Diuretic-induced hypokalaemia in patients with hypertension.

We describe a 55 year-old woman with longstanding hypertension who developed hypokalaemia following diuretic treatment. Investigations revealed primary hyperaldosteronism due to an adrenal adenoma, and normal blood pressure was restored after surgical removal of the tumour. Primary hyperaldosteronism is a potentially curable cause of hypertension and should be considered in hypertensive patients who present with diuretic - induced hypokalaemia.

Anti-Chlamydophila immunoglobulin prevalence in sarcoidosis and usual interstitial pneumoniae.

Sarcoidosis and usual interstitial pneumoniae (UIP) are diseases of unknown aetiology affecting the lower respiratory tract. Although there are a number of studies investigating the causal role of these disorders, no micro-organism could be identified as the causal agent. The high incidence of Chlamydophila pneumoniae infections associated with lung injury encouraged the present investigations to screen patients with sarcoidosis and with UIP for their Chlamydophila-specific immune response. Thirty-nine patients with sarcoidosis, 26 patients with UIP and 34 controls were tested for the prevalence of Chlamydophila-specific antibodies in bronchoalveolar lavage fluids (BALF) and sera. Samples were tested for the presence of antibodies in a genus-specific test for Chlamydophila-lipopolysaccharide (LPS) and in a species-specific test for C. pneumoniae. This study revealed a significantly higher prevalence of Chlamydophila LPS-specific immunoglobulin (Ig)-G in the BALF of sarcoidosis patients (36.8%) compared to controls (8.8%) and patients with UIP (12.0%). Similar findings were observed in sera. The prevalence of C. pneumoniae-specific antibodies in BALF was significantly higher in sarcoidosis patients for IgG and IgA (IgG: 74.4%; IgA: 46.2%) and in UIP for IgG (IgG: 50.0%; IgA: 11.5%) compared to controls (IgG: 14.7%; IgA: 14.7%). The elevated prevalence of Chlamydophila-specific antibodies in sarcoidosis patients might implicate Chlamydophila as a causal agent. However, considering the high prevalence of Chlamydophila antibodies in the healthy population, the data presented might reflect Chlamydophila co-infections in pre-injured lungs seen in these patients.

Localization of ceramide and glucosylceramide in human epidermis by immunogold electron microscopy.

Ceramides and glucosylceramides are pivotal molecules in multiple biologic processes such as apoptosis, signal transduction, and mitogenesis. In addition, ceramides are major structural components of the epidermal permeability barrier. The barrier ceramides derive mainly from the enzymatic hydrolysis of glucosylceramides. Recently, anti-ceramide and anti-glucosylceramide anti-sera have become available that react specifically with several epidermal ceramides and glucosylceramides, respectively. Here we demonstrate the detection of two epidermal covalently bound omega-hydroxy ceramides and one covalently bound omega-hydroxy glucosylceramide species by thin-layer chromatography immunostaining. Moreover, we show the ultrastructural distribution of ceramides and glucosylceramides in human epidermis by immunoelectron microscopy on cryoprocessed skin samples. In basal epidermal cells and dermal fibroblasts ceramide was found: (i) at the nuclear envelope; (ii) at the inner and outer mitochondrial membrane; (iii) at the Golgi apparatus and the endoplasmic reticulum; and (iv) at the plasma membrane. The labeling density was highest in mitochondria and at the inner nuclear membrane, suggesting an important role for ceramides at these sites. In the upper epidermis, ceramides were localized: (i) in lamellar bodies; (ii) in trans-Golgi network-like structures; (iii) at the cornified envelope; and (viii) within the intercellular space of the stratum corneum, which is in line with the known analytical data. Glucosylceramides were detected within lamellar bodies and in trans-Golgi network-like structures of the stratum granulosum. The localization of glucosylceramides at the cornified envelope of the first corneocyte layer provides further proof for the existence of covalently bound glucosylceramides in normal human epidermis.

Crystallization and preliminary X-ray diffraction analysis of two homologous antigen-binding fragments in complex with different carbohydrate antigens.

The antigen-binding fragments (Fab) of two murine monoclonal antibodies (mAb) S25-2 and S45-18, specific for carbohydrate epitopes in the lipopolysacchaide of the bacterial family Chlamydiaceae, have been crystallized in the presence and absence of synthetic oligosaccharides corresponding to their respective haptens. Crystals of both Fabs show different morphology depending on the presence of antigens. The sequence of mAb S45-18 was determined and shows a remarkable homology to that reported for mAb S25-2. These crystals offer an unparalleled opportunity to compare the structure and modes of binding of two homologous antibodies to similar but distinct carbohydrate epitopes.

A monoclonal antibody with specificity for the genus Klebsiella binds to a common epitope located in the core region of Klebsiella lipopolysaccharide.

A mouse monoclonal antibody (mAb) which has been obtained after immunization of mice with heat-killed Klebsiella pneumoniae strain R20/O1(-) followed by standard plasmacytoma cell fusion protocols was investigated for its ability to identify various species of the genus Klebsiella. Based on the published observation that the antibody binds to an epitope located in the core region of lipopolysaccharide (LPS) of strain R20/O1(-), we tested whether this epitope is shared and exposed by other species of the genus Klebsiella. The antibody was able to bind to LPS of clinical isolates of K. pneumoniae (n = 77), K. oxytoca (n = 50), K. terrigena (n = 49) and K. planticola (n = 50) in 93%, 98%, 96% and 100%, respectively, but did not bind to LPS of other Gram-negative genera (n = 159) as tested by Western blots and dot blots using proteinase K-digested whole cell lysates as antigens. Western blot analyses indicated that the antibody bound only to those LPS molecules which did not carry an O-antigen and that the antibody is thus different from those already published.

Synthesis and immunochemical characterization of neoglycoproteins containing epitopes of the inner core region of Pseudomonas aeruginosa RNA group I lipopolysaccharide.

The disaccharide allyl 2-acetamido-2-deoxy-alpha-D-galactopyranosyl-(1-->3)-7-O-carbamoyl-L-glycero-alpha-D-manno-heptopyranoside 5 (GalNAc-cmHep-allyl) was synthesized starting from 1 and 2. Compound 5, cmHep-allyl and the disaccharide cmHep-(1-->3)-Hep-allyl were converted into cysteamine-spacered derivatives and conjugated to bovine serum albumin (BSA) to yield the neoglycoconjugates 7--9, respectively. These conjugates were used to immunize mice and to prepare monoclonal antibodies (mAbs) which were characterized in comparison to mAbs obtained after immunization with heat-killed Pseudomonas aeruginosa strain H4. Two antibodies obtained after immunization with the neoglycoconjugates bound strongly to cmHep-BSA and with lower affinity to cmHep-Hep-BSA but did not bind to GalNAc-cmHep-BSA or to H4 LPS. Another antibody obtained after immunization with heat-killed bacteria bound to LPS and GalNAc-cmHep-BSA but not to cmHep-Hep-BSA or cmHep-BSA

Mouse anti-ceramide antiserum: a specific tool for the detection of endogenous ceramide.

Ceramide is a pivotal molecule in signal transduction and an essential structural component of the epidermal permeability barrier. The epidermis is marked by a high concentration of ceramide and by a unique spectrum of ceramide species: Besides the two ceramide structures commonly found in mammalian tissue, N-acylsphingosine and N-2-hydroxyacyl-sphingosine, six additional ceramides differing in the grade of hydroxylation of either the sphingosine base or the fatty acid have been identified in the epidermis. Here we report on the characterization of an IgM-enriched polyclonal mouse serum against ceramide. In dot blot assays with purified epidermal lipids the antiserum bound to a similar extent to N-acyl-sphingosine (ceramide 2), N-acyl-4-hydroxysphinganine (ceramide 3), and N-(2-hydroxyacyl)-sphingosine (ceramide 5), whereas no specific reaction was detected with glycosylceramides, sphingomyelin, free sphingosine, phospholipids, or cholesterol. In contrast, a monoclonal IgM antibody, also claimed to be specific for ceramide, was shown to bind specifically to sphingomyelin and therefore was not further investigated. In thin-layer chromatography immunostaining with purified lipids a strong and highly reproducible reaction of the antiserum with ceramide 2 and ceramide 5 was observed, whereas the reaction with ceramide 1 and ceramide 3 was weaker and more variable. Ceramide 2 and ceramide 5 were detected in the nanomolar range at serum dilutions of up to 1:100 by dot blot and thin-layer immunostaining. In thin-layer chromatography immunostaining of crude lipid extracts from human epidermis, the antiserum also reacted with N-(2-hydroxyacyl)-4-hydroxysphinganine (ceramide 6) and N-(2-hydroxyacyl)-6-hydroxysphingosine (ceramide 7). Furthermore, the suitability of the antiserum for the detection of endogenous ceramide by immunolight microscopy was demonstrated on cryoprocessed human skin tissue. Double immunofluorescence labeling experiments with the anti-ceramide antiserum and the recently described anti-glucosylceramide antiserum (Brade et al., 2000, Glycobiology 10, 629) showed that both lipids are concentrated in separate epidermal sites. Whereas anti-ceramide stained the dermal and basal epidermal cells as well as the corneocytes, anti-glucosylceramide staining was concentrated in the stratum granulosum. In conclusion, the specificity and sensitivity of the reagent will enable studies on the subcellular distribution and biological functions of endogenous ceramide.

Generation and serological characterization of murine monoclonal antibodies against O antigens from Acinetobacter reference strains.

O-antigen-specific monoclonal antibodies were generated against Acinetobacter strains from international type culture collections and characterized by enzyme immunoassay and Western and colony blotting. The antibodies aid in the further completion of an O-serotyping scheme for Acinetobacter and, due to their high specificity, are especially useful to all working with these strains.

O-antigen diversity among Acinetobacter baumannii strains from the Czech Republic and Northwestern Europe, as determined by lipopolysaccharide-specific monoclonal antibodies.

O-antigen-specific monoclonal antibodies (MAbs) are currently being generated to develop an O-serotyping scheme for the genus Acinetobacter and to provide potent tools to study the diversity of O-antigens among Acinetobacter strains. In this report, Acinetobacter baumannii strains from the Czech Republic and from two clonal groups identified in Northwestern Europe (termed clones I and II) were investigated for their reactivity with a panel of O-antigen-specific MAbs generated against Acinetobacter strains from various species. The bacteria were characterized for their ribotype, biotype, and antibiotic susceptibility and the presence of the 8.7-kb plasmid pAN1. By using the combination of these typing profiles, the Czech strains could be classified into four previously defined groups (A. Nemec, L. Janda, O. Melter, and L. Dijkshoorn, J. Med. Microbiol. 48:287-296, 1999): two relatively homogeneous groups of multiresistant strains (termed groups A and B), a heterogeneous group of other multiresistant strains, and a group of susceptible strains. O-antigen reactivity was observed primarily with MAbs generated against Acinetobacter calcoaceticus and Acinetobacter baumannii strains. A comparison of reaction patterns confirmed the previously hypothesized clonal relationship between group A and clone I strains, which are also similar in other properties. The results show that there is limited O-antigen variability among strains with similar geno- and phenotypic characteristics and are suggestive of a high prevalence of certain A. baumannii serotypes in the clinical environment. It is also shown that O-antigen-specific MAbs are useful for the follow-up of strains causing outbreaks in hospitals.

Identification of a novel dendritic cell-like subset of CD64(+) / CD16(+) blood monocytes.

Human monocytes (Mo) consist of a major subset of Fcgamma-receptor I (CD64)-positive typical low accessory phagocytes, and a minor CD64(-) DC-like subset with high T cell-accessory and IFN-alpha-releasing activity. Both populations also differentially express CD16 (Fcgamma-receptor III). Double labeling with anti-CD64 and anti-CD16 mAb, as performed here, identified four different subsets. The CD64(-) subset consists of CD64(-) / 16(+) cells with high antigen-presenting cell (APC) function and macrophage-like phenotype, and a CD64(-) / 16(-) subset of less active APC but which exhibits a higher mixed lymphocyte reaction (MLR) stimulating and IFN-alpha-producing capacity, possibly resembling plasmacytoid dendritic cell type II (DC2) blood precursors. As well as the majority of CD64(+) cells that appeared CD64(+) / 16(-) and represent typical low-accessory, CD14(high) Mo, we could identify and describe a novel minor subset of CD64(+) / 16(+) cells which is unique in combining typical DC and Mo characteristics in the same cell. These are high IL-12 production, high accessory capacity for antigen- or allogen-activated lymphocytes, and high expression of HLA-DR, CD86, and CD11c.

Synthesis of neoglycoproteins containing Kdo epitopes specific for Chlamydophila psittaci lipopolysaccharide.

The oligosaccharides alpha-Kdop-(2-->8)-alpha-Kdop-(2-->6)-beta-D- GlcpNAc-(1-->OAll) 4, alpha-Kdop-(2-->4)-alpha- Kdop-(2-->4)-alpha-Kdop-(2-->6)-beta-D-GlcpNAc-(1-->OAll+ ++) 10, and the branched Kdo tetrasaccharide alpha- Kdop-(2-->4)-[alpha-Kdop-(2-->8)]-alpha-Kdop-(2-->4)-a lpha-Kdop-(2-->OAll) 21 have been prepared using en bloc transfer of Kdo oligosaccharide bromide donors to protected mono- or disaccharide acceptors. Radical addition of cysteamine to the anomeric allyl glycosides afforded good yields of the corresponding 3-(2-aminoethylthio)propyl glycosides 5, 11 and 22. The spacer ligands were activated with thiophosgene and reacted with bovine serum albumin to give the neoglycoconjugates 6, 12 and 23 which were used to prepare solid-phase antigens in enzyme immuno-assays for the characterization of monoclonal antibodies against chlamydial LPS. The data showed that the (2-->8)-linked Kdo disaccharide and the (2-->8)-(2-->4)-linked Kdo trisaccharide portion of the neoglycoconjugate 23 were not available for binding of antibodies which recognize these structures as di- and trisaccharide, respectively.

Mapping the binding of synthetic disaccharides representing epitopes of chlamydial lipopolysaccharide to antibodies with NMR.

A NMR study of the binding of the synthetic disaccharides alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl 1 (Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid) and alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl 2, representing partial structures of the lipopolysaccharide epitope of the intracellular bacteria Chlamydia, to corresponding monoclonal antibodies (mAbs) S23-24, S25-39, and S25-2 is presented. The conformations of 1 bound to mAbs S25-39 and of 2 bound to mAbs S23-24 and S25-39 were analyzed by employing transfer-NOESY (trNOESY) and QUIET-trNOESY experiments. A quantitative analysis of QUIET-trNOESY buildup curves clearly showed that S25-39 recognized a conformation of 1 that was similar to the global energy minimum of 1, and significantly deviated from the conformation of 1 bound to mAb S25-2. For disaccharide 2, only a qualitative analysis was possible because of severe spectral overlap. Nevertheless, the analysis showed that all mAbs most likely bound to only one conformational family of 2. Saturation transfer difference (STD) NMR experiments were then employed to analyze the binding epitopes of the disaccharide ligands 1 and 2 when binding to mAbs S23-24, S25-39, and S25-2. It was found that the nonreducing pyranose unit was the major binding epitope, irrespective of the mAb and the disaccharide that were employed. Individual differences were related to the engagement of other portions of the disaccharide ligands.

In vitro characterization of anti-glucosylceramide rabbit antisera.

Glucosylceramides (GlcCer) are biosynthetic precursors of glycosphingolipids. They are widely distributed in biological systems where they exhibit numerous biological functions. Studies on the localization of glucosylceramides in different tissues have used biochemical methods only since specific antibodies against GlcCer were not previously available. We have characterized two commercially available rabbit antisera which were prepared against GlcCer of plant origin (1-O-(beta-D-glucopyranosyl)-N-acyl-4-hydroxysphinganine; GlcCer-3) or human origin (1-O-(beta-D-glucopyranosyl)-N-acyl-sphingosine; GlcCer-2) and claimed to be specific for GlcCer. The antisera were also able to detect specifically GlcCer species in crude lipid extracts from human epidermis after separation by thin-layer chromatography. The reagents are sensitive since both antisera reacted at dilutions higher than 1:500 with their homologous antigen in the nanogram range in thin layer immunostaining or dot-blot assays. The antisera are specific for GlcCer although they did not differentiate between GlcCer-2 and GlcCer-3 containing sphingosine or 4-hydroxysphinganine. The antisera also reacted with N-stearoyl-DL-dihydroglucocere-broside indicating that the naturally occurring structural variations in the amino alcohol moiety are not determining the specificity. No crossreactivity was observed with other mono- or diglycosylceramides (galactosylceramides, lactosyl-ceramide), free ceramides or structurally unrelated lipids (cholesterol, sphingomyelin, or phospholipids). Therefore, the glycosylmoiety seems to represent the major antigenic determinant. Finally, the antisera also proved to be useful for the immunohistochemical localization of GlcCer in human epidermis by which earlier biochemical data on the distribution of GlcCer in the various epidermal layers were confirmed.

Antibody response to lipopolysaccharide in patients colonized or infected with an endemic strain of Acinetobacter genomic species 13 sensu Tjernberg and Ursing.

The levels of antilipopolysaccharide (anti-LPS) antibodies in patients colonized with an endemic Acinetobacter strain were compared to those in patients with bloodstream infections. Seropositivity and seronegativity correlated with positive and negative blood cultures, respectively, indicating that determination of the level of anti-LPS antibodies is useful for diagnosing Acinetobacter infections.

Characterization of high affinity monoclonal antibodies specific for chlamydial lipopolysaccharide.

Pathogens belonging to the genus Chlamydia contain lipopolysaccharide with a 3-deoxy-D- manno- oct-2-ulosonic acid (Kdo) trisaccharide of the sequence alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. This lipopolysaccharide is recognized in a genus-specific pattern by murine monoclonal antibodies (mAbs), S25-23 and S25-2 (both IgG1kappa), which bind as the minimal structures the trisaccharide and the terminal Kdo-disaccharide, respectively. The variable domains of these mAbs were reverse transcribed from mRNA which was isolated from hybridomas and cloned as single-chain variable fragments (scFvs) in E.coli TG1. The kinetics of binding of whole antibodies, Fab fragments and scFvs to natural and synthetically modified ligands were determined by surface plasmon resonance (SPR) using synthetic neoglycoconjugates. As examples of an antibody-carbohydrate interaction involving anionic carboxyl groups on the ligand, we report that the affinities of these antibodies are higher than usually observed in carbo-hydrate-protein interactions (K(D)of 10(-3)to 10(-5)M). SPR analy-ses of monovalent Fab and scFv binding to the natural trisaccharide epitope gave dissociation constants of 770 nM for S25-2 and 350 nM for S25-23, as determined by global fitting (simultaneous fitting of several measurements at different antibody concentrations) of sensorgram data to a one-to-one interaction model. Local fitting (separate fitting of individual sensorgram data at different antibody concentrations) and Scatchard analysis of the data gave kinetic and affinity constants that were in good agreement with those obtained by global fitting. The SPR data also showed that while S25-2 bound well to several Kdo disaccharides and carboxyl-reduced Kdo ligands, S25-23 did not. Identification of amino acids in the complementarity determining regions revealed the presence of a large number of positively charged amino acids which were located towards the center of the combining site, thus suggesting a different recognition mechanism than that observed for neutral ligands. The latter mainly involves aromatic amino acids for hydrophobic stacking inter-actions and hydrogen bonds.

Binding of lipopolysaccharide (LPS) to CHO cells does not correlate with LPS-induced NF-kappaB activation.

Activation of myeloid cells by lipopolysaccharide (LPS) is a key event in the development of gram-negative sepsis. One crucial step within this process is the binding of LPS to CD14. CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein requiring at least one additional membrane-spanning molecule for signal transduction. It is not clear whether the function of CD14 is to merely catalyze LPS binding, followed by the interaction of LPS with the signal transducer, or whether CD14 has a more specific function and may be a part of the signaling complex. To address this question we generated Chinese hamster ovary (CHO) cells expressing a human GPI-anchored form of LPS-binding protein (mLBP) to substitute for CD14 as LPS acceptor molecule. By comparison of CHO / mLBP with CHO / vector and CHO / CD14 cells we found that expression of GPI-linked LBP results in an enhanced binding of LPS but not in an increase in cell activation as determined by translocation of NF-kappaB. Furthermore, excess of recombinant soluble LBP resulted also in increased LPS binding without affecting NF-kappaB translocation. These data show that LPS binding alone is not sufficient to induce signaling. We conclude that CD14 is more than a catalyst for LPS binding: it seems to be directly involved in LPS signaling and thus appears to be an essential part of the signaling complex.

A monoclonal antibody recognizing the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) trisaccharide alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo of Chlamydophila psittaci 6BC lipopolysaccharide.

A monoclonal antibody (mAb) S45-18 was generated against a synthetic neoglycoconjugate containing the trisaccharide alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo (Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid) which represents a structure of the lipopolysaccharide (LPS) from Chlamydophila psittaci 6BC. The antibody was characterized by binding and inhibition assays in ELISA using: (i) the immunizing antigen and chemically synthesized derivatives thereof; (ii) chlamydial elementary bodies (EB); and (iii) LPS of Chl. psittaci 6BC and Chlamydia trachomatis L2. The specificity was determined in comparison to that of mAb S25-23 recognizing the alphaKdo(2-->8)alphaKdo(2-->4)alphaKdo trisaccharide which represents an epitope shared by all species of the family. MAb S45-18 bound to an epitope of the structure alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo, with lower reactivity with the (2-->8)-(2-->4)-linked analog. Using chlamydial EB or LPS, mAb S45-18 bound preferentially to LPS and EB of Chl. psittaci. Therefore, Chl. psittaci LPS contains, in addition to the known genus-specific epitope, a species-specific epitope.

Assay of anti-endotoxin antibodies.

Lipopolysaccharides (LPS) constitute components of the outer membrane of Gram-negative bacteria. Chemically, they consist of a heteropolysaccharide and a covalently linked lipid, termed lipid A. The polysaccharide region is made up of the O-specific chain (built from repeating units of three to eight sugars) and the core part, divided into the inner core (the part linked to the lipid) and the outer core (the part linked to the O-specific chain). LPSs possessing an O-specific chain are called smooth LPS (S-LPS), those not having an O-chain are termed rough (R-LPS). The latter type of LPS may be observed in mutants that have lost the ability to synthesize the O-chain, or in wild-type bacteria without known genetic defect. LPS also represent the endotoxin of Gram-negative bacteria. In mammals, including humans, LPS exhibits a variety of biological effects that may be beneficial if administered in low amounts but harmful when present in higher concentrations as in the case of Gram-negative infection and Gram-negative septicemia.

Chemical and antigenic structure of the O-polysaccharide of the lipopolysaccharides from two Acinetobacter haemolyticus strains differing only in the anomeric configuration of one glycosyl residue in their O-antigens.

In a previous study [Pantophlet, R., Brade, L., Dijkshoorn, L., and Brade, H. (1998) J. Clin. Microbiol. 36, 1245-1250] the O-polysaccharide of the lipopolysaccharides (LPS) from Acinetobacter haemolyticus strains 57 and 61 exhibited indistinguishable banding-patterns following Western blot and immunostaining with homologous or heterologous rabbit antiserum. In this report, the molecular basis for the observed cross-reactivity was elucidated, by determining the chemical structure of the polysaccharides by compositional analysis and NMR spectroscopy. The structures are: [sequence: see text] for strain 61 [GulpNAcA, 2-acetamido-2-deoxy-gulopyranosyluronic acid; ManpNAcA, 2-acetamido-2-deoxy-mannopyranosyluronic acid; QuipN4N, 2,4-diamino-2,4,6-trideoxy-glucopyranose; acyl (S)-3-hydroxybutyryl], thus, differing only in the anomeric configuration of the QuipN4N residue. The antigenic structures were determined by generating murine monoclonal antibodies, which were characterized by Western blot using LPS as antigen, by ELISA using LPS and de-O-acylated LPS as solid-phase antigens, and by ELISA inhibition studies using LPS, polysaccharide, and de-O-acylated LPS as inhibitors. Of the four antibodies selected, two were specific for the respective LPS moieties and two were cross-reactive. All antibodies were found to require the presence of the O-acetyl group for reactivity.

NMR experiments reveal distinct antibody-bound conformations of a synthetic disaccharide representing a general structural element of bacterial lipopolysaccharide epitopes.

The recognition reactions between a synthetic disaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl and two monoclonal antibodies (mAbs) were studied by NMR, yielding two distinct bound conformations of the carbohydrate ligand. One mAb, S23-24, recognizes the disaccharides alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl and alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl with similar affinities, whereas mAb S25-2 binds to the disaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl with an approximately 10-fold higher affinity than to the disaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl. Compared to S25-2, S23-24 binds to alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl with an approximately 50-fold increased affinity. We used NMR experiments that are based on the transferred NOE effect, specifically, trNOESY, trROESY, QUIET-trNOESY, and MINSY experiments, to show that the (2-->8)-specific mAb, S25-2, stabilizes a conformation of the alpha-(2-->4)-linked disaccharide that is not highly populated in solution. S23-24 recognizes two conformations of alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl, one that is highly populated in aqueous solution and another conformation that is similar to the one bound by S25-2. This is the first example where it is experimentally shown that a carbohydrate ligand may adopt different bioactive conformations upon interaction with mAbs with different fine specificities. Our NMR studies indicate that a careful examination of spin diffusion is critical for the analysis of bioactive conformations of carbohydrate ligands.