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
Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2ß2γ2). Upon proteolytic cleavage by site-1 protease, the α/ß-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/ß-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.
Assuntos
Enzimas/metabolismo , Lisossomos/metabolismo , Mucolipidoses/metabolismo , Mucolipidoses/patologia , Proteoma/metabolismo , Animais , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Marcação por Isótopo , Manosefosfatos/metabolismo , Camundongos Knockout , Subunidades Proteicas/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
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
The process of natural selection favours germ-line gene segments that encode CDRs that have the ability to recognize a range of structurally related antigens. This presents an immunological advantage to the host, as it can confer protection against a common pathogen and still cope with new or changing antigens. Cross-reactive and polyspecific antibodies also play a central role in autoimmune responses, and a link has been shown to exist between auto-reactive B cells and certain bacterial infections. Bacterial DNA, lipids, and carbohydrates have been implicated in the progression of autoimmune diseases such as systemic lupus erythematosus. As well, reports of anti-lipid A antibody polyspecificity towards single-stranded DNA together with the observed sequence homology amongst isolated auto- and anti-lipid A antibodies has prompted further study of this phenomenon. Though the lipid A epitope appears cryptic during Gram-negative bacterial infection, there have been several reported instances of lipid A-specific antibodies isolated from human sera, some of which have exhibited polyspecificity for single stranded DNA. In such cases, the breakdown of negative selection through polyspecificity can have the unfortunate consequence of autoimmune disease. This review summarizes current knowledge regarding such antibodies and emphasizes the features of S1-15, A6, and S55-5, anti-lipid A antibodies whose structures were recently determined by X-ray crystallography.
Assuntos
Especificidade de Anticorpos , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Autoimunidade , Infecções Bacterianas/imunologia , Lipídeo A/imunologia , Animais , Autoanticorpos/química , Doenças Autoimunes/microbiologia , Linfócitos B/imunologia , Infecções Bacterianas/microbiologia , DNA de Cadeia Simples/imunologia , Humanos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
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 acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 â 2)αMan(1 â 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH â¼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition.
Assuntos
Manosefosfatos/química , Anticorpos de Cadeia Única/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Camundongos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Anticorpos de Cadeia Única/metabolismoRESUMO
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
Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant ß-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The ß-glucuronidase produced in parallel bioreactors was quantified by capillary electrophoresis, the catalytic kinetic parameters were measured using steady-state analysis, and mannose-6-phosphorylation status was assessed using an M6P-specific single-chain antibody fragment. Using this approach, we found that butyrate treatment increased ß-glucuronidase production up to approximately threefold without significantly affecting the catalytic properties of the enzyme. However, M6P content in ß-glucuronidase was inversely correlated with the increased enzyme production induced by butyrate treatment. This assessment demonstrated that although butyrate dramatically increased ß-glucuronidase production in bioreactors, it adversely impacted the mannose-6-phosphorylation of this LSD therapeutic enzyme. This strategy may have utility in evaluating manufacturing process changes to improve therapeutic enzyme yields and CQAs.
Assuntos
Reatores Biológicos , Butiratos/farmacologia , Glucuronidase/biossíntese , Doenças por Armazenamento dos Lisossomos/enzimologia , Animais , Butiratos/química , Células CHO , Cricetinae , Cricetulus , Glucuronidase/uso terapêutico , Humanos , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/patologia , Manosefosfatos/química , Manosefosfatos/farmacologia , Fosforilação , Polissacarídeos/químicaRESUMO
More than two million people worldwide are affected by life-threatening, invasive fungal infections annually. Candida species are the most common cause of nosocomial, invasive fungal infections and are associated with mortality rates above 40%. Despite the increasing incidence of drug-resistance, the development of novel antifungal formulations has been limited. Here we investigate the antifungal mode of action and therapeutic potential of positively charged, synthetic peptide mimics to combat Candida albicans infections. Our data indicates that these synthetic polymers cause endoplasmic reticulum stress and affect protein glycosylation, a mode of action distinct from currently approved antifungal drugs. The most promising polymer composition damaged the mannan layer of the cell wall, with additional membrane-disrupting activity. The synergistic combination of the polymer with caspofungin prevented infection of human epithelial cells in vitro, improved fungal clearance by human macrophages, and significantly increased host survival in a Galleria mellonella model of systemic candidiasis. Additionally, prolonged exposure of C. albicans to the synergistic combination of polymer and caspofungin did not lead to the evolution of tolerant strains in vitro. Together, this work highlights the enormous potential of these synthetic peptide mimics to be used as novel antifungal formulations as well as adjunctive antifungal therapy.
Assuntos
Antifúngicos , Candida albicans , Candidíase , Caspofungina , Sinergismo Farmacológico , Peptídeos , Candida albicans/efeitos dos fármacos , Antifúngicos/farmacologia , Humanos , Caspofungina/farmacologia , Animais , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Peptídeos/farmacologia , Peptídeos/química , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mananas/farmacologia , Mananas/química , Mariposas/microbiologia , Mariposas/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Polímeros/farmacologia , Polímeros/químicaRESUMO
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
BACKGROUND: Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test. METHODS: Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types. RESULTS: We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 102 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts. CONCLUSIONS: Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.
Assuntos
Mycobacterium tuberculosis , Tuberculose dos Linfonodos , Tuberculose Pulmonar , Criança , Humanos , Fenômenos Magnéticos , Mycobacterium tuberculosis/genética , Saliva , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologiaRESUMO
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
GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, alpha(2)beta(2)gamma(2), that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3-10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the gamma-subunit but not the beta-subunit was found to be proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the gamma-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the gamma-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.
Assuntos
Lisossomos/enzimologia , Macrófagos/enzimologia , Manosefosfatos/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Animais , Transporte Biológico , Células COS , Catepsina D/química , Chlorocebus aethiops , Cromatografia/métodos , Células HeLa , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Modelos Biológicos , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transferases (Outros Grupos de Fosfato Substituídos)/fisiologiaRESUMO
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
Newly synthesized soluble lysosomal hydrolases require mannose 6-phosphate (Man6P) residues on their oligosaccharides for their transport to lysosomes. The formation of Man6P residues is catalyzed by the GlcNAc-1-phosphotransferase, which is defective in the lysosomal storage disorders mucolipidosis type II (ML II) and ML III. Both hypersecretion and reduced intracellular level of lysosomal enzymes as well as direct sequencing of GlcNAc-1-phosphotransferase genes are important diagnostic markers for ML II and ML III. A high-affinity Man6P-specific single-chain antibody fragment was generated, allowing the rapid indirect demonstration of defective GlcNAc-1-phosphotransferase. In media and extracts of cultured fibroblasts of healthy controls but not of ML II and ML III patients, several Man6P-containing proteins could be detected by anti-Man6P Western blotting. Immunoprecipitation of Man6P-containing proteins from conditioned media or mouse brain extracts followed by arylsulfatase A and cathepsin D Western blotting confirmed the specificity of the antibody fragment for lysosomal proteins. Application of the antibody fragment in immunohistochemistry of human brain slices from nonaffected patients showed strong neuronal immunoreactivity, which was not observed in cortical sections of an ML II patient. Finally, in brain extracts of a novel GlcNAc-1-phosphotransferase knock-in mouse no Man6P-containing proteins were detectable. Thus, the single-chain antibody fragment against Man6P was demonstrated to allow the specific, rapid, and convenient detection of Man6P-containing proteins and facilitates the diagnosis of ML II and ML III.
Assuntos
Fragmentos de Imunoglobulinas/metabolismo , Manosefosfatos/metabolismo , Mucolipidoses/diagnóstico , Proteínas/química , Anticorpos de Cadeia Única/metabolismo , Animais , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Técnicas de Introdução de Genes , Humanos , Fragmentos de Imunoglobulinas/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Mucolipidoses/enzimologia , Anticorpos de Cadeia Única/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/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.