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1.
N A Nnalue S M Lind A A Lindberg 《Journal of immunology (Baltimore, Md. : 1950)》1992,149(8):2722-2728
We generated a panel of mAb containing at least one specificity against each of the known chemotypes of the Salmonella LPS core domain and used them to investigate the accessibility of core determinants in smooth LPS. Most of the mAb were reactive with at the most three chemotypes of the core as determined by enzyme immunoassay and failed to bind smooth LPS or any of the complete cores of E. coli. One mAb, MASC1-MM3 (MM3), reacted with six different Salmonella core chemotypes, the R2 core of Escherichia coli and a variety of smooth LPS. This mAb reacted equally well with live and heat-killed bacteria. It bound to 123 of 126 clinical isolates of Salmonella and 11 of 73 E. coli strains in a dot-immunoblot assay. Typical ladder-like patterns of bands were observed after immunoblotting of this mAb against electrophoretically resolved smooth LPS from the five major serogroups of Salmonella species (A, B, C1, D1, and E). MM3 had no reactivity with BSA conjugates of O-Ag polysaccharides from the above serogroups confirming specificity for a core epitope. Polysaccharides derived from or synthetic saccharides representative of the various chemotypes of Salmonella LPS core were tested as competitive inhibitors of the binding of MM3 to LPS. The results led to a conclusion that MM3 recognizes the structure, L-alpha-D-Heptose1-->7-L-alpha-D-Heptose1-->disaccharide present as a branch in the Ra, Rb1, Rb2, Rb3 and Rc but lacking in the Rd1, Rd2, and Re chemotypes of the Salmonella LPS core. This disaccharide seems free and accessible on the basis of the previously calculated conformations of the Salmonella (Ra) and E. coli complete cores (R1, R2, R3, R4, and K12). It therefore defines or contains an epitope within the inner core subdomain of Salmonella LPS that is accessible to antibody in long-chained LPS and in intact bacteria with complete LPS. 相似文献
2.
G W Bruse R Wollin S Oscarson P E Jansson A A Lindberg 《Journal of molecular recognition : JMR》1991,4(4):121-128
Phage G13 binds to the carbohydrate part of lipopolysaccharides from rough mutants of Salmonella and Escherichia coli as the first event of infection. Equilibrium dialysis inhibition studies with native and synthetic trisaccharides as inhibitors suggested that phage G13 recognizes branched oligosaccharides having 6-O-alpha- or 7-O-alpha-glycosyl groups with alpha-Man(1----3) [alpha-Man(1----6)]Man (Man[Man]Man) and alpha-Glc(1----3)-[alpha-Hep(1----7)] alpha-Hep(1----3) alpha-Hep(1----5)Kdo as the smallest saccharides with inhibitory activity (Wollin et al., 1989). Of four synthetic analogues to Man[Man]Man only Man(1----3)[alpha-Gal(1----6)]alpha-Man-OMe (Man[Gal]-Man) and alpha-Glc(1----3)[alpha-Hep(1----7)]alpha-Hep-OMe (Glc[Hep]Hep) inhibited the binding of labelled E. coli C core nonasaccharide ligand to G13 with activities which were 10- and 15-fold lower than Man[Man]Man. The trisaccharides alpha-Man(1----3)[alpha-Glc(1----6)[alpha-Man-OMe (Man[Glc]Mann) and alpha-Man(1---3)[alpha-Tal(1----6)]alpha-Man-OMe (Man[Tal]Man) showed no inhibition at concentrations 75-fold higher than Man[Man]Man. Minimum energy conformation calculations of the saccharides using the GESA method showed that the 6-O-alpha-Man group in Man[Man]Man and the 7-O-alpha-Hep group SL805 pentasaccharide expose their OH-2 and OH-3 groups in a similar way and these are postulated to be key structural features for binding activity. The importance of hydroxy groups at certain positions is implied from the fact that both manno- and galacto-isomers are active. We also conclude that the O6-C6-C5-O5-C1 region of the 3-O-alpha-glycosyl group in the Man[Man]Man trisaccharide, or part of it, is important for the G13 binding activity. 相似文献
3.
A modified methylation analysis is described which allows the elucidation of the structure of the inner core region [heptose/3-deoxy-D-manno-2-octulosonic acid (KDO)] of enterobacterial lipopolysaccharides (LPS) of Salmonella minnesota rough mutants (Re, strain R595; and Rd2P-, strain R4). Methylation, carboxyl-reduction, remethylation, hydrolysis, carbonyl-reduction, and acetylation of the Re-mutant LPS yielded the 2,6-di-O-acetyl and 2,4,6-tri-O-acetyl derivatives of partially methylated 3-deoxyoctitol in equimolar amounts, indicating the presence of a terminal and a 4-linked pyranosidic KDO residue. For Rd2P- LPS, the hydrolysis step involved 0.1M trifluoroacetic acid at 100 degrees for 1 h which cleaved ketosidic linkages, and the final products included the foregoing acetyl derivatives in the molar ratio of 1:02 and a partially methylated and acetylated 3-deoxyoctitol derivative which was substituted at O-5 by a methylated heptopyranosyl residue. Trideuteriomethylation of the latter product followed by methanolysis and acetylation gave 5-O-acetyl-3-deoxy-1,7,8-tri-O-methyl-2,4,6-tri-O-trideuteriomethyl++ +-D- glycero-D-talo/galacto-octitol and 1,5-di-O-acetyl-2,3,4,6,7-penta-O-methyl-L-glycero-D-manno-heptitol++ +. These results prove the presence of a (2----4)-linked KDO disaccharide in Re LPS and show that the core region of Rd2P- LPS contains a terminal alpha-L-glycero-D-manno-heptopyranosyl group and a non-substituted, a 4-O-, and a 4,5-di-O-substituted pyranosidic KDO residue in the molar ratios 1:1:0.2:1. 相似文献
4.
J M Luk S M Lind R S Tsang A A Lindberg 《The Journal of biological chemistry》1991,266(34):23215-23225
Four murine monoclonal antibodies reactive with distinctive regions of the hexose core domain of Salmonella lipopolysaccharide (LPS) were generated and their epitope specificities were delineated. MAST 56 (IgG1) and MAST 50 (IgG3) antibodies elicited by immunizations with Salmonella typhimurium Rb1 and Rb2 mutants, reacted selectively in enzyme immunoassay with the LPS from rough mutants. In contrast, MATy 1 (IgM) and MATy 2 (IgG2b) antibodies raised by an attenuated Salmonella typhi 620 Ty strain were reactive with LPS from both smooth and rough Salmonellae. Immunoblotting analysis showed that MATy 1 distinguished only the bottom bands (naked LPS core) among the heterogeneous LPS populations, whereas MATy 2 gave a ladder pattern (reactive with both naked and O-chain-substituted LPS cores). Differential binding specificities of MATy 1 and MATy 2 antibodies to the naked and capped LPS cores were further analyzed utilizing S. typhimurium polysaccharide fractions with different O-chain:core ratios which were obtained after separation by Sephacryl S-200 chromatography. Steric effects on the antibody reactivity by the bulky O-polysaccharide chain were detected. The use of chemically defined native and synthetic saccharides as inhibitors, in combination with the conformation of the Salmonella core oligosaccharide, permitted the definition of antigenic determinants carried in the core domain recognized by each antibody: (i) the branches I and VIII are essential for MATy 1 recognition, (ii) the backbone III-IV-V for MATy 2, (iii) the backbone II-III-IV-V for MAST 56, and (iv) the backbone plus the branch III-IV-V-VIII for MAST 50. (formula; see text) 相似文献
5.
In vitro antigenic reactivity of synthetic lipid A analogues as determined by monoclonal and conventional antibodies 总被引:3,自引:0,他引:3
N Kasai S Arata J Mashimo K Okuda Y Aihara S Kotani H Takada T Shiba S Kusumoto 《Biochemical and biophysical research communications》1985,128(2):607-612
Cross-reactivities of synthetic lipid A analogues with monoclonal and conventional antibodies against Salmonella lipid A were studied. It was shown that the in vitro antigenicity of a synthetic compound 506, beta-(1----6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at 2'-amino and 3'-hydroxyl groups with (R)-3-dodecanoyloxytetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl groups, respectively, and has (R)-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, was practically indistinguishable from that of the natural E. coli lipid A preparation, and that both phosphates in positions 1 and 4' as well as ester- and amide-linked fatty acyl residues, particularly 3-acyloxyacyl group, of the glucosamine disaccharide are involved in the cross-reactivity of lipid A as important antigenic determinants. 相似文献
6.
Haralambieva IH Iankov ID Petrov DP Mladenov IV Mitov IG 《FEMS immunology and medical microbiology》2002,33(2):71-76
A murine monoclonal antibody (MAb) 202D7 of IgG3 isotype recognizes a lipopolysaccharide (LPS) epitope of Chlamydia spp. and cross-reacts with the Re chemotype LPS of Salmonella and Escherichia coli. The antibody exhibits strong complement activating properties and stimulates phagocytosis of Salmonella enterica serovar Minnesota Re mutant by murine macrophages. Salmonella Re mutants are non-invasive for cell monolayers but still can enter and replicate in L-929 murine fibroblast cells. The entry of bacteria within the cells increases five-fold in the presence of MAb 202D7. The antibody mediates attachment and enhances five-fold the infectivity of Chlamydia pneumoniae into L-929 cells, which suggests a possible IgG-mediated mechanism of entry and survival of the pathogen in fibroblast cells. 相似文献
7.
Chlamydiae possess a genus-specific epitope that is located on the lipopolysaccharide (LPS) and is composed of a 3-deoxy-d -manno-octulosonic acid (Kdo) trisaccharide of the sequence αKdo-(2→8)–αKdo–(2→4)-αKdo. In Chlamydia trachomatis, this trisaccharide is biosynthetically generated through the action of a multi-functional Kdo-transferase encoded by the gene gseA. gseA of Chlamydia psittaci 6BC was cloned and expressed in a rough mutant (Re chemotype) of Escherichia coli (strain F515) that contains an LPS with only two α2→4-linked Kdo residues. Recombinant strains were able to add the immunodominant Kdo residue in a α2→8-linkage to the parental LPS, as determined by SDS–PAGE and Western blot analysis using a monoclonal antibody against the genus-specific epitope. The DNA sequence of gseA was determined and aligned to that published recently for C. trachomatis serovar L2. Most surprisingly, the two deduced amino acid sequences shared only an overall homology of 67%. Thus, gseA exhibits species specificity at the DNA level, whereas its gene product results in the synthesis of a carbohydrate antigen with genus specificity. 相似文献
8.
To develop a convenient method for the preparation of an alpha-Kdo derivative carrying a functional spacer at the reducing end, we examined anomeric O-acylation using Kdo and halogenated alkyl/aryl isocyanates as nucleophile and electrophiles, respectively. Reaction of a Kdo derivative with 2-chloroethyl isocyanate in the presence of DMAP gave an alpha-spiro product (82%) and an alpha-Kdo derivative of a dimeric isocyanate adduct (10%). Similar reaction with 4-(chloromethyl)phenyl isocyanate gave only the corresponding alpha-spiro product (81%). The NMR data show that the pyranose rings of both the alkyl and aryl spiro products adopt the 5C2 conformation. Thus, we accomplished alpha-selective anomeric O-acylation by coupling the Kdo derivative with alkyl and aryl isocyanates. 相似文献
9.
Anna Swierzko Lore Brade Elisabeth C. Höffgen Helmut Brade 《FEMS immunology and medical microbiology》1993,7(3):265-270
Abstract Rabbit polyclonal antibodies against the lipopolysaccharide (LPS) of the Rd1 P− mutant strain R7 of Salmonella minnesota were serologically characterized using R7 LPS, dephosphorylated LPS, deacylated LPS, deacylated, dephosphorylated and reduced LPS, and synthetic partial structures. The latter comprised partial structures of the core region of Rd1 P− LPS bound to the β 1 → 6-linked glucosamine disaccharide with two amide-linked 3-hydroxytetradecanoic acid residues or artificial glycoconjugates comprised of the synthetic oligosaccharides coupled to bovine serum albumin. Using a passive hemolysis and an enzyme immunoassay, absorption and inhibition experiments, the antibody specificities present could be determined. One group of antibodies required components of the core region and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The majority of phosphate-independent antibodies was directed against the trisaccharide l -glycero-α- d -manno-heptopyranose(1 → 3)- l -glycero-α- d -manno-heptopyranose(1 → 5)3-deoxy- d -manno-octulosonic acid. Antibodies against the 1 → 3- and 1 → 7-linked heptose disaccharides and against a single heptose were also detected, however, with low titers. No antibodies were found which required the presence of fatty acids. 相似文献
10.
Antibodies recognizing the Re core or lipid A structures of lipopolysaccharide (LPS) derived from Salmonella minnesota R595 and Pseudomonas vesicularis JCM1477 were tested for the ability to neutralize the preparatory activity of endotoxin using the local Shwartzman reaction. Shwartzman-inducing activity of R595 LPS (Re-form) was strongly suppressed when the LPS was incubated with the rabbit anti-R595 antiserum or the purified IgG antibody which recognizes core region of the LPS. The antiserum also suppressed the preparatory activity of LPS from S. typhimurium SL1102 (Re) and Escherichia coli F515 (Re), but not that of either S. typhimurium LT-2 (S) LPS or R595 lipid A. Moreover, it was found that the murine monoclonal antibody (MAb), SmRe100G (IgG2a) which recognizes the core region of R595 LPS, significantly suppressed the preparatory activity of R595 LPS. Both conventional antibodies specific to R595 lipid A, which contains a 1,4'-bisphosphorylated beta-D-glucosaminyl-alpha-D-glucosamine disaccharide structure, and JCM1477 lipid A, which contains a monophosphorylated 3-amino-D-glucosamine disaccharide structure, neutralized the preparatory activity of homologous and a closely related lipid A, but not that of LPS. In addition, it was observed that MAb Sm5G (IgG2b) specific to enterobacterial lipid A preparations (especially R595 lipid A) neutralized the preparatory activity of R595 lipid A, although the effect was somewhat weak as compared with that of rabbit antiserum. These results suggest that anti-Re LPS antibody binding to the core of Re LPS is involved in suppressing the endotoxic activity of Re LPS, and that the direct binding of anti-lipid A antibody to some specific epitopes of lipid A is important in neutralizing the endotoxic activity. 相似文献
11.
Occurence of antibodies against chlamydial lipopolysaccharide in human sera as measured by ELISA using an artificial glycoconjugate antigen 总被引:1,自引:0,他引:1
Lore Brade Helga Brunnemann Martin Ernst Yucai Fu Otto Holst Paul Kosma Helmut Näher Kenneth Persson Helmut Brade 《FEMS immunology and medical microbiology》1994,8(1):27-42
Abstract An artificial glycoconjugate containing, as a ligand, the deacylated carbohydrate backbone of a recombinant Chlamydia -specific lipopolysaccharide was used as a solid-phase antigen in ELISA to measure antibodies against chlamydial LPS. The specificity and reproducibility of the assay was shown by using a panel of prototype monoclonal antibodies representing the spectrum of antibodies also occuring in patient sera. These mAbs recognized Chlamydia -specific epitopes [ α 2→8-linked disaccharide of 3-deoxy- d - manno -octulosonic acid (Kdo) or the trisaccharide α Kdo-(2→8)-→Kdo] or those shared between chlamydial and Re-type LPS ( α Kdo, α →4-linked Kdo disacccharide). The assay was used to measure IgG, IgA and IgM antibodies against chlamydial LPS in patients with genital or respiratory tract infections. In comparison to the results obtained with sera from blood donors, it became evident that both types of infection result in significant changes in the profile of LPS antibodies. 相似文献
12.
Mass spectrometric analyses of lipopolysaccharide (LPS) from isogenic Escherichia coli strains with nonpolar mutations in the waa locus or overexpression of their cognate genes revealed that waaZ and waaS are the structural genes required for the incorporation of the third 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) linked to Kdo disaccharide and rhamnose, respectively. The incorporation of rhamnose requires prior sequential incorporation of the Kdo trisaccharide. The minimal in vivo lipid A-anchored core structure Kdo(2)Hep(2)Hex(2)P(1) in the LPS from ΔwaaO (lacking α-1,3-glucosyltransferase) could incorporate Kdo(3)Rha, without the overexpression of the waaZ and waaS genes. Examination of LPS heterogeneity revealed overlapping control by RpoE σ factor, two-component systems (BasS/R and PhoB/R), and ppGpp. Deletion of RpoE-specific anti-σ factor rseA led to near-exclusive incorporation of glycoforms with the third Kdo linked to Kdo disaccharide. This was accompanied by concomitant incorporation of rhamnose, linked to either the terminal third Kdo or to the second Kdo, depending upon the presence or absence of phosphoethanolamine on the second Kdo with truncation of the outer core. This truncation in ΔrseA was ascribed to decreased levels of WaaR glycosyltransferase, which was restored to wild-type levels, including overall LPS composition, upon the introduction of rybB sRNA deletion. Thus, ΔwaaR contained LPS primarily with Kdo(3) without any requirement for lipid A modifications. Accumulation of a glycoform with Kdo(3) and 4-amino-4-deoxy-l-arabinose in lipid A in ΔrseA required ppGpp, being abolished in a Δ(ppGpp(0) rseA). Furthermore, Δ(waaZ lpxLMP) synthesizing tetraacylated lipid A exhibited synthetic lethality at 21-23°C pointing to the significance of the incorporation of the third Kdo. 相似文献
13.
Silvia Camprubí Susana Merino Vicente-Javier Benedí Juan M. Tomás 《FEMS microbiology letters》1991,83(3):291-297
FC3-10 is a Klebsiella spp. specific bacteriophage isolated on a rough mutant (strain KT707, chemotype Rd) of K. pneumoniae C3. The bacteriophage receptor for this phage was shown to be the low-molecular mass lipopolysaccharide (LPS) fraction (LPS-core oligosaccharides), specifically the heptose content of the LPS inner-core. This is the first phage isolated on Klebsiella, the receptor for which is the LPS-core. This phage was unable to plate on Salmonella typhimurium LPS mutants with chemotypes Rd2 or Re showing incomplete or no heptose content on their LPS-core, respectively. Spontaneous phage-resistant mutants from different Klebsiella strains were deep-rough LPS mutants or encapsulated revertants from unencapsulated mutant strains. 相似文献
14.
The branched Kdo trisaccharide sodium (3-deoxy-α-d-manno-oct-2-ulopyranosyl)onate-(2→8)-[sodium (3-deoxy-α-d-manno-oct-2-ulopyranosyl)onate-(2→4)]-sodium (allyl 3-deoxy-α-d-manno-oct-2-ulopyranosid)onate has been prepared utilizing the regioselective glycosylation of the C-7, C-8 diol entity of a Kdo monosaccharide acceptor with a Kdo bromide donor followed by the attachment of the third Kdo unit to O-4 of the disaccharide intermediate. Deacetylation and hydrolysis of the methyl ester groups furnished the trisaccharide allyl glycoside which was converted into the corresponding 3-(2-aminoethylthio)propyl glycoside. Subsequent covalent attachment to bovine serum albumin furnished a neoglycoconjugate serving as an antigen for the induction of Chlamydophila psittaci-specific monoclonal antibodies. 相似文献
15.
Georg Sixta Kurt Wimmer Andreas Hofinger Helmut Brade Paul Kosma 《Carbohydrate research》2009,344(13):1660-1669
In order to define binding interactions of Kdo-specific monoclonal antibodies directed against the chlamydial α-(2→8)-linked Kdo disaccharide epitope on a molecular level, modifications at the 7-position of the proximal and distal Kdo unit were investigated. The synthesis of 7-O-methyl and 7-azido-7-deoxy-7-epi-Kdo monosaccharide derivatives was achieved via an 8-O-TBS protected derivative, whereas methylation of O-7 at the proximal Kdo unit of the α-(2→8)-linked Kdo disaccharide was conveniently accomplished via a 4,5; 4′,5′; 7′,8′-tri-O-carbonyl-protected disaccharide intermediate. Attempted epimerization at C-5 of the inner unit of a α-(2→4)-linked Kdo disaccharide, however, resulted in formation of the corresponding 5,6-dehydro derivative, which was fully deprotected. Treatment of unprotected α-(2→8)- as well as α-(2→4)-linked Kdo disaccharides in neat acetic acid furnished the corresponding interresidue lactone derivatives. The lactones displayed limited stability under neutral conditions and were hydrolyzed at pH 7 within 3 days. Access to the lactones, however, provides a means for selective derivatization of the carboxylic group located at the distal Kdo residue, which was demonstrated by methanolysis of the lactone to afford the monomethyl ester of the α-(2→8)-linked Kdo disaccharide. ELISA inhibition experiments of the ligands with two Kdo-specific monoclonal antibodies showed slightly reduced reactivity for the binding of the α-(2→8) Kdo-specific antibody S25-2, whereas the 7-O-methyl disaccharide antigen displayed high binding affinity toward the Kdo monosaccharide-specific antibody S67-27. 相似文献
16.
The 1H- and 13C-NMR parameters, chemical shifts and coupling constants, for the pentasaccharide of the genus-specific epitope of Chlamydia lipopolysaccharide and related di-, tri-, and tetra-saccharides have been measured and assigned completely using 1D and 2D techniques, and their structures have been confirmed. NOE experiments indicated the preferred conformation of the pentasaccharide and the component oligosaccharides. The 3JH,H demonstrate a change in conformation by rotation of the C-6-C-7 bond of the side chain of the (2----8)-linked Kdo (unit b) in alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcN-(1--- -6)- GlcNol, alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1- ---O)- allyl, and alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl relative to that preferred in alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, alpha-Kdo-(2----8)-alpha-Kdo-(2----O)-allyl, alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl, and alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, irrespective of the size of the aglycon, e.g., allyl or beta-D-GlcN residues. The conformational results have been substantiated by computer calculations using the HSEA approach. 相似文献
17.
Abstract Hybrid cell lines producing monoclonal antibodies with specificity for the lipopolysaccharide (LPS) from the deep rough mutant Salmonella minnesota R595 have been established. Spleen cells from BALB/c mice immunized with live R595 bacteria were fused with Sp 2/0 myeloma cells and three hybridomas producing antibodies specific for heptoseless LPS from Salmonella were selected. All three monoclonal antibodies were shown to bind only to heptoseless, but 3-deoxy- d -manno-octulosonic acid (KDO) containing LPS when tested in enzyme-linked immunosorbent assay (ELISA) against a set of structurally defined LPS and lipid A from Salmonella, Shigella and Escherichia coli . Synthetic KDO was an efficient inhibitor of the antibody-R595 LPS interaction defining that KDO is in an immunodeterminant position interacting with the monoclonal antibodies. 相似文献
18.
J C Jacquinet 《Carbohydrate research》1990,199(2):153-181
3,4,6-Tri-O-acetyl-D-galactal was transformed into methyl 6-O-acetyl-2-azido-4-O-benzyl-2-deoxy-beta-D-galactopyranoside and its 4-O-acetyl-6-O-benzyl analogue, each of which was glycosylated with activated, O-acetylated derivatives of methyl D-glucopyranosyluronate. The resulting beta-(1----3)-linked disaccharide derivatives were each reductively N-acetylated, hydrogenolysed, O-sulfated, and saponified to afford the disodium salts of methyl 2-acetamido-2-deoxy-3-O-(beta-D-glucopyranosyluronic acid)-4-O-sulfo-beta-D-galactopyranoside and the 6-O-sulfo analogue. D-Galactal was also transformed into activated derivatives of 2-azido-3,6-di-O-benzyl-2-deoxy-D-galactopyranose and their 3,4-di-O-benzyl analogues with various substituents at O-4 and O-6. These glycosyl donors were condensed with 6-O-protected derivatives of methyl 2,3-di-O-benzyl-beta-D-glucopyranoside to give the beta-(1----4)-linked disaccharide derivatives, which were selectively deprotected, then oxidised at C-6 of the gluco unit, reductively N-acetylated, selectively deprotected, O-sulfated at C-4 or C-6 of the galacto unit, and hydrogenolysed to give the disodium salts of methyl 4-O-(2-acetamido-2-deoxy-4-O-sulfo-beta-D-galactopyranosyl)-beta-D- glucopyranosiduronic acid and the 6-O-sulfo analogue. 相似文献
19.
Lipopolysaccharide (LPS) of Chlamydophila psittaci but not of Chlamydophila pneumoniae or Chlamydia trachomatis contains a tetrasaccharide of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid (Kdo) of the sequence Kdo(2-->8)[Kdo(2-->4)] Kdo(2-->4)Kdo. After immunization with the synthetic neoglycoconjugate antigen Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4) Kdo-BSA, we obtained the mouse monoclonal antibody (mAb) S69-4 which was able to differentiate C. psittaci from Chlamydophila pecorum, C. pneumoniae, and C. trachomatis in double labeling experiments of infected cell monolayers and by enzyme-linked immunosorbent assay (ELISA). The epitope specificity of mAb S69-4 was determined by binding and inhibition assays using bacteria, LPS, and natural or synthetic Kdo oligosaccharides as free ligands or conjugated to BSA. The mAb bound preferentially Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4)Kdo(2-->4) with a K(d) of 10 microM, as determined by surface plasmon resonance (SPR) for the monovalent interaction using mAb or single chain Fv. Cross-reactivity was observed with Kdo(2-->4)Kdo(2-->4) Kdo but not with Kdo(2-->8)Kdo(2-->4)Kdo, Kdo disaccharides in 2-->4- or 2-->8-linkage, or Kdo monosaccharide. MAb S69-4 was able to detect LPS on thin-layer chromatography (TLC) plates in amounts of <10 ng by immunostaining. Due to the high sensitivity achieved in this assay, the antibody also detected in vitro products of cloned Kdo transferases of Chlamydia. The antibody can therefore be used in medical and veterinarian diagnostics, general microbiology, analytical biochemistry, and studies of chlamydial LPS biosynthesis. Further contribution to the general understanding of carbohydrate-binding antibodies was obtained by a comparison of the primary structure of mAb S69-4 to that of mAb S45-18 of which the crystal structure in complex with its ligand has been elucidated recently (Nguyen et al., 2003, Nat. Struct. Biol., 10, 1019-1025). 相似文献
20.
Wang H Head J Kosma P Brade H Müller-Loennies S Sheikh S McDonald B Smith K Cafarella T Seaton B Crouch E 《Biochemistry》2008,47(2):710-720
Lipopolysaccharides (LPS) of Gram-negative bacteria are important mediators of bacterial virulence that can elicit potent endotoxic effects. Surfactant protein D (SP-D) shows specific interactions with LPS, both in vitro and in vivo. These interactions involve binding of the carbohydrate recognition domain (CRD) to LPS oligosaccharides (OS); however, little is known about the mechanisms of LPS recognition. Recombinant neck+CRDs (NCRDs) provide an opportunity to directly correlate binding interactions with a crystallographic analysis of the binding mechanism. In these studies, we examined the interactions of wild-type and mutant trimeric NCRDs with rough LPS (R-LPS). Although rat NCRDs bound more efficiently than human NCRDs to Escherichia coli J-5 LPS, both proteins exhibited efficient binding to solid-phase Rd2-LPS and to Rd2-LPS aggregates presented in the solution phase. Involvement of residues flanking calcium at the sugar binding site was demonstrated by reciprocal exchange of lysine and arginine at position 343 of rat and human CRDs. The lectin activity of hNCRDs was inhibited by specific heptoses, including l-glycero-alpha-d-manno-heptose (l,d-heptose), but not by 3-deoxy-alpha-d-manno-oct-2-ulosonic acid (Kdo). Crystallographic analysis of the hNCRD demonstrated a novel binding orientation for l,d-heptose, involving the hydroxyl groups of the side chain. Similar binding was observed for a synthetic alpha1-->3-linked heptose disaccharide corresponding to heptoses I and II of the inner core region in many LPS. 7-O-Carbamoyl-l,d-heptose and d-glycero-alpha-d-manno-heptose were bound via ring hydroxyl groups. Interactions with the side chain of inner core heptoses provide a potential mechanism for the recognition of diverse types of LPS by SP-D. 相似文献