RESUMEN
Conformational flexibility in antibody-combining sites has been hypothesized to facilitate polyspecificity toward multiple unique epitopes and enable the limited germline repertoire to match an overwhelming diversity of potential antigens; however, elucidating the mechanisms of antigen recognition by flexible antibodies has been understandably challenging. Here, multiple liganded and unliganded crystal structures of the near-germline anticarbohydrate antibodies S25-2 and S25-39 are reported, which reveal an unprecedented diversity of complementarity-determining region H3 conformations in apparent equilibrium. These structures demonstrate that at least some germline or near-germline antibodies are flexible entities sensitive to their chemical environments, with conformational selection available as an evolved mechanism that preserves the inherited ability to recognize common pathogens while remaining adaptable to new threats.
Asunto(s)
Anticuerpos , Regiones Determinantes de Complementariedad , Anticuerpos/química , Sitios de Unión de Anticuerpos , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Cristalografía por Rayos X , Células Germinativas , Conformación Molecular , Conformación ProteicaRESUMEN
Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2â8)αKdo(2â4)αKdo (where Kdo is 3-deoxy-α-d- manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26. We determined the high-resolution crystal structures of S25-23 and S25-5 antigen binding fragments in complex with a pentasaccharide derived from the LPS of Chlamydia and measured the affinity of S25-5 for chlamydial LPS antigens using isothermal titration microcalorimetry. The 1.75 Å resolution structure of S25-23 shows how subtle conservative mutations Arg(L)-27E to lysine and Ser(H)-56 to threonine lead to an order of magnitude increase in affinity. Importantly, comparison between previous S25-26 structures and the 1.99 and 2.05 Å resolution liganded and unliganded structures of S25-5, respectively, shows how a Ser(L)-27E mutation results in an intermediate affinity due to the reduced enthalpic penalty associated with complex formation that would otherwise be required for arginine in this position. This strategy allows for subtle adjustments in the combining site via affinity maturation that have dramatic consequences for the affinity of an antibody for its antigen.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/metabolismo , Chlamydiaceae/inmunología , Epítopos/metabolismo , Oligosacáridos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Monoclonales de Origen Murino/inmunología , Afinidad de Anticuerpos , Sitios de Unión de Anticuerpos , Epítopos/inmunología , Enlace de Hidrógeno , Ratones , Oligosacáridos/inmunología , Unión Proteica , Alineación de SecuenciaRESUMEN
Lipopolysaccharide dispersed in the blood by Gram-negative bacteria can be a potent inducer of septic shock. One research focus has been based on antibody sequestration of lipid A (the endotoxic principle of LPS); however, none have been successfully developed into a clinical treatment. Comparison of a panel of anti-lipid A antibodies reveals highly specific antibodies produced through distinct germ line precursors. The structures of antigen-binding fragments for two homologous mAbs specific for lipid A, S55-3 and S55-5, have been determined both in complex with lipid A disaccharide backbone and unliganded. These high resolution structures reveal a conserved positively charged pocket formed within the complementarity determining region H2 loops that binds the terminal phosphates of lipid A. Significantly, this motif occurs in unrelated antibodies where it mediates binding to negatively charged moieties through a range of epitopes, including phosphorylated peptides used in diagnostics and therapeutics. S55-3 and S55-5 have combining sites distinct from anti-lipid A antibodies previously described (as a result of their separate germ line origin), which are nevertheless complementary both in shape and charge to the antigen. S55-3 and S55-5 display similar avidity toward lipid A despite possessing a number of different amino acid residues in their combining sites. Binding of lipid A occurs independent of the acyl chains, although the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains their inability to recognize LPS. Despite their lack of therapeutic potential, the observed motif may have significant immunological implications as a tool for engineering recombinant antibodies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Regiones Determinantes de Complementariedad/inmunología , Epítopos/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Lípido A/inmunología , Lipopolisacáridos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/metabolismo , Cristalografía por Rayos X , Epítopos/química , Epítopos/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Lípido A/química , Lípido A/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Septic shock is a leading cause of death, and it results from an inflammatory cascade triggered by the presence of microbial products in the blood. Certain LPS from Gram-negative bacteria are very potent inducers and are responsible for a high percentage of septic shock cases. Despite decades of research, mAbs specific for lipid A (the endotoxic principle of LPS) have not been successfully developed into a clinical treatment for sepsis. To understand the molecular basis for the observed inability to translate in vitro specificity for lipid A into clinical potential, the structures of antigen-binding fragments of mAbs S1-15 and A6 have been determined both in complex with lipid A carbohydrate backbone and in the unliganded form. The two antibodies have separate germ line origins that generate two markedly different combining-site pockets that are complementary both in shape and charge to the antigen. mAb A6 binds lipid A through both variable light and heavy chain residues, whereas S1-15 utilizes exclusively the variable heavy chain. Both antibodies bind lipid A such that the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains the lack of LPS recognition. Longstanding reports of polyspecificity of anti-lipid A antibodies toward single-stranded DNA combined with observed homology of S1-15 and A6 and the reports of several single-stranded DNA-specific mAbs prompted the determination of the structure of S1-15 in complex with single-stranded DNA fragments, which may provide clues about the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases.
Asunto(s)
Anticuerpos Monoclonales/química , Glicoconjugados/química , Fragmentos Fab de Inmunoglobulinas/química , Lípido A/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Ascitis/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Sitios de Unión de Anticuerpos , ADN de Cadena Simple/química , ADN de Cadena Simple/inmunología , Glicoconjugados/biosíntesis , Glicoconjugados/inmunología , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/inmunología , Lípido A/inmunología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
The structure of the antigen binding fragment of mAb S25-26, determined to 1.95 Å resolution in complex with the Chlamydiaceae family-specific trisaccharide antigen Kdo(2â8)Kdo(2â4)Kdo (Kdo = 3-deoxy-α-d-manno-oct-2-ulopyranosonic acid), displays a germ-line-coded paratope that differs significantly from previously characterized Chlamydiaceae-specific mAbs despite being raised against the identical immunogen. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germ-line residues to recognize the entire trisaccharide antigen and so confer strict specificity. Interest in S25-23 was sparked by its rare high µm affinity and strict specificity for the family-specific trisaccharide antigen; however, only the related antibody S25-26 proved amenable to crystallization. The structures of three unliganded forms of S25-26 have a labile complementary-determining region H3 adjacent to significant glycosylation of the variable heavy chain on asparagine 85 in Framework Region 3. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the few reported structures of glycosylated mAbs containing these epitopes is the therapeutic antibody Cetuximab; however, unlike Cetuximab, one of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three-dimensional structure of an αGal-containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as it has been implicated in allergic response in patients receiving therapeutic antibodies.
Asunto(s)
Sitios de Unión de Anticuerpos , Chlamydia/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Lipopolisacáridos/química , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/inmunología , Afinidad de Anticuerpos , Chlamydia/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Lipopolisacáridos/inmunología , Simulación del Acoplamiento Molecular , Datos de Secuencia MolecularRESUMEN
Escherichia coli infections, a leading cause of septic shock, remain a major threat to human health because of the fatal action to endotoxin (LPS). Therapeutic attempts to neutralize endotoxin currently focus on inhibiting the interaction of the toxic component lipid A with myeloid differentiating factor 2, which forms a trimeric complex together with Toll-like receptor 4 to induce immune cell activation. The 1.73-Å resolution structure of the unique endotoxin-neutralizing protective antibody WN1 222-5 in complex with the core region shows that it recognizes LPS of all E. coli serovars in a manner similar to Toll-like receptor 4, revealing that protection can be achieved by targeting the inner core of LPS and that recognition of lipid A is not required. Such interference with Toll-like receptor complex formation opens new paths for antibody sepsis therapy independent of lipid A antagonists.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos/química , Escherichia coli/metabolismo , Lipopolisacáridos/química , Receptor Toll-Like 4/química , Animales , Complejo Antígeno-Anticuerpo , Carbohidratos/química , Endotoxinas/metabolismo , Infecciones por Escherichia coli/metabolismo , Enlace de Hidrógeno , Ligandos , Lípidos/química , Ratones , Ratones Endogámicos BALB C , Modelos Químicos , Modelos Moleculares , Unión Proteica , Choque Séptico/metabolismoRESUMEN
The near-germline antibody S25-2 exhibits a remarkable cross-reactivity for oligosaccharides containing the bacterial lipopolysaccharide carbohydrate 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The recent synthesis of a variety of Kdo analogues permits a detailed structural analysis of the importance of specific interactions in antigen recognition by S25-2. The Kdo disaccharide analogue Kdo-(2â4)-5,6-dehydro-Kdo lacks a 5-OH group on the second Kdo residue and has been cocrystallized with S25-2. The structure reveals that the modification of the Kdo residue at position 5 results in a rearrangement of intramolecular hydrogen bonds in the antigen that allows it to assume a novel conformation in the antibody-combining site. The cross-reactive binding of S25-2 to this synthetic ligand highlights the adaptability of this antibody to non-natural synthetic analogues.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Azúcares Ácidos/química , Azúcares Ácidos/inmunología , Anticuerpos Monoclonales/metabolismo , Reacciones Antígeno-Anticuerpo , Sitios de Unión de Anticuerpos/inmunología , Conformación de Carbohidratos , Reacciones Cruzadas , Enlace de Hidrógeno , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Ligandos , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Modelos Moleculares , Oligosacáridos/química , Oligosacáridos/inmunología , Conformación Proteica , Azúcares Ácidos/metabolismoRESUMEN
The crystal structures of the antigen-binding fragment of the murine monoclonal antibody (mAb) S25-39 in the presence of several antigens representing chlamydial lipopolysaccharide (LPS) epitopes based on the bacterial sugar 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) have been determined at resolutions from 2.4 to 1.8 Å. The antigen-binding site of this antibody differs from the well-characterized antibody S25-2 by a single mutation away from the germline of asparagine H53 to lysine, yet this one mutation results in a significant increase in avidity across a range of antigens. A comparison of the two antibody structures reveals that the mutated Lys H53 forms additional hydrogen bonds and/or charged-residue interactions with the second Kdo residue of every antigen having two or more carbohydrate residues. Significantly, the NH53K mutation results from a single nucleotide substitution in the germline sequence common among a panel of antibodies raised against glycoconjugates containing carbohydrate epitopes of chlamydial LPS. Like S25-2, S25-39 displays significant induced fit of complementarity determining region (CDR) H3 upon antigen binding, with the unliganded structure possessing a conformation distinct from those reported earlier for S25-2. The four different observed conformations for CDR H3 suggest that this CDR has evolved to exploit the recognition potential of a flexible loop while minimizing the associated entropic penalties of binding by adopting a limited number of ordered conformations in the unliganded state. These observations reveal strategies evolved to balance adaptability and specificity in the germline antibody response to carbohydrate antigens.
Asunto(s)
Sitios de Unión de Anticuerpos/inmunología , Chlamydia/inmunología , Lipopolisacáridos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Antígenos Bacterianos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Epítopos/genética , Epítopos/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Cadenas Pesadas de Inmunoglobulina/genética , RatonesRESUMEN
The structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide has been determined by x-ray diffraction to 2.6 Å resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater affinity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high affinity and specificity independently of CDR H3.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Chlamydia/química , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Complejo Antígeno-Anticuerpo , Conformación de Carbohidratos , Chlamydia/inmunología , Cristalografía por Rayos X , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
Platelet-activating factor (PAF) induces pulmonary edema and has a key role in acute lung injury (ALI). Here we show that PAF induces pulmonary edema through two mechanisms: acid sphingomyelinase (ASM)-dependent production of ceramide, and activation of the cyclooxygenase pathway. Agents that interfere with PAF-induced ceramide synthesis, such as steroids or the xanthogenate D609, attenuate pulmonary edema formation induced by PAF, endotoxin or acid instillation. Our results identify acid sphingomyelinase and ceramide as possible therapeutic targets in acute lung injury.
Asunto(s)
Ceramidas/metabolismo , Factor de Activación Plaquetaria/metabolismo , Edema Pulmonar/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Anticuerpos/metabolismo , Anticuerpos/uso terapéutico , Hidrocarburos Aromáticos con Puentes/metabolismo , Dexametasona/metabolismo , Femenino , Glucocorticoides/metabolismo , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Norbornanos , Inhibidores de Fosfodiesterasa/metabolismo , Edema Pulmonar/tratamiento farmacológico , Ratas , Ratas Wistar , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Tiocarbamatos , Tionas/metabolismoRESUMEN
The structures of antigen-binding fragments from two related monoclonal antibodies have been determined to high resolution in the presence of several carbohydrate antigens raised against chlamydial lipopolysaccharide. With the exception of CDR H3, antibodies S54-10 and S73-2 are both derived from the same set of germline gene segments as the previously reported structures S25-2 and S45-18. Despite this similarity, the antibodies differ in specificity and the mechanism by which they recognize their cognate antigen. S54-10 uses an unrelated CDR H3 to recognize its antigen in a fashion analogous to S45-18; however, S73-2 recognizes the same antigen as S45-18 and S54-10 in a wholly unrelated manner. Together, these antibody-antigen structures provide snapshots into how the immune system uses the same set of inherited germline gene segments to generate multiple possible specificities that allow for differential recognition of epitopes and how unrelated CDR H3 sequences can result in convergent binding of clinically relevant bacterial antigens.
Asunto(s)
Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales/química , Antígenos Bacterianos/química , Chlamydiaceae/inmunología , Epítopos/química , Lipopolisacáridos/inmunología , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Sitios de Unión , Epítopos/inmunología , Lipopolisacáridos/química , Modelos Moleculares , Conformación ProteicaRESUMEN
Chlamydiae contain a rough-type lipopolysaccharide (LPS) of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid residues (Kdo). Two Kdo trisaccharides, 2.8/2.4- and 2.4/2.4-linked, and a branched 2.4[2.8]2.4-linked Kdo tetrasaccharide occur in Chlamydiaceae. While the 2.8/2.4-linked trisaccharide contains a family-specific epitope, the branched Kdo oligosaccharide occurs only in Chlamydophila psittaci and antibodies against it will be useful in human and veterinarian diagnostics. To overcome the generation of cross-reactive antibodies that bind with high affinity to a dominant epitope formed by 2.4/2.4-linked Kdo, we immunized mice with a synthetic 2.4[2.8]-linked branched Kdo trisaccharide and used phage display of scFv to isolate recombinant antibody fragments (NH2240-31 and SAG506-01) that recognize the branched Kdo oligosaccharide with a K(D) of less than 10 nM. Importantly, although these antibodies used germline genes coding for an inherited Kdo recognition site, they were able clearly to distinguish between 2.4[2.8]2.4- and 2.4/2.4-linked Kdo. Sequence determination, binding data, and X-ray structural analysis revealed the basis for the improved discrimination between similar Kdo ligands and indicated that the alteration of a stacking interaction from a phenylalanine residue in the center of the combining site to a tyrosine residue facing away from the center favors recognition of branched 2.4[2.8]2.4-linked Kdo residues. Immunofluorescence tests of infected cell monolayers using this antibody show specific staining of C. psittaci elementary bodies that allow it to be distinguished from other pathogenic chlamydiae.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Carbohidratos/inmunología , Chlamydophila psittaci/inmunología , Lipopolisacáridos/inmunología , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos Antiidiotipos/genética , Carbohidratos/genética , Chlamydiaceae/genética , Chlamydiaceae/inmunología , Chlamydophila psittaci/química , Chlamydophila psittaci/genética , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Lipopolisacáridos/análisis , Lipopolisacáridos/química , Ratones , Oligosacáridos/genética , Oligosacáridos/inmunología , Proteínas Recombinantes/inmunología , Trisacáridos/genética , Trisacáridos/inmunología , Rayos XRESUMEN
In order to explore the structural basis for adaptability in near germline monoclonal antibodies (mAb), we have examined the specificity of the promiscuous mAb S67-27 to both naturally derived carbohydrate antigens and a variety of synthetic nonnatural antigens based on the bacterial lipopolysaccharide component 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (Kdo). One such analog, a 7-O-methyl (7-O-Me) Kdo disaccharide, was found to bind to the antibody with at least 30-fold higher affinity than any other antigen tested. The structure of S67-27 in complex with this analog and three other naturally occurring Kdo antigens revealed that the enhanced affinity of the mAb for the synthetic analog was accomplished by the strategic positioning of CDR H3 away from a conserved Kdo binding pocket that allowed the formation of new antibody-antigen contacts. Furthermore, the comparison of this structure with the structures of related mAbs revealed how the position and structure of CDR H3 influence the specificity or promiscuity of near-germline carbohydrate-recognizing antibodies by altering the architecture of the combining site.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Lipopolisacáridos/inmunología , Azúcares Ácidos/inmunología , Animales , Reacciones Antígeno-Anticuerpo , Antígenos Bacterianos/química , Lipopolisacáridos/síntesis química , Lipopolisacáridos/química , Ratones , Ratones Endogámicos BALB C , Azúcares Ácidos/síntesis química , Azúcares Ácidos/químicaRESUMEN
Chemical analyses, NMR spectroscopy, and mass spectrometry were used to elucidate the structure of the rough lipopolysaccharide (LPS) isolated from Acinetobacter lwoffii F78. As a prominent feature, the core region of this LPS contained the disaccharide alpha-Kdo-(2-->8)-alpha-Kdo (Kdo=3-deoxy-d-D-manno-oct-2-ulopyranosonic acid), which so far has been identified only in chlamydial LPS. In serological investigations, the anti-chlamydial LPS monoclonal antibody S25-2, which is specific for the epitope alpha-Kdo-(2-->8)-alpha-Kdo, reacted with A. lwoffii F78 LPS. Thus, an LPS was identified outside Chlamydiaceae that contains a Chlamydia-specific LPS epitope in its core region.
Asunto(s)
Acinetobacter calcoaceticus/química , Chlamydia/inmunología , Chlamydiaceae/química , Epítopos/inmunología , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Cromatografía en Capa Delgada , Inmunoquímica , Lipopolisacáridos/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Estructura Molecular , Oligosacáridos/química , Espectrometría de Masa por Ionización de Electrospray , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría de Masas en TándemRESUMEN
Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria belongs to the most potent activators of the mammalian immune system. Its lipid moiety, lipid A, the 'endotoxic principle' of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition. The two negative charges of lipid A, represented by the two phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups. We hypothesized that the phosphate groups could be substituted by other negatively charged groups without changing the endotoxic properties of lipid A. To test this hypothesis, we synthesized carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-CM-506) and of tetraacyl lipid A (Bis-CM-406) and correlated their physicochemical with their endotoxic properties. We found that, similarly to compounds 506 and 406, also for their carboxymethyl derivatives a particular molecular ('endotoxic') conformation and with that, a particular aggregate structure is a prerequisite for high cytokine-inducing capacity and antagonistic activity, respectively. In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3-deoxy-D-manno-oct-2-ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed. These data allow a better understanding of endotoxic activity and its structural prerequisites.
Asunto(s)
Lípido A/química , Anticuerpos Monoclonales/metabolismo , Humanos , Lípido A/análogos & derivados , Lípido A/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Lípidos de la Membrana/química , Conformación Molecular , Transición de Fase , Fosfolípidos/química , Relación Estructura-Actividad , Transferasas/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
BACKGROUND: Microarray technology has been used extensively over the past 10 years for assessing gene expression, and has facilitated precise genetic profiling of everything from tumors to small molecule drugs. By contrast, arraying cell membranes in a manner which preserves their ability to mediate biochemical processes has been considerably more difficult. RESULTS: In this article, we describe a novel technology for generating cell membrane microarrays for performing high throughput biology. Our robotically-arrayed supported membranes are physiologically fluid, a critical property which differentiates this technology from other previous membrane systems and makes it useful for studying cellular processes on an industrialized scale. Membrane array elements consist of a solid substrate, above which resides a fluid supported lipid bilayer containing biologically-active molecules of interest. Incorporation of transmembrane proteins into the arrayed membranes enables the study of ligand/receptor binding, as well as interactions with live intact cells. The fluidity of these molecules in the planar lipid bilayer facilitates dimerization and other higher order interactions necessary for biological signaling events. In order to demonstrate the utility of our fluid membrane array technology to ligand/receptor studies, we investigated the multivalent binding of the cholera toxin B-subunit (CTB) to the membrane ganglioside GM1. We have also displayed a number of bona fide drug targets, including bacterial endotoxin (also referred to as lipopolysaccharide (LPS)) and membrane proteins important in T cell activation. CONCLUSION: We have demonstrated the applicability of our fluid cell membrane array technology to both academic research applications and industrial drug discovery. Our technology facilitates the study of ligand/receptor interactions and cell-cell signaling, providing rich qualitative and quantitative information.
Asunto(s)
Biotecnología/métodos , Membrana Celular/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis por Matrices de Proteínas/métodos , Animales , Sitios de Unión , Diferenciación Celular , Toxina del Cólera/química , Dimerización , Diseño de Fármacos , Industria Farmacéutica , Gangliósido G(M1)/química , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Ligandos , Membrana Dobles de Lípidos/química , Lipopolisacáridos/metabolismo , Membranas/metabolismo , Ratones , Ratones Transgénicos , Unión Proteica , Mapeo de Interacción de Proteínas , Robótica , Salmonella enterica/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Tecnología FarmacéuticaRESUMEN
Monoclonal antibodies (mAbs) were obtained after immunization of mice with neoglycoconjugates containing as a ligand the disaccharide Kdo(2-->4)Ko or Ko(2-->4)Kdo, representing structural elements of the core region of lipopolysaccharides (LPSs) from Acinetobacter haemolyticus and Burkholderia cepacia, respectively. One antibody, S67-9, bound with high specificity to Ko(2-->4)Kdo-BSA showing no cross reactivity with Kdo(2-->4)Kdo-BSA and the other antigens tested. A second mAb, S68-12, bound preferentially to Kdo(2-->4)Ko-BSA but cross reacted with Ko(2-->4)Kdo-BSA and Kdo(2-->4)Kdo-BSA. A third mAb, S67-27, was found to bind Kdo monosaccharide. Although mAbs S67-9 and S68-12 did not bind to LPS of Burkholderia or Acinetobacter as expected, the mAbs will be useful tools in studying the biosynthesis of LPS containing Ko.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Oligosacáridos/química , Oligosacáridos/inmunología , Animales , Burkholderia cepacia/química , Secuencia de Carbohidratos , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB CRESUMEN
We report a novel strategy for the preparation of neoglycoconjugates of oligosaccharides which are obtained after complete deacylation of bacterial deep rough lipopolysaccharides (LPS) isolated from recombinant Escherichia coli bacteria synthesizing a Kdo di-[alpha-Kdo-(2-->4)-alpha-Kdo-(2-->] and a Kdo trisaccharide [alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->] of Re-type and chlamydial LPS, respectively. Unlike acylated LPS, such oligosaccharides can be obtained in pure form and thus lead to well-defined neoglycoconjugates. Cleavage of the 1-phosphate of the lipid A moiety by alkaline phosphatase treatment leads to a free reducing glucosamine which can be further reacted with allylamine. After reductive amination, spacer elongation of the allyl group with cysteamine and activation with thiophosgene, the ligands were reacted with BSA. We have compared the immunological reactivity of such defined neoglycoconjugates obtained from natural sources with those obtained by chemical synthesis and report that such neoglycoconjugates are immunogenic and well suited as antigens for the study of epitope specificities of monoclonal antibodies. In addition, we have compared these conjugates with those in which ligands were coupled by glutardialdehyde to BSA. Our approach proved to be superior since the latter led upon immunization of mice to a relatively high percentage of antibodies that reacted with glutardialdehyde derivatized BSA without the carbohydrate ligand. This was not the case for cysteamine-spacered ligands coupled via their isothiocyanate-derivatives.
Asunto(s)
Escherichia coli/inmunología , Glicoproteínas/síntesis química , Lipopolisacáridos/química , Oligosacáridos/química , Acetilación , Conformación de Carbohidratos , Secuencia de Carbohidratos , Escherichia coli/genética , Glicoproteínas/química , Glicoproteínas/inmunología , Lipopolisacáridos/inmunología , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Oligosacáridos/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Extraction of dry bacteria of Acinetobacter baumannii strain 24 by phenol-water yielded a lipopolysaccharide (LPS) that was studied by serological methods and fatty acid analysis. After immunisation of BALB/c mice with this strain, monoclonal antibody S48-3-13 (IgG(3) isotype) was obtained, which reacted with the LPS in western blot and characterized it as S-form LPS. Degradation of the LPS in aqueous 1% acetic acid followed by GPC gave the O-antigenic polysaccharide, whose structure was determined by compositional analyses and NMR spectroscopy of the polysaccharide and O-deacylated polysaccharide as [carbohydrate structure: see text] where QuiN4N is 2,4-diamino-2,4,6-trideoxyglucose and GalNAcA 2-acetamido-2-deoxygalacturonic acid. The amino group at C-4 of the QuipN4N residues is acetylated in about 2/3 of LPS molecules and (S)-3-hydroxybutyrylated in the rest.
Asunto(s)
Acinetobacter baumannii/química , Lipopolisacáridos/química , Antígenos O/química , Polisacáridos Bacterianos/química , Animales , Anticuerpos Monoclonales/química , Secuencia de Carbohidratos , Electroforesis en Gel de Poliacrilamida , Ácidos Grasos/química , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Oligosacáridos/química , Fenol/química , Agua/químicaRESUMEN
The immune response of cows against the core oligosaccharide of Escherichia coli rough mutants (core types R1-R4, K-12 and J-5) was investigated after immunization with a synthetic glycoconjugate composed of deacylated LPS conjugated to hemocyanine (22 animals). Ab formation was measured by ELISA using LPS or deacylated LPS conjugated to BSA as an Ag. The glycoconjugate immunogens were used to vaccinate cows (36 animals), which were then challenged intramammarily with E. coli O 157 (K1 negative, R1 core type). Compared with control groups no protection was observed, although high titers against the R1 core type were detected in vaccinated animals. Western blots using the immune sera showed that the Ab response was directed against the core region and not against the O-antigen, which may explain the failure of the vaccine.