RESUMO
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.
Assuntos
Anticorpos , Regiões Determinantes de Complementaridade , Anticorpos/química , Sítios de Ligação de Anticorpos , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Cristalografia por Raios X , Células Germinativas , Conformação Molecular , Conformação ProteicaRESUMO
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.
Assuntos
Anticorpos Monoclonais Murinos/metabolismo , Chlamydiaceae/imunologia , Epitopos/metabolismo , Oligossacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Afinidade de Anticorpos , Sítios de Ligação de Anticorpos , Epitopos/imunologia , Ligação de Hidrogênio , Camundongos , Oligossacarídeos/imunologia , Ligação Proteica , Alinhamento de SequênciaRESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Regiões Determinantes de Complementaridade/imunologia , Epitopos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Lipídeo A/imunologia , Lipopolissacarídeos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/metabolismo , Cristalografia por Raios X , Epitopos/química , Epitopos/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Anticorpos Monoclonais/química , Glicoconjugados/química , Fragmentos Fab das Imunoglobulinas/química , Lipídeo A/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Ascite/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Sítios de Ligação de Anticorpos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/imunologia , Glicoconjugados/biossíntese , Glicoconjugados/imunologia , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/imunologia , Lipídeo A/imunologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
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.
Assuntos
Sítios de Ligação de Anticorpos , Chlamydia/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Lipopolissacarídeos/química , Sequência de Aminoácidos , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Afinidade de Anticorpos , Chlamydia/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Lipopolissacarídeos/imunologia , Simulação de Acoplamento Molecular , Dados de Sequência MolecularRESUMO
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.
Assuntos
Anticorpos Monoclonais/química , Anticorpos/química , Escherichia coli/metabolismo , Lipopolissacarídeos/química , Receptor 4 Toll-Like/química , Animais , Complexo Antígeno-Anticorpo , Carboidratos/química , Endotoxinas/metabolismo , Infecções por Escherichia coli/metabolismo , Ligação de Hidrogênio , Ligantes , Lipídeos/química , Camundongos , Camundongos Endogâmicos BALB C , Modelos Químicos , Modelos Moleculares , Ligação Proteica , Choque Séptico/metabolismoRESUMO
It is well established that lipopolysaccharide (LPS) often carries nonstoichiometric substitutions in lipid A and in the inner core. In this work, the molecular basis of inner core alterations and their physiological significance are addressed. A new inner core modification of LPS is described, which arises due to the addition of glucuronic acid on the third heptose with a concomitant loss of phosphate on the second heptose. This was shown by chemical and structural analyses. Furthermore, the gene whose product is responsible for the addition of this sugar was identified in all Escherichia coli core types and in Salmonella and was designated waaH. Its deduced amino acid sequence exhibits homology to glycosyltransferase family 2. The transcription of the waaH gene is positively regulated by the PhoB/R two-component system in a growth phase-dependent manner, which is coordinated with the transcription of the ugd gene explaining the genetic basis of this modification. Glucuronic acid modification was observed in E. coli B, K12, R2, and R4 core types and in Salmonella. We also show that the phosphoethanolamine (P-EtN) addition on heptose I in E. coli K12 requires the product of the ORF yijP, a new gene designated as eptC. Incorporation of P-EtN is also positively regulated by PhoB/R, although it can occur at a basal level without a requirement for any regulatory inducible systems. This P-EtN modification is essential for resistance to a variety of factors, which destabilize the outer membrane like the addition of SDS or challenge to sublethal concentrations of Zn(2+).
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Etanolaminas/metabolismo , Ácido Glucurônico/metabolismo , Heptoses/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/genética , Configuração de Carboidratos , Sequência de Carboidratos , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Glicosiltransferases/genética , Lipopolissacarídeos/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Anotação de Sequência Molecular , Dados de Sequência Molecular , Transcrição GênicaRESUMO
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.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Açúcares Ácidos/química , Açúcares Ácidos/imunologia , Anticorpos Monoclonais/metabolismo , Reações Antígeno-Anticorpo , Sítios de Ligação de Anticorpos/imunologia , Configuração de Carboidratos , Reações Cruzadas , Ligação de Hidrogênio , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Ligantes , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Modelos Moleculares , Oligossacarídeos/química , Oligossacarídeos/imunologia , Conformação Proteica , Açúcares Ácidos/metabolismoRESUMO
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.
Assuntos
Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Ramnose/química , Western Blotting , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Lipopolissacarídeos/química , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Pequeno RNA não Traduzido/genética , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Sítios de Ligação de Anticorpos/imunologia , Chlamydia/imunologia , Lipopolissacarídeos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Antígenos de Bactérias/imunologia , Regiões Determinantes de Complementaridade/imunologia , Epitopos/genética , Epitopos/imunologia , Fragmentos Fab das Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , CamundongosRESUMO
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.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Chlamydia/química , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo , Configuração de Carboidratos , Chlamydia/imunologia , Cristalografia por Raios X , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência MolecularRESUMO
Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease are Escherichia coli and Staphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection. Here, we report the O-antigen (OPS, O-specific polysaccharide) structure of the LPS from bovine mastitis isolate E. coli 1303. The structure was determined utilizing chemical analyses, mass spectrometry, and 1D and 2D NMR spectroscopy methods. The O-repeating unit was characterized as -[â4)-ß-D-Quip3NAc-(1â3)-α-L-Fucp2OAc-(1â4)-ß-D-Galp-(1â3)-α-D-GalpNAc-(1â]- in which the O-acetyl substitution was non-stoichiometric. The nucleotide sequence of the O-antigen gene cluster of E. coli 1303 was also determined. This cluster, located between the gnd and galF genes, contains 13 putative open reading frames, most of which represent unknown nucleotide sequences that have not been described before. The O-antigen of E. coli 1303 was shown to substitute O-7 of the terminal LD-heptose of the K-12 core oligosaccharide. Interestingly, the non-OPS-substituted core oligosaccharide represented a truncated version of the K-12 outer core - namely terminal LD-heptose and glucose were missing; however, it possessed a third Kdo residue in the inner core. On the basis of structural and genetic data we show that the mastitis isolate E. coli 1303 represents a new serotype and possesses the K-12 core type, which is rather uncommon among human and bovine isolates.
Assuntos
Escherichia coli/química , Lipopolissacarídeos/química , Mastite Bovina/microbiologia , Antígenos O/química , Animais , Sequência de Bases , Sequência de Carboidratos , Bovinos , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Feminino , Humanos , Lipopolissacarídeos/genética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Família Multigênica , Antígenos O/genética , Análise de Sequência de DNARESUMO
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.
Assuntos
Ceramidas/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Edema Pulmonar/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Anticorpos/metabolismo , Anticorpos/uso terapêutico , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Dexametasona/metabolismo , Feminino , Glucocorticoides/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Norbornanos , Inibidores de Fosfodiesterase/metabolismo , Edema Pulmonar/tratamento farmacológico , Ratos , Ratos Wistar , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Tiocarbamatos , Tionas/metabolismoRESUMO
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.
Assuntos
Anticorpos Antibacterianos/química , Anticorpos Monoclonais/química , Antígenos de Bactérias/química , Chlamydiaceae/imunologia , Epitopos/química , Lipopolissacarídeos/imunologia , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Sítios de Ligação , Epitopos/imunologia , Lipopolissacarídeos/química , Modelos Moleculares , Conformação ProteicaRESUMO
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.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Carboidratos/imunologia , Chlamydophila psittaci/imunologia , Lipopolissacarídeos/imunologia , Animais , Anticorpos/genética , Anticorpos/imunologia , Anticorpos Anti-Idiotípicos/genética , Carboidratos/genética , Chlamydiaceae/genética , Chlamydiaceae/imunologia , Chlamydophila psittaci/química , Chlamydophila psittaci/genética , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Imunofluorescência , Humanos , Lipopolissacarídeos/análise , Lipopolissacarídeos/química , Camundongos , Oligossacarídeos/genética , Oligossacarídeos/imunologia , Proteínas Recombinantes/imunologia , Trissacarídeos/genética , Trissacarídeos/imunologia , Raios XRESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Regiões Determinantes de Complementaridade/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Lipopolissacarídeos/imunologia , Açúcares Ácidos/imunologia , Animais , Reações Antígeno-Anticorpo , Antígenos de Bactérias/química , Lipopolissacarídeos/síntese química , Lipopolissacarídeos/química , Camundongos , Camundongos Endogâmicos BALB C , Açúcares Ácidos/síntese química , Açúcares Ácidos/químicaRESUMO
Recent findings have implicated interleukin-1beta (IL-1beta) as an important mediator of the inflammatory response in the female genital tract during chlamydial infection. But how IL-1beta is produced and its specific role in infection and pathology are unclear. Therefore, our goal was to determine the functional consequences and cellular sources of IL-1beta expression during a chlamydial genital infection. In the present study, IL-1beta(-/-) mice exhibited delayed chlamydial clearance and decreased frequency of hydrosalpinx compared to wild-type (WT) mice, implying an important role for IL-1beta both in the clearance of infection and in the mediation of oviduct pathology. At the peak of IL-1beta secretion in WT mice, the major producers of IL-1beta in vivo are F4/80(+) macrophages and GR-1(+) neutrophils, but not CD45(-) epithelial cells. Although elicited mouse macrophages infected with Chlamydia muridarum in vitro secrete minimal IL-1beta, in vitro prestimulation of macrophages by Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) purified from Escherichia coli or C. trachomatis L2 prior to infection greatly enhanced secretion of IL-1beta from these cells. By using LPS-primed macrophages as a model system, it was determined that IL-1beta secretion was dependent on caspase-1, potassium efflux, and the activity of serine proteases. Significantly, chlamydia-induced IL-1beta secretion in macrophages required bacterial viability but not growth. Our findings demonstrate that IL-1beta secreted by macrophages and neutrophils has important effects in vivo during chlamydial infection. Additionally, prestimulation of macrophages by chlamydial TLR ligands may account for the elevated levels of pro-IL-1beta mRNA observed in vivo in this cell type.
Assuntos
Infecções por Chlamydia/imunologia , Chlamydia muridarum/imunologia , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Animais , Infecções por Chlamydia/microbiologia , Contagem de Colônia Microbiana , Tubas Uterinas/patologia , Feminino , Células HeLa , Humanos , Interleucina-1beta/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologiaRESUMO
A difference of seven conservative amino-acid substitutions between two single-chain antibodies (scFvs) specific for chlamydial lipopolysaccharide does not significantly affect their molecular structures or packing contacts, but dramatically affects their crystallization. The structure of the variable domain (Fv) of SAG173-04 was solved to 1.86 A resolution and an R(cryst) of 18.9% in space group P2(1)2(1)2(1). Crystals of the homologous SAG506-01 diffracted to 1.95 A resolution and appeared at first to have Patterson symmetry I4/m or P4/mmm; however, no solution could be found in space groups belonging to the former and refinement in the only solution corresponding to the latter (in space group P4(3)2(1)2) stalled at R(free) = 30.0%. Detailed examination of the diffraction data revealed that the crystal was likely to be twinned and that the correct space group was P2(1)2(1)2(1). Both translational pseudo-symmetry and pseudo-merohedral twinning were observed in one crystal of SAG506-01 and pseudo-merohedral twinning was observed for a second crystal. The final R factor for SAG506-01 after refinement in P2(1)2(1)2(1) was 20.5%.
Assuntos
Chlamydiaceae , Regiões Determinantes de Complementaridade/química , Lipopolissacarídeos/química , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Clonagem Molecular , Regiões Determinantes de Complementaridade/imunologia , Cristalização , Epitopos , Lipopolissacarídeos/imunologia , Dados de Sequência Molecular , Mutação , Biblioteca de Peptídeos , Estrutura Terciária de Proteína , Especificidade da Espécie , Homologia Estrutural de Proteína , Relação Estrutura-AtividadeRESUMO
The neuronal ceroid lipofuscinoses comprise a group of inherited severe neurodegenerative lysosomal disorders characterized by lysosomal dysfunction and massive accumulation of fluorescent lipopigments and aggregated proteins. To examine the role of lipids in neurodegenerative processes of these diseases, we analysed phospho- and glycolipids in the brains of ctsd-/- and nclf mice, disease models of cathepsin D and CLN6 deficiency, respectively. Both ctsd-/- and nclf mice exhibited increased levels of GM2 and GM3 gangliosides. Immunohistochemically GM2 and GM3 staining was found preferentially in neurons and glial cells, respectively, of ctsd-/- mice. Of particular note, a 20-fold elevation of the unusual lysophospholipid bis(monoacylglycero)phosphate was specifically detected in the brain of ctsd-/- mice accompanied with sporadic accumulation of unesterified cholesterol in distinct cells. The impaired processing of the sphingolipid activator protein precursor, an in vitro cathepsin D substrate, in the brain of ctsd-/- mice may provide the mechanistic link to the storage of lipids. These studies show for the first time that cathepsin D regulates the lysosomal phospho- and glycosphingolipid metabolism suggesting that defects in the composition, trafficking and/or recycling of membrane components along the late endocytic pathway may be critical for the pathogenesis of early onset neuronal ceroid lipofuscinoses.
Assuntos
Modelos Animais de Doenças , Gangliosídeos/metabolismo , Lisofosfolipídeos/metabolismo , Monoglicerídeos/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Animais , Catepsina D/deficiência , Catepsina D/genética , Células Cultivadas , Gangliosídeos/genética , Hipocampo/química , Hipocampo/metabolismo , Hipocampo/patologia , Membranas Intracelulares/química , Membranas Intracelulares/patologia , Lipídeos/biossíntese , Lipídeos/fisiologia , Lisofosfolipídeos/fisiologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoglicerídeos/fisiologia , Lipofuscinoses Ceroides Neuronais/etiologia , Lipofuscinoses Ceroides Neuronais/patologia , Neurônios/química , Neurônios/metabolismo , Neurônios/patologiaRESUMO
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.