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The β-configuration of the rhamnosidic linkage in salmonella serogroups C2 and C3, lipopolysaccharide is important for the immunochemistry of the o-antigen 8
Molecular Immunology, Vol. 30, No. 10, Printed in Great Britain.
0161-5890/93
pp. 887-893, 1993
%6.00 + 0.00
0 1993 Pergamon Press Ltd
THE /l-CONFIGURATION OF THE RHAMNOSIDIC LINKAGE IN SALMONELLA SEROGROUPS C, AND C, LIPOPOLYSACCHARIDE IS IMPORTANT FOR THE IMMUNOCHEMISTRY OF THE O-ANTIGEN 8 ANATOLY YA. CHERNYAK,* ANDREJ WEIN’mAuqt
and
NIKOLAY K. KOCHETKOV*
ALF A. LINDBERGt$
*N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 117913 Moscow, Russia and tKarolinska Institute, Department of Clinical Bacteriology, Huddinge Hospital, S-141 86 Huddinge, Sweden
(First received 23 December 1992; accepted in revised form 5 March 1993) Abstract-Pairs of synthetic di- and trisaccharide-polyacrylamide (PAA) conjugates, isomers in configuration of the rhamnose residue and related to the sequence abequosyl-(a 1-3)-rhamnosyl-(/l l2)-mannose (ARM), found in Salmonella serogroup C2 (O-antigens 6,8) and C, (O-antigens 8,20) lipopolysaccharides, have been used as coating antigens and inhibitors in enzyme immunoassay to evaluate the immunochemical importance of the b-rhamnosidic linkage in the O-antigen 8. In each pair, the reaction with the factor 0 : 6,8 serum was more pronounced for the synthetic antigen with the p-rhamnosidic linkage. The ARM-PAA conjugate with the /?-rhamnosidic linkage was found to be 2000 fold more efficient as inhibitor of binding of the factor 0: 6,8 serum to the AR/?M-PAA conjugate as compared to the a-linked analogue. The discrepancy in immunochemical behaviour of the a and /?-rhamnose containing ARM oligosaccharides can be explained by conformational differences of the oligosaccharides. A slight cross-reactivity observed in the interaction of antiserum against abequosyl-(a I-3)-mannose, representative of Salmonella O-antigen 4, coupled to BSA, with Salmonella O-factor 8 specific abequosyl-(a I-3)-rhamnose containing conjugates is due to the common terminal immunodominant sugar, abequose.
berg, 1979; Lindberg and Le Minor, 1984). In structural studies of the Salmonella serogroup C2 and C3 LPS, the
INTRODUCTION of Salmonella bacteria into different serogroups is based on the antigenic properties of the polysaccharide chain in the lipopolysaccharide (LPS) of
The classification
the cell envelope. The polysaccharide chains consist of polymerized oligosaccharide repeating units made up of 3 to 6 mbnosaccharide residues (Lindberg and Le Minor, 1984). The structures of the repeating units of the 0-antigenic polysaccharide chains of the LPSs in Salmonella serogroups C2 and C3 are shown in Fig. 1 (Hellerqvist et al., 1970, 1971, 1972). The immunodominant epitope 0: 8 in Salmonella
serogroups CZ and CJ is most likely the disaccharide abequosyl-(a 1 + 3)-rhamnose (Svenungson and Lind-
IAuthor to whom correspondence should be addressed. Abbreviations: EIA, enzyme immunoassay; LPS, lipopolysaccharide; PAA, polyacrylamide; BSA, bovine serum albumin; C,, -CH2-CH,-; C6, -CH,-(CH,),-CH2-; ARa Abe(a 1+ 3) Rha(a 1+; C,-PAA, ARB -C,-PAA, Abe(a 1 + 3)RhaAbe(a 1 + 3)Rha@ 1 +; ARa -C,-PAA, (a l+; AMa-C,-PAA, Abe(a 1-+3)Man(a l+; PMa-C,PAA, Par(a 1+3)Man(a 1+; ARa M-C,-PAA, Abe(a 1 -+ 3)Rha(a 1+2)Man(a l--+; ARB M-C,-PAA, Abe(a 1 + 3)Rha(fi 1+2)Man(a 1 -+; RB M-C,-PAA, Rha(B 1+ Z)Man(a l+; Abe(a 1+3)Rha(a 1-+OCsH,ARa-BSA, NHCSNH-BSA; AMa-BSA, Abe(a 1+3)Man(a l-+ OC,H,NHCSNH-BSA; Abe, abequose; Rha, rhamnose; Par, paratose; Man, mannose.
L-rhamnose residue was considered to be in a configuration based on optical rotation versus time on mild acid hydrolysis (Hellerqvist et al., 1970, 1971). Further studies revised the structure and the L-rhamnosidic linkage was proven to be in the /?-configuration using oxidation of the fully acetylated LPS with chromium trioxide in acetic acid (Hellerqvist et al., 1972). Recently, ‘H and I3C-NMR studies of oligosaccharide fragments isolated from the O-specific polysaccharide of S. kentucky strain 98/39 by Smith degradation, confirmed that the configuration of the L-rhamnose residue is /3 (Torgov et al., 1989). The disaccharide abequosyl-(a 1+3)-rhamnose, representative of the 0:8 epitope was synthesized as the u-glycoside (Garegg and Hultberg, 1979) and coupled to bovine serum albumin (BSA) (Svenungsson and Lindberg, 1979). The rabbit antiserum against this synthetic disaccharide-protein conjugate was successfully used for correct identification of Salmonella serogroup C2 (0 : 6,8) and C, (0: 8,20) bacteria by indirect immunofluorescence and by co-agglutination using sensitized protein A-containing staphylococci (Svenungsson and Lindberg, 1979). Some questions still remain: (i) is the configuration of the rhamnosidic linkage of importance for the immunochemistry of the O-antigen 8, and (ii) are antibodies specific for the O-antigen 8 able to recognize configuration changes of the rhamnosidic linkage.
887
A. YA. CHERNYAKet al.
888
These questions prompted us to synthesize the disaccharide determinant 0: 8 in the form of both c( and /?-glycosides [Abe(cr 1-+3)Rha(a 1 --, and Abe(cr 1 + 3)Rha(P I-] (Chernyak et al., 1989a) as well as longer oligosaccharide sequences comprising the L-rhamnose residue 1x-1,2 or p-1,2-linked to the a-D-mannose [Abe(a 1-+3)Rha(a 1-+2)Man(a l+ and Abe(cr 1 -+ 3)Rha(/? 1-*2)Man(cc l-1 (Chernyak et al., 1989b). The oligosaccharides were obtained in the form of 2-(acrylamido)ethyl glycosides and converted by radical co-polymerization with acrylamide to high-molecularweight neoglycoconjugates (Kochetkov et al., 1982~. 6, c). The neoglycoconjugates were shown to be serologically active and we report now their use in immunochemical studies of Salmonella O-antigen 8 by enzyme immunoassay (EIA). MATERIALS
Synthetic
AND METHODS
neoglycoconjugates
The syntheses of the hapten disaccharide abequosyl(a l-+3)-rhamnose (AR) as c1- and /?-glycosides have been described previously (Chernyak et al., 1989~). The key stage was condensation of 3,6-dideoxy-2,4-di-O-(pnitrobenzoyl)-a-D-xylo-hexopyranosyl bromide (Eklind et al., 1976) with 2-(trifluoroacetamido)ethyl 2,4-di0-benzoyl-a-L-rhamnopyranoside or 2-(trifluoroacetamido)ethyl 2,4-di-O-benzoyl-/?-L-rhamnopyranoside, promoted by mercury(I1) salts in respectively, dichloromethane. Condensation of the same abequosyl bromide with 6-(trifluoroacetamido)hexyl 2-O-acetyl40 -benzoyl-cr -r,-rhamnopyranoside under conditions described above, gave the corresponding or-linked disaccharide with a longer (C,) aglycon-spacer. The o-(trifluoroacetamido)alkyl aglycon was easily converted (by treatment with aqueous ammonia followed by N-acryloylation) to the UI-(acrylamido)alkyl group suitable for further transformation of haptens to neoglycocopolymerization with acrylamide conjugates via (Kochetkov et al., 1982a, 6, c). The synthesis of the hapten disaccharides abequosyl-(cr I-3)-mannose (AM) and paratosyl-(a I-3)-mannose as 2-(acryl(PM) amido)ethyl a-glycosides and immunochemical properties of the corresponding polyacrylamide conjugates have been described previously (Chernyak et al., 1988, 1992). Synthesis of the isomeric trisaccharides abequosyl(a l--+3)-rhamnosyl-(cr or /3 l-+2)-mannose, has been described previously (Chernyak et al., 19896). Condensation of 2-(benzyloxycarbonylamino)ethyl 3,4,6-tri-O-benzyl-a-D-mannopyranoside with 3-0acetyl-2,4-di-0 -benzyl-cr -L-rhamnopyranosyl bromide (Chernyak et al., 1989~) under condition of heterogeneous catalysis (silver carbonate and molecular sieve 4A in toluene) give a mixture of protected anomeric disaccharides. After de-0-acetylation, they were separately glycosylated with abequosyl bromide as described above. Removal of protective groups (de-0-acylation and catalytic hydrogenolysis) followed by N-acryloylation resulted in conversion of the 2-(benzyloxycarbonylamino)ethyl group into the 2-(acrylamido)ethyl aglycon
spacer. Hydrogenolysis (over PdjC in acetic acid) was accompanied by partial splitting of the abequosylrhamnosidic linkage to give the 2-(acrylamido)ethyl glycoside of rhamnosyl-(P 1+2)-mannose. All haptens (obtained as glycosides) were separately copolymerized with acrylamide in water with the radicalprocess promoters, ammonium persulfate and N,N,N’,N’,-tetramethyl-ethylenediamine (TEMED) (Horejsi et al., 1978). The high-molecular-weight copolymerit neoglycoconjugates were isolated by elution from a column of Sephadex G-50 with pyridine-acetic acid buffer (pH 5.5). The integral intensities of corresponding signals in “C-NMR spectra of the neoglycoconjugates showed that on an average every 8th acrylamide residue in the polymeric chain was substituted by a carbohydrate hapten. Judging from ultrafiltration data (Amicon Diaflo XM 100 and XM 300 membranes), the molecular masses of the copolymers were assigned in the range of 100 kD. The neoglycoconjugates described above will be referred to as Abe(cr 1-+3)Rha(P 1 + = AR/?-C,-PAA; Abe(cc l-+3)ARu -C,-PAA; Rha(a 1+ = ARa-QPAA Par(a 1+3)Abe(cr l-+3)-Man(a 1 + = AMu-(?~~PAA: Abe(cr 1-+3)Rha(a 1 -+ Mama 1 -+ = PMcr -C,-PAA; 2)Man(a l+ = ARclM-C,-PAA; Abe-(@ 1-+3)Rha(B 1 -+ Rha-(b 1+ 2)Man(cc l+ = ARPM-C,-PAA and 2)Man(cr 1 -+ = RPM-C,-PAArespectively. The oligosaccharides were linked to polyacrylamide either via a -CH,CH,-spacer as indicated by CZ or via a -CH,(CH,),CH,-spacer as indicated by C,. The disaccharide ARcl as p-nitrophenyl glycoside was synthesized by Garegg and Hultberg (1979) the hapten obtained was coupled to BSA by the isothiocyanate method (Svenungsson and Lindberg, 1979). The synthetic disaccharide abequosyl-(cr l-3)-mannose (AM, as p-nitrophenyl glycoside) (Eklund et al., 1976), representative of the O-antigen 4 determinant in Salmonella serogroup B, was similarly converted into the neoglycoprotein AMa-BSA (Svenungsson and Lindberg, 1977). Bacterial
lipopolysaccharides
Salmonella typhimurium strain SH4809 (serogroup B, O-antigens 4,5,12), S. newport IS50 (serogroup CZ, 0: 6,8) and S. kentucky IS98 (serogroup C,, 0: 8,20) LPSs were extracted from submerged cultures with hot phenol-water (Westphal et al., 1952). The S. kentucky LPS-OH was prepared by mild alkaline hydrolysis (0.2 M NaOH, lOO”C, 1 hr). In the resulting LPS, the 0-acetyl group linked to the terminal glucose residue in the repeating unit was removed. In addition, all esterlinked fatty acids in the lipid A portion of the LPS molecule were hydrolyzed off. The S. typhimurium SH4809 LPS-OH was prepared as above. This preparation lack the O-antigen 5 which is defined as the 0-acetyl group linked to the carbon atom 2 in the terminal abequosyl residue. Enzyme
immunoassay
(EZA)
The enzyme immunoassay as described by Karlsson
was performed essentially et al. (1986). The coating
Immunochemistry
of SuZmonelZaO-antigen
dose for all polyacrylamide neoglycoconjugates was 0.01 pgg/ml. The BSA-conjugates were used at 5 pg/ml and LPSs at lOpg/ml. Anti-Salmonella 0 antigen factor sera 0:4; 0: 8 and 0 : 6,8 were prepared as described by Kauffmann (1966) and obtained from the National Bacteriology Laboratory, Solna, Sweden. The preparation and characterization of rabbit anti-AMcc-BSA and of anti-ARcc-BSA were described by Svenungsson and Lindberg (1977, 1979). The antibodies were used at ten-fold dilutions ranging from 10m3 to 10e6. Alkaline phosphatase conjugated goat anti-rabbit (whole molecule) IgG (Sigma Chemical Company, St Louis, MO, U.S.A.) was used at the dilution recommended by the manufacturer. In inhibition experiments, the ARPM-C,-PAA was used as a coating antigen, with either rabbit factor 0: 6,8 antiserum or with rabbit anti-ARcc-BSA serum. ARPMC,-PAA and ARuM-C,-FAA were used as inhibitors. Serum was preincubated for 1 hr at room temp with different concns of ARflM-C,-PAA or ARctM-C,-PAA (0.001 pg-1OOpg). The 50% inhibitory value is the concn of inhibitor needed to obtain a 50% lowering of the optical density at 405 nm as compared with control wells with no inhibitor added. In another experiment, the interaction between anti AMa-BSA rabbit antibodies and AMu-PAA glycoconjugate was inhibited with different LPSs and glycoconjugates. The inhibitors: AMu -C,-PAA; ARct-C,-PAA; ARc( -C,-PAA; ARcr M-C,-PAA; AR/3 M-C,-PAA; S. ty phimurium SH4809 LPS; S. typhimurium SH4809 LPSOH; S. newport LPS; S. kentucky LPS and PAA were preincubated at different concn (10 pg-100 pg/ml) with rabbit anti-AMcl -BSA antibodies at room temp for 1 hr. The 50% inhibitory value is the concn of inhibitor needed to obtain a 50% lowering of the optical density at 405 nm as compared with control wells with no inhibitor added.
Conformational
889
8 analyses
Conformations ated using the parameters.
of oligosaccharides have been generCHARMM programme with default
RESULTS Enzyme
immunoassay
(EZA)
The specificity and activity of conventional Salmonella factor 0: 8, 0: 6,8 and 0: 4 rabbit sera and the rabbit anti-ARcr-BSA and anti-AMcr-BSA sera was tested EIA using synthetic oligosaccharidein the polyacrylamide and disaccharide-protein conjugates, LPS from S. newport (0 : 6,8), S. kentucky (0 : 8,20) and S. typhimurium (0: 4,5,12) and BSA as coating antigens (Table 1). The rabbit polyclonal factor 0:8 serum reacted efficiently with LPSs and LPS-OH from Salmonella serogroups C2 and C, (S. newport and S. kentucky) but did not bind to the synthetic conjugates representative of the O-antigen 8. With the rabbit factor 0: 6,8 serum a pronounced binding to LPSs from serogroups C, and C, was seen and a weak reaction with 0 : 8 epitope-containing conjugates. For each isomeric oligosaccharide pair (disaccharides or trisaccharides), the absorbance value of the isomer with a fl-rhamnosidic linkage (AR/?-C,PAA or ARPM-C,-PAA, respectively) was higher than that of the corresponding Lx-isomer (Table 1). This was a first indication of the importance of the /I-configuration of the rhamnosidic linkage for interaction with 0 : 8 antibodies. The rabbit anti-ARM-BSA serum reacted with both LPS from S. newport and S. kentucky as well as with the synthetic 0 : 8 antigens with comparable absorbance values. Apparently, the anti-ARcr-BSA antibodies did not discriminate between the a- and /3-rhamnosidic linkages in these synthetic conjugates.
Table 1. Absorbance values at 405 nm after 100 min in EIA for different Salmonella specific antibodies against the polyacrylamide neoglycoconjugates; BSA-conjugates and lipopolysaccharides Antisera” Antigen ARB-C,-PAA ARa -C,-PAA ARu -C,-PAA ARfl M-C,-PAA ARci M-C,-PAA RP M-C,-PAA AMci -C,-PAA PMci -C,-PAA AMci-BSA ARa-BSA BSA S. newport LPS S. kentucky IS98 LPS S. kentucky IS98 LPS-OH S. typhimurium SH4809 LPS
Factor 0: 8
Factor 0: 6,8
0.11 0.11 0.13 0.25
0.31 0.26 0.34 0.90 0.20 0.11
Anti ARa-BSA
“All antisera were used at 10m3 dilution except the anti-ARu-BSA ‘nd-not done.
1.79 1.88 1.77 1.90 2.00 2.0 1.88 1.17 1.19 0.11 (10m4).
Factor 0: 4
Anti AMu-BSA 0.51 0.48 1.28 0.67 0.43 0.13 3.55 0.45 nd nd > 2.0 0.43 0.13 0.15 3.05
890
A. YA. CHERNYAK et al. CONH2
Antibodies raised against AMa-BSA bound to the AR-disaccharide moiety of the synthetic glycoconjugates (Table 1). The absorbance observed against the ARa-C2PAA conjugate was, however, only 14% of that observed against the homologous AMa-C,-PAA conjugate. Inhibition
1
I-~CHzCH),-CH2CH-(CHn2CH),-]n
CONHR
R =
of EIA
In order to define immunochemically important structural elements of the 0: 8 determinant, EIA-inhibition experiments were performed with LPSs from S. newport, S. ken&&y, and the S. kentucky LPS-OH as coating antigens. As homologous antisera, conventional factor 0: 8 and 0: 6,8 sera at an appropriate dilution (l/4000 to l/40,000) were used. Inhibition assays were done with synthetic conjugates in concns from 0.001 to 100 pg/ml and LPS in concns from 0.1 to 50 pg/ml. None of the synthetic neoglycoconjugates used inhibited the binding of antibodies to LPS but LPS themselves were efficient inhibitors (data not shown). In another experiment, the ARflM-C,-PAA conjugate was used as coating antigen with polyclonal 0: 6,8 antibodies. The homologous neoglycoconjugate, i.e. AR/3M-C,-PAA was an efficient inhibitor; 50% inhibition at 0.05 ,ug/ml. In contrast, the isomeric neoglycoconjugate, i.e. ARaM-C,-PAA showed a very poor inhibitory activity with a 50% inhibition value of > 100 pgg/ml (20% inhibition at 100 pgg/ml-the highest dose used) (Fig. 3A). In a corresponding experiment using rabbit antibodies elicited against ARa-BSA, no difference in inhibitory activity between the AR/?M-C,PAA (50% inhibition at 0.008 pg/ml) and the ARaMC,-PAA (50% inhibition at 0.015 pg/ml) was observed (Fig. 3B). Inhibition experiment using anti AMa-BSA antibodies and the AMa -C,-PAA conjugate showed that the AMa-C,-PAA conjugate was the best inhibitor (50%
CONE42
/
Abc(ul~3)Rha(al_tOCH2CHz-
(ARa-C2-PAA)
Abe(al+3)Rha(~lbOCHzCHz-
(ARP-CZ-PAA)
A~~(~I~~)R~~(~I~OCH~(CHZ)~CH~
(ARa-C6-PAA)
Abe(al~3)Rha(al~Z)Man(al~OCHzCH2-
(ARaM-CZ-PAA)
Abe(ul~3)Rha(~l~2)Man(al--1OCH~CH2-
(ARPM-CZ-PAA)
Rha(Pl~Z)Man(aIjOCH2CH2-
(RPM-CZ-PAA)
Fig. 2. Structure of synthetic oligosaccharide-polyacrylamide conjugates related to Salmonella O-antigen determinant 8. inhibition at 0.0008 pug/ml). The LPS isolated from S. typhimurium SH4809 as well as its de-0-acetylated derivative also inhibited the reaction between anti AMaBSA antibodies and the AMa-C,-PAA conjugate (50% inhibition at 3 pg/ml for both preparations). A lower activity (50% inhibition at 12 pgg/ml) was observed with the ARa-C,-PAA (Table 2). None of the other inhibitors GO
~4)~-Rhap(~l~Z)-~-Manp(al~2)-~-Manp(al-13)-~-~~(~1-, 3
3
7
7
INHIBITOR
CONCENTRATION
pglml
2AcGlcpal
Akpal
~)_LRhap(~l~Z)-~-M~(al~2)-~-Manp(al~3)-~-G~(~l~ 4
3
t
f 2AcGlcpal
Akpal
INHIBITOR CONCENTRATION
II Fig. 1. Structures of the O-antigen repeating units in the LPS from S. newport, serogroup C2 (I), and S. ken&&y, serogroup C, (II).
pglml
Fig. 3. EIA-inhibition. Antibodies preincubated with ARjJMC,-PAA (0) or ARaM-C,-PAA (0) before binding to AR/3M-C,-PAA. (a) Factor 0:6,8 rabbit serum. (b) AntiARa-BSA rabbit serum.
Immunochemistry
of Salmonella O-antigen
891
8
Table 2. Concentration of inhibitor @g/ml) needed to obtain 50% inhibition of binding of anti-AMa-BSA rabbit antibodies to AMu-Cl-PAA glycoconjugate Concentration needed for 50% inhibition @s/ml)
Inhibitor AMa -C,-PAA S. typhimurium SH4809 LPS S. typhimurium SH4809 LPS-OH” ARci -C,-PAA ARci -C,-PAA ARa M-C,-PAA ARP M-C,-PAA S. newport LPS S. kentucky LPS PAA
> > > > > >
“LPS-OH is de-0-acetylated LPS. bFigures in parentheses are the inhibition highest concn tested. reached 50% concn tested).
of inhibition
0.0008 3.0 2.9 12 100 (30%)b 100 (10%) 100 (10%) 100 (0%) 100 (0%) 100 (0%) value with the
at 100 pgg/ml (the
highest (b)
Conformational analyses Conformational analyses of the disaccharides Abe(cr 1+3)Rha and Abe(a 1+3)Man generated using the CHARMM-programme did not show any difference in the terminal, non-reducing abequosyl residue (Fig. 4). Conformational analysis of both trisaccharide sequences (ARaM and ARBM) are shown in Fig. 5. The result clearly shows a significant difference in the rhamnose-tomannose linkage region reflecting in &Jand I,$angles. The Abe-Rha linkage conformation is practically the same in both trisacharides (ARclM, 4 = - 65”, $ = -44”; AR/?M, 4 = - 60”, $ = -45”), whereas the conformation of the Rha(fll-+2)Man linkage (4 = - 71”, $ = 18”) is distinct from that of the Rha(a 1+2)Man linkage (4 = 58”, $ = 39”). DISCUSSION Synthetic Salmonella O-antigen determinants as haptens, either as polyacrylamide conjugates (Tendetnik et al., 1991) or as BSA-conjugates (Lindberg et al., 1983) have been shown previously to be useful in immunochemical studies. In this investigation polyacrylamide conjugates, prepared from synthetic oligosaccharides related to O-antigen 8, were used in evaluation of the importance of configuration of rhamnosidic linkage for immunochemistry of this antigen. In EIA, synthetic oligosaccharide-polyacrylamide conjugates (ARB-C,-PAA, ARa -C,-PAA, ARu-C,PAA, ARfl M-C,-PAA, ARcl M-C,-PAA) reacted weakly with the conventional factor 0 : 8 serum but binding was seen with the factor 0: 6,8 serum. The reason for this difference could be a lower concn of antibodies in the factor 0 : 8 serum prepared by absorption (Kauffmann, 1966). The reaction of the synthetic conjugates with the factor 0: 6,8 serum was weak. It should be noted, however, that the interaction of the serum with the con-
Fig.
4. Conformations
Abe(cz 1-+3)Man(u
of Abe(a1+3)Rha(a
1-+ (b) generated programme.
using
I-,
(a) and
the CHARMM-
jugates with a B-rhamnosidic linkage was stronger, than to conjugates with the corresponding u-isomer (Table 1). The importance of the /I -configuration of the rhamnosidic linkage for the immunochemical specificity of the O-antigen 8 was unambiguously demonstrated by the inhibition of the interaction of the factor 0: 6,8 serum with the ARfiM-C,-PAA-conjugate (Fig. 3A). Among the synthetic conjugates and LPS used as inhibitors, the only conjugate with inhibitory activity was ARBM-C2PAA. The isomeric conjugate with a-rhamnosidic linkage (ARaM-C,-PAA) was shown to be at least 2000-fold less efficient as an inhibitor (Fig. 3A). This finding confirmed the importance of the anomeric configuration of the subterminal sugar (Rha) in the antigen-antibody interaction. Conformational analysis of both trisaccharide sequences (ARaM and AR/?M) using the CHARMM-programme clearly show drastic changes in the rhamnose-to-mannose linkage region. Rabbit antibodies elicited by the ARa-BSA conjugate revealed in EIA approximately the same activity in the interaction with conjugates comprising the AR-fragment (Table 1). The polyclonal antibodies are probably not able to discriminate between the two configurations of the rhamnosidic linkage. The EIA results were confirmed in the EIA inhibition experiments. Both glycoconjugates, i.e. ARBM-C,-PAA and ARclM-C,-PAA were able to inhibit the anti-ARcr-BSA antibodies in an almost superimposable way (Fig. 3B). The reason for this could be that the combining site of the antibodies in this serum is complementary to a structure equal to or smaller than the disaccharide, or differs in the cc-rhamnose-to-BSA linkage region because of the aromatic character of the spacer.
892
A. YA. CHERNYAKet al.
lb)
Fig. 5. Conformations of Abe(a 1+3)Rha(a 1-+2)Man(a 1+ (a) and Abe(a 1+3)Rha(/? 1-+2)Man(a 1+ (b) generated using the CHARMM-programme.
The length of a spacer (C, or C,) did not affect the binding of the 0 : 8 antibodies to the synthetic conjugates (Table 1). The previous investigation of the specificity of the anti-ARcc-BSA serum in EIA (Svenungsson and Lindberg, 1979), revealed a very weak reaction to the serum with S. typhimurium LPS (0:4,5,12) comprising AMafragment representative of Salmonella O-antigen 4. In this investigation we used the neoglucoconjugates AMaC,-PAA and PMa-C,-PAA with the anti-ARcr-BSA serum. The reaction of the anti-ARcr-BSA antibodies with the heterologous neoglycoconjugates was very weak (Table 1). However, the cross-reactivity between AMcr- and ARa-moieties in the corresponding BSAconjugates was seen when rabbit factor 0 : 4 serum was used in EIA (Table 1). The intensity of the binding of the cross-reacting antigen ARu-BSA to antibodies was comparable with that of the homologous antigens AMcrBSA and AMor-C,-PAA. It should be noted that no reaction with this serum was seen with the heterologous AR-containing PAA-conjugates or with PM@-C,-PAA (Table 1). The antibodies raised against the synthetic disaccharide-protein conjugate (AMa-BSA) interacted with AMcr-C,-PAA as well as with the AR-C,-PAA conjugate. This finding was confirmed by inhibition EIA where AMc(+PAA inhibited the binding of anti AMa-BSA antibodies to the AMa-C,-PAA conjugate coated to the solid phase. The ARcr-C,-PAA also pos-
sessed inhibitory activity but an approximately 4000 fold higher concn was needed to obtain 50% inhibition (Table 2). Conformational analyses of the disaccharides Abe(cr 1+3)Rha and Abe(cr 1-+3)Man did not show any difference in the terminal, non-reducing abequosyl residue which probably has an immunodominant role. However, the difference between the mannosyl and rhamnosyl residues adopting approximately the same chair conformation is evident. Different arrangement of the functional groups in residues of the same mannoconfiguration is due to the 6-deoxy and L-character of rhamnose (Fig. 4). Although different, the rabbit antibodies against AMa-BSA are able to partially recognize the heterologous AR disaccharide in the ARa-C,-PAA where the sugar moiety is more accessible than in the ARa-C2-PAA conjugate. The results of inhibition EIA using a series of natural and chemically well defined synthetic antigens show that polyclonal 0: 8 specific antibodies are able to recognize the anomeric configuration of the subterminal residue (Rha) in the trisaccharide sequence Abe-Rha-Man. This coincides in a way with the classical investigation of Kabat (1966), about the relative contribution of different sugars in an antigenic oligosaccharide sequence to binding with an antibody. The ability of the anti-AMa-BSA antibodies to partially recognize the heterologous AR sequence can be explained by the presence of the same terminal immunodominant sugar, abequose. Acknowledgements-This
work was supported by a grant “Swedish-Soviet Scientific Cooperation” from the Royal Swedish Academy of Sciences and by grant 16X656 from the Swedish Medical Research Mrs K. Bergman and acknowledged. We thank Stenutz (Department of Stockholm, Sweden) for
Council. The technical assistance of Mrs Monica Jansson is gratefully Dr Per-Erik Jansson and Mr Ronald Organic Chemistry, University of the generation of Figs 4 and 5.
REFERENCES Chernyak A. Ya., Demidov I. V., Karmanova I. B., Chernyak N. V. and Kochetkov N. K. (1989a) Synthesis of 2-acrylamidoethyl 3-O-(3,6-dideoxy-a -o-xylo -hexopyranosyl)-aand -fl-L-rhamnopyranosides for preparation of polyacrylamide copolymers with the specificity of 0: 8 factor of SalmonelIa. Bioorg. Khim. 15, 111-122. Chernyak A. Ya., Demidov I. V. and Kochetkov N. K. (19896) Synthesis of the trisaccharide fragment from O-specific polysaccharides of Salmonella (serological groups CZ and
C,) and its conversion into the artificial antigen of copolymer type. Bioorg. Khim. 15, 1673-1685. Chernyak A. Ya., Levinsky A. B. and Kochetkov N. K. (1988) Synthesis of determinant disaccharides of the Salmonella O-antigens (serological groups A, B, and D,) in the form of glycosides suitable for conversion into artificial antigens via copolymerisation. Bioorg. Khim. 14, 104771058. Chernyak A. Ya., Levinsky A. B., Tendetnik Yu. Ya., Kochetkov N. K. and Pokrovsky V. I. (1992) Mono- and polyspecific glycoconjugates prepared from synthetic Salmonella 0-antigenic determinants and serological characterization thereof. Bioorg. Khim. 18, 680-688.
Immunochemistry
of Salmonella O-antigen 8
Eklind K., Garegg P. J. and Gotthammar B. (1976) Synthesis of p-isothiocyanatophenyl 3-O-(3,6-dideoxy-a -D-xylo -hexopyranosyl)-a-D-manno-pyranoside. Acta Chem. Stand. Section B. 30, 305-308. Garegg P. J. and Hultberg H. (1979) Synthesis ofp-nitrophenyl 3-0-(3,6-dideoxy-o:-D-xylO-hexopyranosyl)-a-L-rhamnopyranoside for making artificial antigens corresponding to the Salmonella O-factor 8. Carbohydr. Res. 72, 276-279. Hellerqvist C.-G., Hoffman J., Lindberg A. A., Lindberg B. and Svensson S. (1972) Sequence analysis of the polysaccharides from Salmonella newport and S. kentucky. Acta Chem. Stand. 26, 3282-3286.
Hellerqvist C.-G., Lindberg B., Svensson S., Holme T. and Lindberg A. A. (1970) Structural studies on the O-specific side-chains of the cell-wall lipopolysaccharides from Salmonella newport and S. kentucky. Carbohydr. Res. 14, 17-26.
Hellerqvist C.-G., Lindberg B., Svensson S., Lijnngren J. and Lindberg A. A. (1971) Fragmentation analysis of the Salmonella newport lipopolysaccharide. Acta Chem. Stand. 25, 601-606.
Horejsi V., Smolek P. and Kocourek J. (1978) Studies of lectins. XXXV. Water-soluble 0-glycosyl polyacrylamide derivatives for specific precipitation of lectins. Biochim. Biophys. Acta 538, 293-298.
Kabat E. A. (1966) The nature of antigenic determinant. J. Immun. 97, l-11. Karlsson K., Granstrbm M. and Lindberg A. A. (1986) Salmonella sp. Antibodies. In Methods of Enzymatic Analysis. Vol. XI. Antigens and Antibodies 2 (Edited by Bergmeyer H. U.), pp. 85-98. VCH, Verlagsgesellschaft, Weinheim, Germany. Kauffmann F. (1966) The Bacteriology of Enterobacteriaceae. Munksgaard, Copenhagen. Kochetkov N. K., Dmitriev B. A., Chernyak A. Ya. and Levinsky A. B. (1982a) A new type of carbohydratecontaining synthetic antigen: synthesis of carbohydratecontaining polyacrylamide copolymers with the specificity of 0: 3 and 0:4 factors of Salmonella. Carbohydr. Res. 110, cl6c20. Kochetkov N. K., Dmitriev B. A., Chernyak A. Ya., Pokrovsky V. I. and Tendetnik Yu. Ya. (19826) New type of carbohydrate-containing antigens. Synthesis and immunological properties of a carbohydrate-containing co-
893
polymer with the specificity of a factor 0: 3 of Salmonella bacteria of serological group E. Dokl. Akad. Nauk SSSR 263, 1277-1280.
Kochetkov N. K., Dmitriev B. A., Chemyak A. Ya., Pokrovsky V. I., Tendetnik Yu. Ya and Ovcharova N. M. (1982~) Copolymer of ally1 3-0-[4-O-(/l -D-mannopyranosyl)-u-L-rhamnopyranosyl]-B-D-galactopyranoside with acrylamide with serological specificity of O-factor 3 of Salmonella genus bacteria belonging to serological group E (USSR patent SU 879.970). Otkrytiya, Zzobret., Prom. Obraztsy, Tovarnye Znaki (26) 316; Chem. Abstr. 98,17777e
(1983). Lindberg A. A. and Le Minor L. (1984) Serology of Salmonella. In Methods in Microbiology vol. 15 (Edited by Bergan T.), pp. I-141. Academic Press, London and New York. Lindberg A. A., Wollin R., Bruse G., Ekwall E. and Svenson S. B. (1983) Immunology and immunochemistry of synthetic and semisynthetic Salmonella O-antigen-specific glycoconjugates. In ACS Symposium Series, No. 231. Bacterial Lipopolysaccharides. Structure, Synthesis, and Biological Activities (Edited by Anderson L. and Unger F. M.), pp.
83-187. American Chemical Society, Washington. Tendetnik Yu. Ya., Pokrovsky V. I., Chernyak A. Ya., Levinsky A. B. and Kochetkov N. K. (1991) The use of synthetic O-antigens of Salmonella for improvement of serological diagnosis of enteric infections. FEMS Microbial Immun. 76, 93-98.
Torgov V. I., Shibaev V. N., Shashkov A. S. and Rozhnova S. Sh. (1989) The structure of the 0-antigenic polysaccharide of gram-negative bacteria Salmonella kentucky strain 98/39 (0:8,H:i,Z6). Bioorg. Khim. 15, 1664-1672. Svenungsson B. and Lindberg A. A. (1977) Synthetic disaccharide-protein antigens for production of specific 04 and 09 antisera for immunofluorescence diagnosis of Salmonella. Med. Microbial. Immun. 163, l-l 1. Svenungsson B. and Lindberg A. A. (1979) Diagnosis of Salmonella bacteria: antibodies against synthetic Salmonella O-antigen 8 for immunofluorescence and co-agglutination using sensitized protein A-containing staphylococci. Acta Pathol. Microbial. Stand. Sect B. 87, 29-36. Westphal O., Liideritz 0. and Bister F. (1952) Uber die Extraction von Bacterien mit Phenol/Wasser. Z. Naturforsch. 7, 148-155.
0161-5890/93
pp. 887-893, 1993
%6.00 + 0.00
0 1993 Pergamon Press Ltd
THE /l-CONFIGURATION OF THE RHAMNOSIDIC LINKAGE IN SALMONELLA SEROGROUPS C, AND C, LIPOPOLYSACCHARIDE IS IMPORTANT FOR THE IMMUNOCHEMISTRY OF THE O-ANTIGEN 8 ANATOLY YA. CHERNYAK,* ANDREJ WEIN’mAuqt
and
NIKOLAY K. KOCHETKOV*
ALF A. LINDBERGt$
*N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 117913 Moscow, Russia and tKarolinska Institute, Department of Clinical Bacteriology, Huddinge Hospital, S-141 86 Huddinge, Sweden
(First received 23 December 1992; accepted in revised form 5 March 1993) Abstract-Pairs of synthetic di- and trisaccharide-polyacrylamide (PAA) conjugates, isomers in configuration of the rhamnose residue and related to the sequence abequosyl-(a 1-3)-rhamnosyl-(/l l2)-mannose (ARM), found in Salmonella serogroup C2 (O-antigens 6,8) and C, (O-antigens 8,20) lipopolysaccharides, have been used as coating antigens and inhibitors in enzyme immunoassay to evaluate the immunochemical importance of the b-rhamnosidic linkage in the O-antigen 8. In each pair, the reaction with the factor 0 : 6,8 serum was more pronounced for the synthetic antigen with the p-rhamnosidic linkage. The ARM-PAA conjugate with the /?-rhamnosidic linkage was found to be 2000 fold more efficient as inhibitor of binding of the factor 0: 6,8 serum to the AR/?M-PAA conjugate as compared to the a-linked analogue. The discrepancy in immunochemical behaviour of the a and /?-rhamnose containing ARM oligosaccharides can be explained by conformational differences of the oligosaccharides. A slight cross-reactivity observed in the interaction of antiserum against abequosyl-(a I-3)-mannose, representative of Salmonella O-antigen 4, coupled to BSA, with Salmonella O-factor 8 specific abequosyl-(a I-3)-rhamnose containing conjugates is due to the common terminal immunodominant sugar, abequose.
berg, 1979; Lindberg and Le Minor, 1984). In structural studies of the Salmonella serogroup C2 and C3 LPS, the
INTRODUCTION of Salmonella bacteria into different serogroups is based on the antigenic properties of the polysaccharide chain in the lipopolysaccharide (LPS) of
The classification
the cell envelope. The polysaccharide chains consist of polymerized oligosaccharide repeating units made up of 3 to 6 mbnosaccharide residues (Lindberg and Le Minor, 1984). The structures of the repeating units of the 0-antigenic polysaccharide chains of the LPSs in Salmonella serogroups C2 and C3 are shown in Fig. 1 (Hellerqvist et al., 1970, 1971, 1972). The immunodominant epitope 0: 8 in Salmonella
serogroups CZ and CJ is most likely the disaccharide abequosyl-(a 1 + 3)-rhamnose (Svenungson and Lind-
IAuthor to whom correspondence should be addressed. Abbreviations: EIA, enzyme immunoassay; LPS, lipopolysaccharide; PAA, polyacrylamide; BSA, bovine serum albumin; C,, -CH2-CH,-; C6, -CH,-(CH,),-CH2-; ARa Abe(a 1+ 3) Rha(a 1+; C,-PAA, ARB -C,-PAA, Abe(a 1 + 3)RhaAbe(a 1 + 3)Rha@ 1 +; ARa -C,-PAA, (a l+; AMa-C,-PAA, Abe(a 1-+3)Man(a l+; PMa-C,PAA, Par(a 1+3)Man(a 1+; ARa M-C,-PAA, Abe(a 1 -+ 3)Rha(a 1+2)Man(a l--+; ARB M-C,-PAA, Abe(a 1 + 3)Rha(fi 1+2)Man(a 1 -+; RB M-C,-PAA, Rha(B 1+ Z)Man(a l+; Abe(a 1+3)Rha(a 1-+OCsH,ARa-BSA, NHCSNH-BSA; AMa-BSA, Abe(a 1+3)Man(a l-+ OC,H,NHCSNH-BSA; Abe, abequose; Rha, rhamnose; Par, paratose; Man, mannose.
L-rhamnose residue was considered to be in a configuration based on optical rotation versus time on mild acid hydrolysis (Hellerqvist et al., 1970, 1971). Further studies revised the structure and the L-rhamnosidic linkage was proven to be in the /?-configuration using oxidation of the fully acetylated LPS with chromium trioxide in acetic acid (Hellerqvist et al., 1972). Recently, ‘H and I3C-NMR studies of oligosaccharide fragments isolated from the O-specific polysaccharide of S. kentucky strain 98/39 by Smith degradation, confirmed that the configuration of the L-rhamnose residue is /3 (Torgov et al., 1989). The disaccharide abequosyl-(a 1+3)-rhamnose, representative of the 0:8 epitope was synthesized as the u-glycoside (Garegg and Hultberg, 1979) and coupled to bovine serum albumin (BSA) (Svenungsson and Lindberg, 1979). The rabbit antiserum against this synthetic disaccharide-protein conjugate was successfully used for correct identification of Salmonella serogroup C2 (0 : 6,8) and C, (0: 8,20) bacteria by indirect immunofluorescence and by co-agglutination using sensitized protein A-containing staphylococci (Svenungsson and Lindberg, 1979). Some questions still remain: (i) is the configuration of the rhamnosidic linkage of importance for the immunochemistry of the O-antigen 8, and (ii) are antibodies specific for the O-antigen 8 able to recognize configuration changes of the rhamnosidic linkage.
887
A. YA. CHERNYAKet al.
888
These questions prompted us to synthesize the disaccharide determinant 0: 8 in the form of both c( and /?-glycosides [Abe(cr 1-+3)Rha(a 1 --, and Abe(cr 1 + 3)Rha(P I-] (Chernyak et al., 1989a) as well as longer oligosaccharide sequences comprising the L-rhamnose residue 1x-1,2 or p-1,2-linked to the a-D-mannose [Abe(a 1-+3)Rha(a 1-+2)Man(a l+ and Abe(cr 1 -+ 3)Rha(/? 1-*2)Man(cc l-1 (Chernyak et al., 1989b). The oligosaccharides were obtained in the form of 2-(acrylamido)ethyl glycosides and converted by radical co-polymerization with acrylamide to high-molecularweight neoglycoconjugates (Kochetkov et al., 1982~. 6, c). The neoglycoconjugates were shown to be serologically active and we report now their use in immunochemical studies of Salmonella O-antigen 8 by enzyme immunoassay (EIA). MATERIALS
Synthetic
AND METHODS
neoglycoconjugates
The syntheses of the hapten disaccharide abequosyl(a l-+3)-rhamnose (AR) as c1- and /?-glycosides have been described previously (Chernyak et al., 1989~). The key stage was condensation of 3,6-dideoxy-2,4-di-O-(pnitrobenzoyl)-a-D-xylo-hexopyranosyl bromide (Eklind et al., 1976) with 2-(trifluoroacetamido)ethyl 2,4-di0-benzoyl-a-L-rhamnopyranoside or 2-(trifluoroacetamido)ethyl 2,4-di-O-benzoyl-/?-L-rhamnopyranoside, promoted by mercury(I1) salts in respectively, dichloromethane. Condensation of the same abequosyl bromide with 6-(trifluoroacetamido)hexyl 2-O-acetyl40 -benzoyl-cr -r,-rhamnopyranoside under conditions described above, gave the corresponding or-linked disaccharide with a longer (C,) aglycon-spacer. The o-(trifluoroacetamido)alkyl aglycon was easily converted (by treatment with aqueous ammonia followed by N-acryloylation) to the UI-(acrylamido)alkyl group suitable for further transformation of haptens to neoglycocopolymerization with acrylamide conjugates via (Kochetkov et al., 1982a, 6, c). The synthesis of the hapten disaccharides abequosyl-(cr I-3)-mannose (AM) and paratosyl-(a I-3)-mannose as 2-(acryl(PM) amido)ethyl a-glycosides and immunochemical properties of the corresponding polyacrylamide conjugates have been described previously (Chernyak et al., 1988, 1992). Synthesis of the isomeric trisaccharides abequosyl(a l--+3)-rhamnosyl-(cr or /3 l-+2)-mannose, has been described previously (Chernyak et al., 19896). Condensation of 2-(benzyloxycarbonylamino)ethyl 3,4,6-tri-O-benzyl-a-D-mannopyranoside with 3-0acetyl-2,4-di-0 -benzyl-cr -L-rhamnopyranosyl bromide (Chernyak et al., 1989~) under condition of heterogeneous catalysis (silver carbonate and molecular sieve 4A in toluene) give a mixture of protected anomeric disaccharides. After de-0-acetylation, they were separately glycosylated with abequosyl bromide as described above. Removal of protective groups (de-0-acylation and catalytic hydrogenolysis) followed by N-acryloylation resulted in conversion of the 2-(benzyloxycarbonylamino)ethyl group into the 2-(acrylamido)ethyl aglycon
spacer. Hydrogenolysis (over PdjC in acetic acid) was accompanied by partial splitting of the abequosylrhamnosidic linkage to give the 2-(acrylamido)ethyl glycoside of rhamnosyl-(P 1+2)-mannose. All haptens (obtained as glycosides) were separately copolymerized with acrylamide in water with the radicalprocess promoters, ammonium persulfate and N,N,N’,N’,-tetramethyl-ethylenediamine (TEMED) (Horejsi et al., 1978). The high-molecular-weight copolymerit neoglycoconjugates were isolated by elution from a column of Sephadex G-50 with pyridine-acetic acid buffer (pH 5.5). The integral intensities of corresponding signals in “C-NMR spectra of the neoglycoconjugates showed that on an average every 8th acrylamide residue in the polymeric chain was substituted by a carbohydrate hapten. Judging from ultrafiltration data (Amicon Diaflo XM 100 and XM 300 membranes), the molecular masses of the copolymers were assigned in the range of 100 kD. The neoglycoconjugates described above will be referred to as Abe(cr 1-+3)Rha(P 1 + = AR/?-C,-PAA; Abe(cc l-+3)ARu -C,-PAA; Rha(a 1+ = ARa-QPAA Par(a 1+3)Abe(cr l-+3)-Man(a 1 + = AMu-(?~~PAA: Abe(cr 1-+3)Rha(a 1 -+ Mama 1 -+ = PMcr -C,-PAA; 2)Man(a l+ = ARclM-C,-PAA; Abe-(@ 1-+3)Rha(B 1 -+ Rha-(b 1+ 2)Man(cc l+ = ARPM-C,-PAA and 2)Man(cr 1 -+ = RPM-C,-PAArespectively. The oligosaccharides were linked to polyacrylamide either via a -CH,CH,-spacer as indicated by CZ or via a -CH,(CH,),CH,-spacer as indicated by C,. The disaccharide ARcl as p-nitrophenyl glycoside was synthesized by Garegg and Hultberg (1979) the hapten obtained was coupled to BSA by the isothiocyanate method (Svenungsson and Lindberg, 1979). The synthetic disaccharide abequosyl-(cr l-3)-mannose (AM, as p-nitrophenyl glycoside) (Eklund et al., 1976), representative of the O-antigen 4 determinant in Salmonella serogroup B, was similarly converted into the neoglycoprotein AMa-BSA (Svenungsson and Lindberg, 1977). Bacterial
lipopolysaccharides
Salmonella typhimurium strain SH4809 (serogroup B, O-antigens 4,5,12), S. newport IS50 (serogroup CZ, 0: 6,8) and S. kentucky IS98 (serogroup C,, 0: 8,20) LPSs were extracted from submerged cultures with hot phenol-water (Westphal et al., 1952). The S. kentucky LPS-OH was prepared by mild alkaline hydrolysis (0.2 M NaOH, lOO”C, 1 hr). In the resulting LPS, the 0-acetyl group linked to the terminal glucose residue in the repeating unit was removed. In addition, all esterlinked fatty acids in the lipid A portion of the LPS molecule were hydrolyzed off. The S. typhimurium SH4809 LPS-OH was prepared as above. This preparation lack the O-antigen 5 which is defined as the 0-acetyl group linked to the carbon atom 2 in the terminal abequosyl residue. Enzyme
immunoassay
(EZA)
The enzyme immunoassay as described by Karlsson
was performed essentially et al. (1986). The coating
Immunochemistry
of SuZmonelZaO-antigen
dose for all polyacrylamide neoglycoconjugates was 0.01 pgg/ml. The BSA-conjugates were used at 5 pg/ml and LPSs at lOpg/ml. Anti-Salmonella 0 antigen factor sera 0:4; 0: 8 and 0 : 6,8 were prepared as described by Kauffmann (1966) and obtained from the National Bacteriology Laboratory, Solna, Sweden. The preparation and characterization of rabbit anti-AMcc-BSA and of anti-ARcc-BSA were described by Svenungsson and Lindberg (1977, 1979). The antibodies were used at ten-fold dilutions ranging from 10m3 to 10e6. Alkaline phosphatase conjugated goat anti-rabbit (whole molecule) IgG (Sigma Chemical Company, St Louis, MO, U.S.A.) was used at the dilution recommended by the manufacturer. In inhibition experiments, the ARPM-C,-PAA was used as a coating antigen, with either rabbit factor 0: 6,8 antiserum or with rabbit anti-ARcc-BSA serum. ARPMC,-PAA and ARuM-C,-FAA were used as inhibitors. Serum was preincubated for 1 hr at room temp with different concns of ARflM-C,-PAA or ARctM-C,-PAA (0.001 pg-1OOpg). The 50% inhibitory value is the concn of inhibitor needed to obtain a 50% lowering of the optical density at 405 nm as compared with control wells with no inhibitor added. In another experiment, the interaction between anti AMa-BSA rabbit antibodies and AMu-PAA glycoconjugate was inhibited with different LPSs and glycoconjugates. The inhibitors: AMu -C,-PAA; ARct-C,-PAA; ARc( -C,-PAA; ARcr M-C,-PAA; AR/3 M-C,-PAA; S. ty phimurium SH4809 LPS; S. typhimurium SH4809 LPSOH; S. newport LPS; S. kentucky LPS and PAA were preincubated at different concn (10 pg-100 pg/ml) with rabbit anti-AMcl -BSA antibodies at room temp for 1 hr. The 50% inhibitory value is the concn of inhibitor needed to obtain a 50% lowering of the optical density at 405 nm as compared with control wells with no inhibitor added.
Conformational
889
8 analyses
Conformations ated using the parameters.
of oligosaccharides have been generCHARMM programme with default
RESULTS Enzyme
immunoassay
(EZA)
The specificity and activity of conventional Salmonella factor 0: 8, 0: 6,8 and 0: 4 rabbit sera and the rabbit anti-ARcr-BSA and anti-AMcr-BSA sera was tested EIA using synthetic oligosaccharidein the polyacrylamide and disaccharide-protein conjugates, LPS from S. newport (0 : 6,8), S. kentucky (0 : 8,20) and S. typhimurium (0: 4,5,12) and BSA as coating antigens (Table 1). The rabbit polyclonal factor 0:8 serum reacted efficiently with LPSs and LPS-OH from Salmonella serogroups C2 and C, (S. newport and S. kentucky) but did not bind to the synthetic conjugates representative of the O-antigen 8. With the rabbit factor 0: 6,8 serum a pronounced binding to LPSs from serogroups C, and C, was seen and a weak reaction with 0 : 8 epitope-containing conjugates. For each isomeric oligosaccharide pair (disaccharides or trisaccharides), the absorbance value of the isomer with a fl-rhamnosidic linkage (AR/?-C,PAA or ARPM-C,-PAA, respectively) was higher than that of the corresponding Lx-isomer (Table 1). This was a first indication of the importance of the /I-configuration of the rhamnosidic linkage for interaction with 0 : 8 antibodies. The rabbit anti-ARM-BSA serum reacted with both LPS from S. newport and S. kentucky as well as with the synthetic 0 : 8 antigens with comparable absorbance values. Apparently, the anti-ARcr-BSA antibodies did not discriminate between the a- and /3-rhamnosidic linkages in these synthetic conjugates.
Table 1. Absorbance values at 405 nm after 100 min in EIA for different Salmonella specific antibodies against the polyacrylamide neoglycoconjugates; BSA-conjugates and lipopolysaccharides Antisera” Antigen ARB-C,-PAA ARa -C,-PAA ARu -C,-PAA ARfl M-C,-PAA ARci M-C,-PAA RP M-C,-PAA AMci -C,-PAA PMci -C,-PAA AMci-BSA ARa-BSA BSA S. newport LPS S. kentucky IS98 LPS S. kentucky IS98 LPS-OH S. typhimurium SH4809 LPS
Factor 0: 8
Factor 0: 6,8
0.11 0.11 0.13 0.25
0.31 0.26 0.34 0.90 0.20 0.11
Anti ARa-BSA
“All antisera were used at 10m3 dilution except the anti-ARu-BSA ‘nd-not done.
1.79 1.88 1.77 1.90 2.00
Factor 0: 4
Anti AMu-BSA 0.51 0.48 1.28 0.67 0.43 0.13 3.55 0.45 nd nd > 2.0 0.43 0.13 0.15 3.05
890
A. YA. CHERNYAK et al. CONH2
Antibodies raised against AMa-BSA bound to the AR-disaccharide moiety of the synthetic glycoconjugates (Table 1). The absorbance observed against the ARa-C2PAA conjugate was, however, only 14% of that observed against the homologous AMa-C,-PAA conjugate. Inhibition
1
I-~CHzCH),-CH2CH-(CHn2CH),-]n
CONHR
R =
of EIA
In order to define immunochemically important structural elements of the 0: 8 determinant, EIA-inhibition experiments were performed with LPSs from S. newport, S. ken&&y, and the S. kentucky LPS-OH as coating antigens. As homologous antisera, conventional factor 0: 8 and 0: 6,8 sera at an appropriate dilution (l/4000 to l/40,000) were used. Inhibition assays were done with synthetic conjugates in concns from 0.001 to 100 pg/ml and LPS in concns from 0.1 to 50 pg/ml. None of the synthetic neoglycoconjugates used inhibited the binding of antibodies to LPS but LPS themselves were efficient inhibitors (data not shown). In another experiment, the ARflM-C,-PAA conjugate was used as coating antigen with polyclonal 0: 6,8 antibodies. The homologous neoglycoconjugate, i.e. AR/3M-C,-PAA was an efficient inhibitor; 50% inhibition at 0.05 ,ug/ml. In contrast, the isomeric neoglycoconjugate, i.e. ARaM-C,-PAA showed a very poor inhibitory activity with a 50% inhibition value of > 100 pgg/ml (20% inhibition at 100 pgg/ml-the highest dose used) (Fig. 3A). In a corresponding experiment using rabbit antibodies elicited against ARa-BSA, no difference in inhibitory activity between the AR/?M-C,PAA (50% inhibition at 0.008 pg/ml) and the ARaMC,-PAA (50% inhibition at 0.015 pg/ml) was observed (Fig. 3B). Inhibition experiment using anti AMa-BSA antibodies and the AMa -C,-PAA conjugate showed that the AMa-C,-PAA conjugate was the best inhibitor (50%
CONE42
/
Abc(ul~3)Rha(al_tOCH2CHz-
(ARa-C2-PAA)
Abe(al+3)Rha(~lbOCHzCHz-
(ARP-CZ-PAA)
A~~(~I~~)R~~(~I~OCH~(CHZ)~CH~
(ARa-C6-PAA)
Abe(al~3)Rha(al~Z)Man(al~OCHzCH2-
(ARaM-CZ-PAA)
Abe(ul~3)Rha(~l~2)Man(al--1OCH~CH2-
(ARPM-CZ-PAA)
Rha(Pl~Z)Man(aIjOCH2CH2-
(RPM-CZ-PAA)
Fig. 2. Structure of synthetic oligosaccharide-polyacrylamide conjugates related to Salmonella O-antigen determinant 8. inhibition at 0.0008 pug/ml). The LPS isolated from S. typhimurium SH4809 as well as its de-0-acetylated derivative also inhibited the reaction between anti AMaBSA antibodies and the AMa-C,-PAA conjugate (50% inhibition at 3 pg/ml for both preparations). A lower activity (50% inhibition at 12 pgg/ml) was observed with the ARa-C,-PAA (Table 2). None of the other inhibitors GO
~4)~-Rhap(~l~Z)-~-Manp(al~2)-~-Manp(al-13)-~-~~(~1-, 3
3
7
7
INHIBITOR
CONCENTRATION
pglml
2AcGlcpal
Akpal
~)_LRhap(~l~Z)-~-M~(al~2)-~-Manp(al~3)-~-G~(~l~ 4
3
t
f 2AcGlcpal
Akpal
INHIBITOR CONCENTRATION
II Fig. 1. Structures of the O-antigen repeating units in the LPS from S. newport, serogroup C2 (I), and S. ken&&y, serogroup C, (II).
pglml
Fig. 3. EIA-inhibition. Antibodies preincubated with ARjJMC,-PAA (0) or ARaM-C,-PAA (0) before binding to AR/3M-C,-PAA. (a) Factor 0:6,8 rabbit serum. (b) AntiARa-BSA rabbit serum.
Immunochemistry
of Salmonella O-antigen
891
8
Table 2. Concentration of inhibitor @g/ml) needed to obtain 50% inhibition of binding of anti-AMa-BSA rabbit antibodies to AMu-Cl-PAA glycoconjugate Concentration needed for 50% inhibition @s/ml)
Inhibitor AMa -C,-PAA S. typhimurium SH4809 LPS S. typhimurium SH4809 LPS-OH” ARci -C,-PAA ARci -C,-PAA ARa M-C,-PAA ARP M-C,-PAA S. newport LPS S. kentucky LPS PAA
> > > > > >
“LPS-OH is de-0-acetylated LPS. bFigures in parentheses are the inhibition highest concn tested. reached 50% concn tested).
of inhibition
0.0008 3.0 2.9 12 100 (30%)b 100 (10%) 100 (10%) 100 (0%) 100 (0%) 100 (0%) value with the
at 100 pgg/ml (the
highest (b)
Conformational analyses Conformational analyses of the disaccharides Abe(cr 1+3)Rha and Abe(a 1+3)Man generated using the CHARMM-programme did not show any difference in the terminal, non-reducing abequosyl residue (Fig. 4). Conformational analysis of both trisaccharide sequences (ARaM and ARBM) are shown in Fig. 5. The result clearly shows a significant difference in the rhamnose-tomannose linkage region reflecting in &Jand I,$angles. The Abe-Rha linkage conformation is practically the same in both trisacharides (ARclM, 4 = - 65”, $ = -44”; AR/?M, 4 = - 60”, $ = -45”), whereas the conformation of the Rha(fll-+2)Man linkage (4 = - 71”, $ = 18”) is distinct from that of the Rha(a 1+2)Man linkage (4 = 58”, $ = 39”). DISCUSSION Synthetic Salmonella O-antigen determinants as haptens, either as polyacrylamide conjugates (Tendetnik et al., 1991) or as BSA-conjugates (Lindberg et al., 1983) have been shown previously to be useful in immunochemical studies. In this investigation polyacrylamide conjugates, prepared from synthetic oligosaccharides related to O-antigen 8, were used in evaluation of the importance of configuration of rhamnosidic linkage for immunochemistry of this antigen. In EIA, synthetic oligosaccharide-polyacrylamide conjugates (ARB-C,-PAA, ARa -C,-PAA, ARu-C,PAA, ARfl M-C,-PAA, ARcl M-C,-PAA) reacted weakly with the conventional factor 0 : 8 serum but binding was seen with the factor 0: 6,8 serum. The reason for this difference could be a lower concn of antibodies in the factor 0 : 8 serum prepared by absorption (Kauffmann, 1966). The reaction of the synthetic conjugates with the factor 0: 6,8 serum was weak. It should be noted, however, that the interaction of the serum with the con-
Fig.
4. Conformations
Abe(cz 1-+3)Man(u
of Abe(a1+3)Rha(a
1-+ (b) generated programme.
using
I-,
(a) and
the CHARMM-
jugates with a B-rhamnosidic linkage was stronger, than to conjugates with the corresponding u-isomer (Table 1). The importance of the /I -configuration of the rhamnosidic linkage for the immunochemical specificity of the O-antigen 8 was unambiguously demonstrated by the inhibition of the interaction of the factor 0: 6,8 serum with the ARfiM-C,-PAA-conjugate (Fig. 3A). Among the synthetic conjugates and LPS used as inhibitors, the only conjugate with inhibitory activity was ARBM-C2PAA. The isomeric conjugate with a-rhamnosidic linkage (ARaM-C,-PAA) was shown to be at least 2000-fold less efficient as an inhibitor (Fig. 3A). This finding confirmed the importance of the anomeric configuration of the subterminal sugar (Rha) in the antigen-antibody interaction. Conformational analysis of both trisaccharide sequences (ARaM and AR/?M) using the CHARMM-programme clearly show drastic changes in the rhamnose-to-mannose linkage region. Rabbit antibodies elicited by the ARa-BSA conjugate revealed in EIA approximately the same activity in the interaction with conjugates comprising the AR-fragment (Table 1). The polyclonal antibodies are probably not able to discriminate between the two configurations of the rhamnosidic linkage. The EIA results were confirmed in the EIA inhibition experiments. Both glycoconjugates, i.e. ARBM-C,-PAA and ARclM-C,-PAA were able to inhibit the anti-ARcr-BSA antibodies in an almost superimposable way (Fig. 3B). The reason for this could be that the combining site of the antibodies in this serum is complementary to a structure equal to or smaller than the disaccharide, or differs in the cc-rhamnose-to-BSA linkage region because of the aromatic character of the spacer.
892
A. YA. CHERNYAKet al.
lb)
Fig. 5. Conformations of Abe(a 1+3)Rha(a 1-+2)Man(a 1+ (a) and Abe(a 1+3)Rha(/? 1-+2)Man(a 1+ (b) generated using the CHARMM-programme.
The length of a spacer (C, or C,) did not affect the binding of the 0 : 8 antibodies to the synthetic conjugates (Table 1). The previous investigation of the specificity of the anti-ARcc-BSA serum in EIA (Svenungsson and Lindberg, 1979), revealed a very weak reaction to the serum with S. typhimurium LPS (0:4,5,12) comprising AMafragment representative of Salmonella O-antigen 4. In this investigation we used the neoglucoconjugates AMaC,-PAA and PMa-C,-PAA with the anti-ARcr-BSA serum. The reaction of the anti-ARcr-BSA antibodies with the heterologous neoglycoconjugates was very weak (Table 1). However, the cross-reactivity between AMcr- and ARa-moieties in the corresponding BSAconjugates was seen when rabbit factor 0 : 4 serum was used in EIA (Table 1). The intensity of the binding of the cross-reacting antigen ARu-BSA to antibodies was comparable with that of the homologous antigens AMcrBSA and AMor-C,-PAA. It should be noted that no reaction with this serum was seen with the heterologous AR-containing PAA-conjugates or with PM@-C,-PAA (Table 1). The antibodies raised against the synthetic disaccharide-protein conjugate (AMa-BSA) interacted with AMcr-C,-PAA as well as with the AR-C,-PAA conjugate. This finding was confirmed by inhibition EIA where AMc(+PAA inhibited the binding of anti AMa-BSA antibodies to the AMa-C,-PAA conjugate coated to the solid phase. The ARcr-C,-PAA also pos-
sessed inhibitory activity but an approximately 4000 fold higher concn was needed to obtain 50% inhibition (Table 2). Conformational analyses of the disaccharides Abe(cr 1+3)Rha and Abe(cr 1-+3)Man did not show any difference in the terminal, non-reducing abequosyl residue which probably has an immunodominant role. However, the difference between the mannosyl and rhamnosyl residues adopting approximately the same chair conformation is evident. Different arrangement of the functional groups in residues of the same mannoconfiguration is due to the 6-deoxy and L-character of rhamnose (Fig. 4). Although different, the rabbit antibodies against AMa-BSA are able to partially recognize the heterologous AR disaccharide in the ARa-C,-PAA where the sugar moiety is more accessible than in the ARa-C2-PAA conjugate. The results of inhibition EIA using a series of natural and chemically well defined synthetic antigens show that polyclonal 0: 8 specific antibodies are able to recognize the anomeric configuration of the subterminal residue (Rha) in the trisaccharide sequence Abe-Rha-Man. This coincides in a way with the classical investigation of Kabat (1966), about the relative contribution of different sugars in an antigenic oligosaccharide sequence to binding with an antibody. The ability of the anti-AMa-BSA antibodies to partially recognize the heterologous AR sequence can be explained by the presence of the same terminal immunodominant sugar, abequose. Acknowledgements-This
work was supported by a grant “Swedish-Soviet Scientific Cooperation” from the Royal Swedish Academy of Sciences and by grant 16X656 from the Swedish Medical Research Mrs K. Bergman and acknowledged. We thank Stenutz (Department of Stockholm, Sweden) for
Council. The technical assistance of Mrs Monica Jansson is gratefully Dr Per-Erik Jansson and Mr Ronald Organic Chemistry, University of the generation of Figs 4 and 5.
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