz
spectrometer in D2O at 25 °C. Two-dimensional NMR
spectra were obtained in a similar manner as described (11-13, 31);
DQF-COSY (number of acquisition = 32, sweep width = ±2000 Hz, the
1 × 4K data points were processed to give the final 2 × 4K
points, and digital resolutions were 0.68 and 1.37 Hz/point in the
From a panel of LOS recognized by mAb 2C7, we selected a DGI
isolate, WG. This strain produces two LOS components whose molecular weights are higher than 15253 LOS, and mAb 2C7 binds to both (Fig. 1). To obtain the molecular mass and
preliminary composition of the WG LOS, we analyzed the
O-de-acylated WG LOS by negative electrospray ion MS
(ESI-MS). Treatment of the LOS with anhydrous hydrazine at room
temperature (~25 °C) for 2 h gave a water-soluble
de-O-acylated LOS preparation. This ESI-MS analysis
indicated that the major O-deacylated WG LOS has the
following general structure:
(Hex)5-(HexNAc)2-(Hep)2-(KDO)2-(de-O-acylated lipid A) with (PEA)0-2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1-4GlcNAc1-3Gal1-4Glc (lactoneotetraose) and
a Gal1-4Glc (lactose), respectively, on Hep[1] and Hep[2] of
the GlcNAc-Hep[2]-Hep[1] trisaccharide core. mAb 2C7 recognizes the
inner 15253 LOS present in WG LOS as a minimum structure and requires
N-linked fatty acids in the lipid A for maximum expression
of its epitope. In addition, mAb 2C7 binds to LOS whose OS are
elongated from the lactose on Hep[1] of the 15253 OS but not on
Hep[2], and the 2C7 epitope is expressed on gonococcal LOS despite
the presence of human carbohydrate epitopes such as lactosamine or its
N-acetylgalactosaminylated (globo) form.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-N-Acetylhexosaminidase (from jack beans) and
-galactosidase (from Aspergillus niger) were purchased
from Sigma.
2 and 1 axes, respectively), HOHAHA
(number of acquisition = 64, sweep width = ±2000 Hz,
Tm = 120 ms, the 0.5 × 2K data points were
processed to give the final 1 × 4K points); two-dimensional NOE
(number of acquisition = 96, sweep width = ± 2000 Hz,
Tm = 200 ms, the 0.5 × 2K data points were
processed to give the final 1 × 4K points).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
PAGE blot analysis of WG LOS and 15253 (100 ng each). Lane 1, WG LOS; lane 2, 15253 LOS.
Apparent molecular weights of the WG LOS components were estimated by
using gonococcal LOS, MS11mk (12), and F62 (11) (data are not shown).
Anti-mouse IgG (alkaline phosphatase conjugate) was used for the
detection of mAb 2C7.
This composition was indicated by the presence of three sets of triply
and doubly charged ions of the LOS and singly charged ions of its OS
(Fig. 2 and Table
I); for example, the most abundant triply
charged ion at m/z 1038.1 and a doubly charged ion at
m/z 1558.1 correspond to the composition of
(Hex)5-(HexNAc)2-(Hep)2-(KDO)2-PEA-(de-O-acylated lipid A). Its OS counterpart (singly charged) is seen in Fig. 2 as a
dehydrated form at m/z 2163.7. The mass differences between the OS-de-O-acylated lipid A with (PEA)0-1 and
their OS (Table I) confirmed that the intact LOS was
de-O-acylated with the hydrazine treatment described
above.
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Out of singly charged ions, the mass of 1820.3 corresponding to (Hex)5-(HexNAc)2-(Hep)2-KDO (Fig. 2) was also detected in ESI/MS-MS (data not shown) of the doubly charged ion at m/z 1496.3 of Fig. 2. This ESI/MS-MS analysis confirmed that the ion at m/z 1820.3 is due to the OS moiety. In addition, the peak at m/z 2146.8 in Fig. 2 suggested the presence of the OS derived from a minor LOS component, an adduct of HexNAc to (Hex)5-(HexNAc)2-(Hep)2-KDO-PEA, whose identity will be described later. The molecular compositions of the major WG LOS predicted with the above ESI-MS was also confirmed by FAB-MS and compositional analysis described below.
FAB-MS analysis (Fig. 3) of a major WG OS
(dephosphorylated), obtained after P-6 gel chromatography, indicated
the presence of the ion at m/z 1837.9 and its dehydrated
form (at m/z 1820.2). This molecular ion at m/z
1837.9 corresponds to
(Hex)5-(HexNAc)2-(Hep)2-KDO, and
this result was consistent with the presence the singly charged ion at
m/z 1820.3 in the ESI/MS analysis (Fig. 2 and Table I). The
FAB-MS spectrum also indicated the following sequence ions together
with their hydrated forms: 1675.7 (-Hex), 1472.6 (-Hex-HexNAc), 1310.6 (-Hex-HexNAc-Hex), and 1148.0 (-Hex-HexNAc-Hex-Hex). Compositional analysis of the major OS showed the molar ratio of
Gal/Glc/GlcNAc/Hep/KDO is 2.8:2.1:1.7:1.8:1, which showed the HexNAc in
the above sequence ions to be GlcNAc. The ESI-MS, FAB-MS, and the
compositional analysis confirmed the molecular mass of the LOS and the
composition of the OS moiety.
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As predicted from the MS and compositional results, the one-dimensional
NMR analysis (Fig. 4) of the major WG OS
(dephosphorylated) showed the presence of nine anomeric protons (Fig.
4) as follows: Hep(I) (5.45 ppm); Glc(II) (5.31 ppm); GlcNAc(III) (5.09 ppm); Hep(IV) (5.07 ppm); Glc(V) (4.72 ppm); Gal(VI) (4.56 ppm);
Gal(VII) (4.48 ppm); GlcNAc(VIII) (4.43 ppm); and Gal(IX) (4.42 ppm).
These nine anomeric protons were identified based upon coherence relay patterns of H-1 of each carbohydrate residue of I-IX (Fig.
5), the coupling constants (Table
II) obtained with DQF-COSY (data not
shown), and the data of methylation analysis as will described later.
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The structure of the major WG OS (dephosphorylated) was determined by
immunochemical and methylation analyses and by comparative analysis of
NOE data of the WG OS with those of OS previously studied, F62 (11),
MS11mk (variant A) (12), and 15253 (13). We found that a
lactoneotetraose (Gal1
4GlcNAc13Gal14Glc) and a
lactose (Gal14Glc), respectively, are linked to Hep[1] and
Hep[2] of the GlcNAc-Hep[2]-Hep[1]-KDO, respectively (see the
structure in Fig. 4).
The lactoneotetraose being 14-linked to Hep(I) was confirmed by
the following results: 1) mAb 3F11, specific for a Gal14GlcNAc (8, 11), bound to the major LOS component (Fig.
6); 2) the presence of inter-residual
NOEs (Fig. 7), Gal14GlcNAc
(VII-1/V-4); GlcNAc13Gal (V-1/IX-3), Gal14Glc (IX-1/VI-4),
and Glc14Hep (VI-1/IV-4); 3) the presence of a terminal Gal,
4-linked GlcNAc, 3-linked Gal, 4-linked Glc, 3,4-linked Hep in the
methylation analysis. Also, The NMR data of the lactoneotetraose part
of the WG OS are almost identical with those of F62 OS (11).
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The identity of a residue V to be GlcNAc or Glc could not be determined
based on the J-coupling data of its endocyclic protons. However, the mAb 3F11 binding to the major WG LOS (Fig. 6) confirmed that the residue V is GlcNAc but not Glc. We determined that the anomeric proton (VI) at 4.56 ppm is Glc but not GlcNAc based upon the
methylation data. The presence of a terminal GlcNAc in the methylation
analysis does not support VI to be GlcNAc, and a terminal Glc should
have been present in the methylation analysis if the residue III were
Glc and not GlcNAc. The lactosamine, Gal14GlcNAc, being linked to
Gal(IX) was determined by the inter-residual NOE (V-1/IX-3). Thus, we
determined the sequence of Gal14GlcNAc13Gal14Glc is on Hep(IV).
We also determined the following: 1) Gal(VIII)-Glc(II) and GlcNAc(III),
respectively, are 
13- and 12-linked to Hep(I), and 2) this
Hep(I) is 13-linked to Hep(IV). Methylation data (terminal Gal,
4-linked Glc, 2,3-di-linked Hep, and terminal GlcNAc) and the
inter-residual NOEs due to VIII-1/II-4, II-1/I-3, and III-1/I-2
confirmed the structure. The H-3 and H-4 of Gal (VIII) were almost
identical to those of the corresponding Gal residue of 15253 OS (12),
which confirmed that the lactosamine described above is not linked to
the Gal (VIII). Similar to the identity of VI as described above, the
presence of a terminal GlcNAc and the absence of a terminal Glc in the
methylation analysis excluded the possibility of the residue II to be
GlcNAc. Therefore, the residues VI and III were assigned to be Glc and
GlcNAc, respectively. The residue III (5.09 ppm) to be GlcNAc was also
supported by previous studies (11-13) which show that the anomeric
proton of GlcNAc linked to the Hep[2] is located at ~5.10 ppm.
Thus, we determined the structure of the major WG OS component; a
lactoneotetraose is 14 linked to Hep[1] and a lactose is
13 linked to Hep[2] of the GlcNAc-Hep[2]-Hep[1] KDO
sequence. In the present study, the structure of the minor WG LOS
component was not fully investigated. However, our data suggest that
the structural difference between the major and minor LOS components is
GalNAc. This suggestion was supported by the following results: mAb
1-1-M, specific for a GalNAc1-3Gal, bound to the minor LOS component (Fig. 6), and mAb 3F11 bound after the LOS component was
cleaved with -hexosaminidase (data not shown). As described earlier,
ESI-MS analysis also showed the peak at m/z 2146.8 (Fig. 2)
which corresponds to
(Hex)5-(HexNAc)3-(Hep)2-KDO-PEA,
and mAb 2C7 also binds to this component (Fig. 1).
These structural data show that strain WG produces elongated forms of 15253 LOS that has a lactose on both Hep[1] and Hep[2] (13). Stain WG elongates only the lactose on Hep[1] of 15253 LOS to express human carbohydrate epitopes, a lactosamine and its N-acetylgalactosaminylated form (globo) at the non-reducing end. Our structural study revealed that mAb 2C7 binds to the 15253 LOS structure contained within the two WG LOS components despite the presence of those human carbohydrate epitopes.
After determining the structures of the WG LOS components, we analyzed the 2C7 epitope. First, we clarified that mAb 2C7 recognizes a specific carbohydrate elongation pattern, OS on both Hep[1] and Hep[2] as represented by 15253 and WG LOS. Second, by analyzing epitope expression of another gonococcal LOS whose carbohydrate elongates from the lactose on Hep[2] of 15253 LOS, we further specified the OS elongation pattern recognized by mAb 2C7. Third, we defined the basic OS structure necessary for expression of the 2C7 epitope. Fourth, we characterized structural requirements other than the OS structure for the maximum expression of the 2C7 epitope.
In order to clarify the specific OS elongation pattern recognized by mAb 2C7, we analyzed a series of LOS having OS only on Hep[1] of the core Hep[1]-Hep[2] diheptose for their binding capability to mAb 2C7. For this purpose, we used previously characterized gonococcal LOS, F62 (11) and MS11mk (variant A) (12), together with their truncated forms that had been prepared previously (10, 12). The results obtained by ELISA and PAGE blot analyses are summarized in Table III. mAb 2C7 bound none of the gonococcal LOS tested, which confirmed partial results obtained by us (18) and by Banerjee et al. (21). In addition, we re-examined the binding of mAb 2C7 to 24-1 LOS. Previous studies (18) showed that mAb 2C7 bound to 24-1 LOS, although carbohydrate elongation of the major 24-1 OS components were found to occur only on Hep[1] (20). We were not be able to detect mAb 2C7 binding to 24-1 LOS at the usual testing level of 100 ng by Western blot; 250 ng of the LOS was necessary for the detection of a minor component whose PAGE mobility was almost identical to 15253 LOS (data not shown). The present analysis indicated that the previous positive binding of mAb 2C7 to 24-1 LOS is due to the presence of a trace amount of LOS whose OS structure is presumably to be same as that of 15253 LOS. Thus, based upon the above results obtained, we concluded that the 2C7 epitope resides on gonococcal LOS that contain OS on both Hep[1] and Hep[2] and that the LOS containing OS only on Hep[1] do not display the 2C7 epitope.
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Then, we further defined the specific OS pattern that mAb 2C7
recognizes and found that the 2C7 epitope is not expressed when the
lactose on Hep[2] of 15253 LOS is elongated. As Fig.
8 shows, mAb 2C7 did not bind to JW31R
LOS (9) but weakly bound to a minor LOS component having the same PAGE
mobility as 15253 LOS. However, the mAb 2C7 binding to JW31R LOS
increased after it was digested sequentially with -hexosaminidase
and -galactosidase, which indicated that the major JW31R LOS
components are also elongated forms of 15253 LOS. The OS of one of the
major JW31R LOS components had been indicated to contain Hex-Hex and
GalNAc-Hex-Hex-Hex on Hep[1] and Hep[2], respectively (9). This
current study suggests that Gal1-4Glc and GalNAc-Gal-Gal1-4Glc,
respectively, are linked to Hep[1] and Hep[2]. The increase of 2C7
binding to JW31R LOS after the -hexosaminidase treatment is probably
due to further cleavage of the enzyme-treated LOS by -galactosidase
present in the -hexosaminidase preparation since this enzyme
preparation of the same unit used for JW31R LOS also cleaved a Gal off
the 4.5-kDa F62 LOS component that contain a lactoneotetraose (data not
shown). The 2C7 epitope was expressed on WG LOS, the lactose-Hep[1] elongated form, but not on JW31R LOS, the lactose-Hep[2] elongated form of 15253 LOS. This finding shows that mAb 2C7 recognizes only the
LOS whose OS elongates from the lactose on Hep[1] of the 15253 LOS
and that the 2C7 epitope is not expressed when the lactose on Hep[2]
is substituted.
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To define the basic OS structure for the 2C7 epitope present within
15253 LOS, we modified 15253 and WG LOS enzymatically and chemically
and then analyzed these modified LOS for binding to mAb 2C7. The
results are shown in Figs. 9 and
10 and summarized in Table
IV. PAGE blot analysis showed that mAb
2C7 bound to the major WG LOS component after the removal of its
-Gal or Gal1-4GlcNAc from the non-reducing end (Table IV), which
confirmed that mAb 2C7 bound to the inner 15253 LOS structure within WG
LOS. However, mAb 2C7 did not bind to the -galactosidase-treated
15253 LOS (marked with an arrow in Fig. 9A, lane
2). The apparent strong binding to the small amount of the intact
LOS in lane 2 is due to the fact that a higher concentration
of mAb 2C7 was used to distinguish the positive binding from the lack
of binding. Although we were not be able to determine which Gal residue
was removed from the two lactosyl moieties present in the LOS, this
lack of 2C7 binding to the -galactosidase-treated 15253 LOS is clear evidence that a -Gal residue at the non-reducing end is
indispensable for the epitope expression. The importance of the -Gal
residue was also supported by the fact that mAb did not bind 15253 lgtE mutant LOS (lane 3) that lacks a Gal residue on each lactose
attached to Hep[1] and Hep[2] of 15253 LOS (21). These binding
studies showed that the -Gal on the non-reducing end of 15253 LOS is essential for the 2C7 epitope to be expressed. As described above, mAb
2C7 did not bind to JW31R LOS until the lactose on Hep[2] (Fig. 8)
was exposed after the enzymatic treatment. Based upon these results we
conclude that mAb 2C7 requires 15253 OS structure as an essential size
for its epitope expression.
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In addition to the basic 2C7 OS structure, we defined other structural requirements necessary for expression of this carbohydrate epitope by immunochemical analysis of chemically modified LOS samples. We modified both 15253 and WG LOS and obtained almost identical results with those LOS. Fig. 10 shows the results obtained with PAGE blot and TLC immunostain of the modified 15253 LOS samples. mAb 2C7 bound the dephosphorylated LOS obtained with usual treatment with aqueous HF (10). In addition, the mAb bound to the partially deacylated LOS obtained after the NaOH or hydrazine treatment. As described earlier, ESI-MS analysis showed that hydrazine treatment cleaved O-linked fatty acids, and NaOH treatment leads to partial de-N- and O-acylation as studied previously (24). As Fig. 10 shows, the hydrazine treatment of 15253 LOS (lane 4) resulted in heavier aggregation than the NaOH treatment (lane 3), and mAb 2C7 bound to those aggregated LOS as well. This aggregation is probably due to hydrophobic interactions of the N-linked fatty acids remained in the lipoidal moiety.
Although the partially deacylated LOS samples described above retained the antigenicity, de-N-acylation (4 M KOH for 4 h at 120 °C) (25) of the de-O-acylated LOS resulted in the loss of epitope expression (Fig. 10B, lane 5). Since the completely de-acylated LOS does not adhere to nitrocellulose membrane or ELISA wells, it was analyzed directly in solid phase only by TLC immunoassay (10). The lack of mAb 2C7 binding showed that the presence of N-linked fatty acids is necessary for the maximum expression of the 2C7 epitope and that the N-acetyl group of GlcNAc on Hep[2] may be necessary.
ELISA inhibition studies confirmed the above results obtained by the
PAGE blot and TLC immunoblot. We analyzed residual mAb 2C7 binding to
15253 LOS in the presence of the chemically modified 15253 LOS samples
(25-800 ng) (Fig. 11). The
dephosphorylated LOS inhibited the binding as well as the intact LOS.
Partially de-acylated 15253 LOS derivatives were slightly less potent
inhibitors than the intact LOS. As expected from the TLC blot results
(Fig. 10B, lane 5), the completely deacylated LOS did not
inhibit mAb 2C7 binding (Fig. 11). However, the 15253 OS that lacks the
lipid A was found to be a potent inhibitor compared with the completely deacylated LOS. Since -N-acetylglucosaminidase is not
commercially available, we were not be able to digest 15253 LOS with
the enzyme to obtain direct evidence for the importance of the GlcNAc.
However, the above results obtained by TLC immunoassays and ELISA
inhibition studies suggested that a GlcNAc moiety on Hep[2] could be
also important for the recognition by mAb 2C7.
|
Taken together, the above immunochemical analyses of chemically modified LOS samples showed 1) phosphate group(s) are not involved in the 2C7 epitope, 2) the O-linked fatty acids are not essential for the 2C7 epitope to be expressed, and 3) in addition to N-linked fatty acids, the GlcNAc moiety linked to Hep[2] could be important for the maximum expression of the 2C7 epitope.
In a previous work, mAb 2C7 was indicated to bind to both 24-1 LOS and its tyraminated OS (18). Very recent mass spectral analysis (20) showed that 24-1 OS elongates only from the Hep[1] of the diheptose. However, 15253 LOS recognized by mAb 2C7 has an OS structure elongating from both Hep[1] and Hep[2]. Therefore, the specific carbohydrate elongation pattern recognized by mAb 2C7 was not clear. In the present study, mAb 2C7 binding to 24-1 LOS was found to be due to the presence of a trace amount of an as yet unidentified LOS component having the same PAGE mobility as 15253 LOS. Also, we found that mAb 2C7 bound to none of the previously characterized gonococcal LOS having carbohydrate elongation only on Hep[1] (Table III). Furthermore, mAb 2C7 did not bind to the major JW31R LOS component (Fig. 8) until the extra disaccharide linked to the lactose on Hep[2] was removed after the sequential enzymatic treatment (Fig. 8). From these results, we conclude that 1) mAb 2C7 binds to 15253 LOS and the LOS whose OS is extended from the lactose on Hep[1] of 15253 LOS and 2) elongation of the lactose on Hep[2] of the 15253 LOS blocks expression of the 2C7 epitope.
Our structural and immunochemical analysis also provided evidence that
15253 LOS contains a basic OS structure necessary for expression of the
2C7 epitope. mAb 2C7 bound to the major WG LOS component after the
removal of a galactose or lactosamine (data not shown but the results
are listed in Table IV). However, it did not bind to 15253 LOS when a
-galactosyl residue was removed from either lactose present at the
non-reducing ends (Fig. 9) (Table IV). The necessity of at least one
(and perhaps both) of the Gal residues for expression of the 2C7
epitope was supported by the fact that mAb 2C7 bound to the 15253 lgtE
mutant LOS that lacks the Gal moiety of each lactose on Hep[1] and
Hep[2] (Table IV). These results do not support the previous
suggestion that the lactose on Hep[2] is the 2C7 epitope (21).
In addition to the importance of either Gal moiety of the lactoses, the
results obtained from TLC immunoassay and ELISA studies (Figs. 9 and
10) suggested that the GlcNAc on Hep[2] could be important for
binding. Direct proof for the importance of the GlcNAc residue could be
obtained by producing a 15253 LOS mutant which lacks the -HexNAc
residue on Hep[2]. Based upon the results described above, we
conclude that mAb 2C7 requires the 15253 LOS structure as a basic
structure for its epitope expression.
Besides the 15253 OS structure, some fatty acids present in the lipoidal moiety are important for expression of the 2C7 epitope. Removal of O-linked fatty acids in the lipoidal moiety of 15253 LOS did not affect expression of the 2C7 epitope (Figs. 9 and 10). However, de-N-acylation of the de-O-acylated 15253 LOS resulted in the loss of epitope expression (Figs. 9 and 10). This showed that the N-linked fatty acids in the lipoidal moiety are important for expression of the 2C7 epitope. The 15253 OS structure is defined by the presence of two lactoses linked to the trisaccharide, GlcNAc-Hep[2]-Hep[1], with N-linked fatty acids present in the lipoidal moiety acting as an anchor to hold the carbohydrate epitope rigid. Removal of the N-linked fatty acids would lead to the conformational changes of the 15253 OS that defines the 2C7 epitope and, as a result, the loss of full expression of the 2C7 epitope.
Our current structural study of WG LOS also provided evidence that
further carbohydrate elongation of the 15253 OS occurs (Fig.
12), which confirmed a previous
suggestion (13). Recent studies have identified genes encoding a set of
glycosyltransferases for the biosynthesis of the specific OS structures
(21, 32, 33). Gonococci add a glycose moiety sequentially on the
lactose of Hep[1] and synthesize a lactoneotetraose and its
N-acetylgalactosaminylated form. Our finding of the tetraose
on the LOS containing a lactose on Hep[2] evokes the question of how
the OS structure represented by WG LOS is synthesized. Gonococci may
elongate either the lactose on Hep[1] after the completion of the
15253 OS structure or Hep[2] after the lactoneotetraose on Hep[1]
is complete. Further molecular biological studies on the regulation of
glycosyl transferases will be necessary to answer this question.
|
The current study also provided an additional insight on
expression of epitopes defined by mAbs 1-1-M and 3F11. The two
mAbs 3F11 and 1-1-M, respectively, have been known to bind to the
Gal1-4GlcNAc and GalNAc1-3Gal residue of the LOS that lacks
elongation on the Hep[2] (10, 11). However, binding of the two mAbs
to WG LOS (Fig. 6) showed that their epitopes are also expressed on LOS
having a lactose attached to Hep[2]. These results indicate that OS
elongation patterns cannot be determined based solely on the binding of
mAbs 1-1-M and 3F11.
In summary, we determined that the major WG LOS has the following
structure: a Gal1-4GlcNAc1-3Gal1-4Glc (lactoneotetraose) and a Gal1-4Glc (lactose), respectively, are linked to Hep[1] and
Hep[2] of the GlcNAc-Hep[2]-Hep[1] trisaccharide core. mAb 2C7
binds to the inner 15253 LOS structure found within WG LOS. In
addition, we found that JW31R LOS is also an elongated form of 15253 LOS containing extra disaccharide linked to the lactose on Hep[2] and
that expression of the 2C7 epitope within JW31R LOS is blocked due to
the substitution of the Hep[2]-lactose. Based on the structural data
of these LOS and the results obtained from immunochemical analyses, we
conclude the following: 1) mAb 2C7 requires both the 15253 OS as a
minimum structure and the N-linked fatty acids in the
lipoidal moiety for full expression of the epitope; 2) mAb 2C7 binds to
LOS whose OS elongate from the lactose on Hep[1] of the 15253 OS but
not on Hep[2]; and 3) the 2C7 epitope is expressed on gonococcal LOS
despite the presence of human carbohydrate epitopes such as lactosamine
or its N-acetylgalactosaminylated (globo) form. Because most
gonococci display the 2C7 epitope in their natural state (in
vivo (18)), the epitope defined by mAb 2C7 could potentially be
used as a safe site for the development of a vaccine candidate.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Uichirou Yabe for reproducing PAGE blot data and Satoko Nishi for technical assistance in PAGE blot analysis of JW31R LOS and its enzyme-treated samples.
| |
FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grants AI01061 and AI32725 and Grant-in-aid for Scientific Research of the Ministry of Education of Japan 10306022.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: Dept. of Biochemistry & Biotechnology, Tottori University, Koyama-Minami-4-101, Tottori City, Tottori-Ken 680-8553, Japan. Tel.: 81-857-31-6751, Fax: 81-857-31-5347; E-mail: yamasaki@muses.tottori-u.ac.jp.
2 To distinguish the two heptoses in the core OS region, we defined the Hep linked to KDO as Hep[1] and the other Hep linked to Hep[1] as Hep[2].
| |
ABBREVIATIONS |
|---|
The abbreviations used are: LOS, lipo-oligosaccharide(s); COSY, chemical shift correlation spectroscopy; DGI, disseminated gonococcal infection; DQF, double quantum-filtered; ELISA, enzyme-linked immunosorbent assay; ESI-MS, electrospray ion mass spectrometry; FAB-MS, fast atomic bombardment-mass spectrometry; Hep, heptose; Hex, hexose; HexNAc, N-acetylhexosamine; HF, hydrofluoric acid; HOHAHA, homonuclear Hartman-Hahn spectroscopy; KDO, 2-keto-3-deoxy-manno-octulosonic acid; mAb, monoclonal antibody; MS, mass spectrometry; NOE, nuclear Overhauser effect; OS, oligosaccharide(s); PEA, phosphoethanolamine; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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m = 120 ms) of the major WG OS (600 MHz at
25 °C). Hep(I) (5.45 ppm); Glc(II) (5.31 ppm); GlcNAc(III)
(5.09 ppm); Hep(IV) (5.07 ppm); GlcNAc(V) (4.72 ppm); Glc(VI) (4.56 ppm); Gal(VII) (4.48 ppm); Gal(VIII) (4.43 ppm); and Gal (IX)(4.42
ppm). For the purpose of obtaining better resolution, the relay peaks
in A-C were plotted along the 
, completely deacylated LOS;
, 15253 OS; 
, partially de-N- and
O-acylated LOS; 
, de-O-acylated LOS; 
,
dephosphorylated LOS; 
, intact LOS.
