RESUMEN
Split inactivated influenza vaccines remain one of the primary preventative strategies against severe influenza disease in the population. However, current vaccines are only effective against a limited number of matched strains. The need for broadly protective vaccines is acute due to the high mutational rate of influenza viruses and multiple strain variants in circulation at any one time. The neuraminidase (NA) glycoprotein expressed on the influenza virion surface has recently regained recognition as a valuable vaccine candidate. We sought to broaden the protection provided by NA within the N1 subtype by computationally engineering consensus NA sequences. Three NA antigens (NA5200, NA7900, NA9100) were designed based on sequence clusters encompassing three major groupings of NA sequence space; (i) H1N1 2009 pandemic and Swine H1N1, (ii) historical seasonal H1N1 and (iii) H1N1 viruses ranging from 1933 till current times. Recombinant NA proteins were produced as a vaccine and used in a mouse challenge model. The design of the protein dictated the protection provided against the challenge strains. NA5200 protected against H1N1 pdm09, a Swine isolate from 1998 and NIBRG-14 (H5N1). NA7900 protected against all seasonal H1N1 viruses tested, and NA9100 showed the broadest range of protection covering all N1 viruses tested. By passive transfer studies and serological assays, the protection provided by the cluster-based consensus (CBC) designs correlated to antibodies capable of mediating NA inhibition. Importantly, sera raised to the consensus NAs displayed a broader pattern of reactivity and protection than naturally occurring NAs, potentially supporting a predictive approach to antigen design.
RESUMEN
AIMS: The aim of this study was to assess whether surgical decompression of nerves in the lower extremity in people with painful diabetic polyneuropathy would have an effect on health-related quality of life and to determine minimal clinically important differences in pain and quality of life scores. METHODS: The design was a randomized controlled trial in which 42 participants with painful diabetic painful neuroapthy underwent unilateral decompression of nerves in their left or right leg, using the other leg as a control, with 12 months follow-up. Surgical decompression was performed at the tibial, superficial, deep and common peroneal nerves. Preoperatively, and at 6 and 12 months post operatively, a visual analogue scale for pain and the 36 item short-form health survey and EuroQual 5 Dimensions questionnaires were completed. RESULTS: At 12 months follow-up, the visual analogue scale was significantly reduced, but decompression surgery did not significantly alter health-related quality of life scores. The minimal clinically important difference for visual analogue scale reduction was determined at 2.9 points decrease, a threshold reached by 42.5% of the study population. CONCLUSIONS: Although decompression surgery does not influence health-related quality of life, it achieves a clinically relevant reduction of pain in ~42.5% of people with diabetic peripheral neuropathy. It can therefore be considered for patients who do not adequately respond to pain medication.
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Descompresión Quirúrgica , Neuropatías Diabéticas/cirugía , Extremidad Inferior/inervación , Extremidad Inferior/cirugía , Percepción del Dolor , Calidad de Vida , Adulto , Anciano , Descompresión Quirúrgica/psicología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Dolor/etiología , Dolor/cirugía , Dimensión del Dolor , Percepción del Dolor/fisiologíaRESUMEN
AIMS: Production of the recombinant Arabidopsis halleri defensin AhPDF1.1 in a native-like form. METHODS AND RESULTS: Mature AhPDF1.1 cDNA was cloned into pET-28-a(+) and expressed in Escherichia coli Rosetta. After a denaturing extraction, purification by metal affinity chromatography and CNBr cleavage of the His-tag, a protein without extra amino acids at the N-terminus was obtained. An oxidative folding step was then required to renature the protein that was then purified to homogeneity by a C18 HPLC separation. Mass spectroscopy and circular dichroism analyses showed that the recombinant AhPDF1.1 has the expected molecular mass and 3D-structure features of a folded defensin with four-disulfide bridges. The recombinant protein is active against the filamentous fungus Fusarium oxysporum with a minimal inhibitory concentration of 0.6 micromol l(-1). CONCLUSION: The proposed purification protocol produces a native-like defensin suitable for tests of new biological roles. SIGNIFICANCE AND IMPACT OF THE STUDY: Plant defensins are essentially known as anti-fungal proteins; however, some unexpected actions on plant cells have recently been discovered. AhPDF1.1, for example, has been shown to confer zinc tolerance. Efficient production of native-like defensins is required to explore the different targets and roles of plant defensins.
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Arabidopsis/metabolismo , Defensinas/metabolismo , Escherichia coli/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Secuencia de Bases , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Defensinas/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Pliegue de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
It is generally believed that asymmetric competition for light plays a predominant role in determining the course of succession by increasing size inequalities between plants. Size-related growth is the product of size-related light capture and light-use efficiency (LUE). We have used a canopy model to calculate light capture and photosynthetic rates of pioneer species in sequential vegetation stages of a young secondary forest stand. Growth of the same saplings was followed in time as succession proceeded. Photosynthetic rate per unit plant mass (P(mass): mol C g(-1) day(-1)), a proxy for plant growth, was calculated as the product of light capture efficiency [Phi(mass): mol photosynthetic photon flux density (PPFD) g(-1) day(-1)] and LUE (mol C mol PPFD(-1)). Species showed different morphologies and photosynthetic characteristics, but their light-capturing and light-use efficiencies, and thus P (mass), did not differ much. This was also observed in the field: plant growth was not size-asymmetric. The size hierarchy that was present from the very early beginning of succession remained for at least the first 5 years. We conclude, therefore, that in slow-growing regenerating vegetation stands, the importance of asymmetric competition for light and growth can be much less than is often assumed.
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Luz , Fotosíntesis , Árboles/crecimiento & desarrollo , Biomasa , Euphorbiaceae/anatomía & histología , Euphorbiaceae/crecimiento & desarrollo , Euphorbiaceae/metabolismo , Helechos/anatomía & histología , Helechos/crecimiento & desarrollo , Helechos/metabolismo , Mallotus (Planta)/anatomía & histología , Mallotus (Planta)/crecimiento & desarrollo , Mallotus (Planta)/metabolismo , Melastomataceae/anatomía & histología , Melastomataceae/crecimiento & desarrollo , Melastomataceae/metabolismo , Poaceae/anatomía & histología , Poaceae/crecimiento & desarrollo , Poaceae/metabolismo , Especificidad de la Especie , Árboles/anatomía & histología , Árboles/metabolismo , VietnamRESUMEN
BACKGROUND AND AIMS: Crown structure and above-ground biomass investment was studied in relation to light interception of trees and lianas growing in a 6-month-old regenerating forest. METHODS: The vertical distribution of total above-ground biomass, height, diameter, stem density, leaf angles and crown depth were measured for individual plants of three short-lived pioneers (SLPs), four long-lived pioneers (LLPs) and three lianas. Daily light interception per individual Phi(d) was calculated with a canopy model. The model was then used to estimate light interception per unit of leaf mass (Phi(leaf mass)), total above-ground mass (Phi(mass)) and crown structure efficiency (E(a), the ratio of absorbed vs. available light). KEY RESULTS: The SLPs Trema and Ochroma intercepted higher amounts of light per unit leaf mass (Phi(leaf mass)) because they had shallower crowns, resulting in higher crown use efficiency (E(a)) than the other species. These SLPs (but not Cecropia) were also taller and intercepted more light per unit leaf area (Phi(area)). LLPs and lianas had considerably higher amounts of leaf mass and area per unit above-ground mass (LMR and LAR, respectively) and thus attained Phi(mass) values similar to the SLPs (Phi(mass)=Phi(area)xLAR). Lianas, which were mostly self-supporting, had light interception efficiencies similar to those of the trees. CONCLUSIONS: These results show how, due to the trade-off between crown structure and biomass allocation, SLPs, and LLPs and lianas intercept similar amount of light per unit mass which may contribute to the ability of the latter two groups to persist.
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Ecosistema , Luz , Árboles/crecimiento & desarrollo , Clima Tropical , Biomasa , Modelos Biológicos , Desarrollo de la Planta , Plantas/anatomía & histología , Árboles/anatomía & histologíaRESUMEN
BACKGROUND: The bioinformatic prediction of protein subcellular localization has been extensively studied for prokaryotic and eukaryotic organisms. However, this is not the case for viruses whose proteins are often involved in extensive interactions at various subcellular localizations with host proteins. RESULTS: Here, we investigate the extent of utilization of human cellular localization mechanisms by viral proteins and we demonstrate that appropriate eukaryotic subcellular localization predictors can be used to predict viral protein localization within the host cell. CONCLUSION: Such predictions provide a method to rapidly annotate viral proteomes with subcellular localization information. They are likely to have widespread applications both in the study of the functions of viral proteins in the host cell and in the design of antiviral drugs.
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Espacio Intracelular/virología , Proteínas Virales/análisis , Biología Computacional/métodos , Citomegalovirus/genética , Citomegalovirus/fisiología , Genoma Humano , Genoma Viral , Humanos , Señales de Clasificación de Proteína/fisiología , Proteínas/análisis , Proteínas/química , Proteínas/fisiología , Virus Vaccinia/genética , Virus Vaccinia/fisiología , Proteínas Virales/química , Proteínas Virales/fisiologíaRESUMEN
Chlamydia pneumoniae is emerging as a significant human pathogen. Infection causes a range of respiratory tract diseases and is associated with atherosclerosis. A vaccine could provide a considerable public health benefit; however, antigens able to elicit a protective immune response are largely unknown. A panel of open-reading frames (ORFs) from the C. pneumoniae genome sequence was screened for ability to elicit protective responses. Balb/c mice immunized with DNA containing the ORFs were tested for their ability to limit lung infection following an intranasal challenge. Immunization with DNA encoding the major outer membrane protein or an ADP/ATP translocase (Npt1(Cp)) of C. pneumoniae resulted in a reduced bacteria load in the lung after challenge. The identification of these antigens as protective is a significant step toward development of a C. pneumoniae vaccine and demonstrates the feasibility of using a DNA immunization strategy to screen the C. pneumoniae genome for other protective ORFs.
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Antígenos Bacterianos/inmunología , Infecciones por Chlamydia/prevención & control , Chlamydophila pneumoniae/inmunología , Modelos Animales de Enfermedad , Enfermedades Pulmonares/prevención & control , Infecciones del Sistema Respiratorio/prevención & control , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Chlamydophila pneumoniae/genética , Humanos , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Sistemas de Lectura Abierta/genética , Sistemas de Lectura Abierta/inmunología , Vacunación , Vacunas de ADN/inmunologíaRESUMEN
Respiratory syncytial virus (RSV) remains a major cause of severe respiratory diseases in infants, young children, and the elderly. However, development of a RSV vaccine has been hampered by the outcome of the infant trials in the 1960s with a formalin-inactivated RSV preparation. Enhanced lung disease was induced by the vaccination post-RSV exposure. Previous studies in mice primed with RSV G protein either formulated in adjuvants or delivered by recombinant vaccinia viruses have indicated that enhanced lung pathology resulted from a Th2-type host immune response against the viral G protein. However, in the present report, we have demonstrated that vaccination with plasmid vectors encoding either a full-length or a secreted G protein (DNA-G) clearly elicited balanced systemic and pulmonary Th1/Th2 cytokine responses in mice and did not induce an atypical pulmonary inflammatory reaction post-RSV challenge in cotton rats. DNA-G immunization also induced marked virus neutralizing antibody responses and protection against RSV infection of the lower respiratory tract of both mice and cotton rats. So far, only genetic immunization has been able to induce a balanced Th1/Th2 response with the RSV G protein, reminiscent of that induced by live RSV. Therefore, DNA-G is a promising immunogen for inclusion in a nucleic acid RSV vaccine.
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Proteína HN , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Vacunas de ADN/inmunología , Proteínas Virales/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunología , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Citocinas/análisis , Citocinas/genética , Citocinas/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inflamación/inmunología , Inflamación/patología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Plásmidos/administración & dosificación , Plásmidos/genética , Plásmidos/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Infecciones por Virus Sincitial Respiratorio/patología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/genética , Sigmodontinae , Bazo/inmunología , Linfocitos T Citotóxicos/inmunología , Células TH1/inmunología , Células Th2/inmunología , Vacunación , Vacunas de ADN/administración & dosificación , Vacunas de ADN/efectos adversos , Vacunas de ADN/genética , Proteínas del Envoltorio Viral , Proteínas Virales/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/efectos adversosRESUMEN
Non-encapsulated or non-typable Haemophilus influenzae (NTHi) is a major cause of middle ear infections in young children. HtrA has been identified as a vaccine candidate antigen from NTHi; therefore physicochemical characterization of this antigen is important for vaccine development. Recombinant NTHi HtrA has been expressed in E. coli and shown to have serine protease activity. Several mutant, recombinant HtrA proteins were expressed and purified to obtain suitable vaccine antigens lacking protease activity. Two mutants with alterations at the putative active site His91 and Ser197, designated H91A and S197A were examined by circular dichroic spectropolarimetry (CD) to evaluate secondary structure. The S197A mutant had a more random secondary structure compared to wild-type rHtrA or H91A. It is likely that improper folding of S197A accounts for its lack of immunoprotective properties in a chinchilla model of otitis media.
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Antígenos Bacterianos/química , Vacunas contra Haemophilus/inmunología , Otitis Media/prevención & control , Vacunas Sintéticas/inmunología , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Chinchilla/inmunología , Dicroismo Circular , Escherichia coli/metabolismo , Vacunas contra Haemophilus/genética , Mutación , Otitis Media/inmunología , Estructura Secundaria de Proteína , Conejos , Vacunas Sintéticas/genéticaRESUMEN
The htrA gene from two strains of nontypeable Haemophilus influenzae has been cloned and sequenced, and the encoded approximately 46-kDa HtrA proteins were found to be highly conserved. H. influenzae HtrA has approximately 55% identity with the Escherichia coli and Salmonella typhimurium HtrA stress response proteins, and expression of the H. influenzae htrA gene was inducible by high temperature. Recombinant HtrA (rHtrA) was expressed from E. coli, and the purified protein was found to have serine protease activity. rHtrA was found to be very immunogenic and partially protective in both the passive infant rat model of bacteremia and the active chinchilla model of otitis media. Immunoblot analysis indicated that HtrA is antigenically conserved in encapsulated and nontypeable H. influenzae species. Site-directed mutagenesis was performed on the htrA gene to ablate the endogenous serine protease activity of wild-type HtrA, and it was found that eight of nine recombinant mutant proteins had no measurable residual proteolytic activity. Two mutant proteins were tested in the animal protection models, and one, H91A, was found to be partially protective in both models. H91A HtrA may be a good candidate antigen for a vaccine against invasive H. influenzae type b disease and otitis media and is currently in phase I clinical trials.
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Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Haemophilus influenzae/inmunología , Proteínas de Choque Térmico , Proteínas Periplasmáticas , Serina Endopeptidasas/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Cobayas , Sueros Inmunes/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes/inmunología , Serina Endopeptidasas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The role of converting enzyme inhibitor enhanced radionuclide investigations in post-transplant hypertension is not clearly defined. Presence of renal failure, chronic rejection and use of cyclosporin A complicates the results. Captopril-induced changes in effective renal plasma flow (ERPF) were studied in 10 patients with severe post-transplant hypertension and no evidence of rejection. Angiographic correlation was available in all. Six patients had a significant increase in ERPF after captopril, and all had a negative angiogram. One patient on CsA with a negative angiogram had no change in ERPF. Three patients had a fall in ERPF, and all 3 had transplant renal artery stenosis. Captopril-induced changes in ERPF can differentiate patients with native-kidney-induced hypertension from those with hypertension secondary to transplant renal artery stenosis in patients without evidence of rejection.
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Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Captopril/farmacología , Hipertensión/etiología , Trasplante de Riñón , Flujo Plasmático Renal Efectivo/efectos de los fármacos , Angiografía , Diagnóstico Diferencial , Humanos , Hipertensión/diagnóstico por imagen , Complicaciones Posoperatorias/diagnóstico por imagen , Estudios Prospectivos , Obstrucción de la Arteria Renal/complicacionesRESUMEN
A disulphide bond was introduced into a single-chain Fv form of the anticarbohydrate antibody, Se155-4 by replacing Ala-L57 of the light chain and Asp-H106 of the heavy chain with cysteines, by site-directed mutagenesis. To maintain the salt-bridge from the latter residue to Arg-H98, Tyr-107 was also altered to Asp. The resulting ds-scFv was shown to retain full antigen-binding activity, by enzyme immunoassay and surface plasmon resonance analysis of binding kinetics. Compared with the parent scFv, the disulphide bonded form was shown to have enhanced thermal stability, by Fourier transform IR spectroscopy. The Tm was raised from 60 degrees C to 69 degrees C. The ds-scFv form thus combines the stable monomeric form of the disulphide form with the expression advantages of the scFv.
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Disulfuros , Calefacción , Fragmentos de Inmunoglobulinas/química , Región Variable de Inmunoglobulina/química , Sitios de Unión , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Mutagénesis , Conformación Proteica , Salmonella/inmunologíaRESUMEN
A conserved proline residue is found at position 331 in the CH2 domains of human IgG subclasses which fix complement. This residue is replaced by a serine in IgG4 which is inactive. To determine the role of residue 331 in the differential ability of human IgGs to activate the complement cascade, a pair of genetically engineered anti-dinitrophenol IgG1 and IgG4 antibodies with reciprocal mutations at position 331 were tested for their hemolytic activity as well as for their ability to bind C1q, activate C1 and cleave C4. The IgG1 Ser331 mutant was virtually unable to mediate the lysis of trinitrobenzene-sulfonic acid-derivatized sheep red blood cells as a result of a marked defect in C1q binding activity. In contrast, the substitution of Pro for Ser331 in IgG4 bestowed partial hemolytic activity (40%) to the IgG4 Pro331 variant. Under low ionic strength conditions, this mutant was found to be approximately 50 and 75% as active as wild-type IgG1 in the C1q binding and C4b deposition assays, respectively. These results indicate that residue Pro331, which folds into close proximity to a previously identified C1q binding motif (Duncan, A. R., and Winter, G. (1988) Nature 332, 738-740), contributes to the architecture of the IgG1 C1q binding site and that its replacement by a serine residue in IgG4 is largely responsible for the functional inactivity of this isotype.
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Activación de Complemento , Complemento C1q/metabolismo , Secuencia Conservada , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Prolina , Conformación Proteica , Serina , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Secuencia de Bases , Hemólisis , Humanos , Inmunoglobulina G/clasificación , Cinética , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Plasmacitoma , Mutación Puntual , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Células Tumorales CultivadasRESUMEN
Although human IgG2 is not cytophilic, we have shown previously that an IgG2 antibody expressing the sequence PLLGG (underline = substitution) spanning CH2 domain residues 233-237 (Eu numbering) displayed IgG1-like Fc gamma RI binding activity. In contrast, IgG1 PLLGG exhibited 3-fold less affinity, whereas IgG2 ELLGG was 3-fold more active than native IgG1. These results suggested that additional site(s) conferred enhanced binding properties to the engineered, cytophilic IgG2 variant. These sites were shown to reside in the IgG2 CH2 domain, since the IgG1 CH2 module did not have enhanced activity in a panel of hybrid IgG1/IgG2 antibodies. To map these sites further, human IgG1 and IgG2 constant region gene segments were modified to allow reciprocal COOH-terminal half segment exchanges of CH2 exons. These were cloned into a pSV2neo expression vector bearing a rearranged MOPC 315 heavy chain variable region gene and transfected into a MOPC 315 heavy chain deletion mutant. The dinitrophenol affinity-purified IgGs were radiolabeled and assessed for Fc gamma RI binding activity in direct binding assays using U937 cells. The COOH terminus of the IgG2 CH2 domain was found to contain accessory site(s) since it enhanced the binding properties of both IgG1 PLLGG and native IgG1. In contrast, grafting of the COOH terminus of the IgG1 CH2 domain onto IgG2 PLLGG and IgG2 ELLGG diminished their cytophilic activity. The amino acid responsible for the enhancing properties of the COOH terminus of the IgG2 CH2 domain was shown to be threonine 339, since IgG1 PLLGG/Thr339 displayed increased Fc gamma RI binding affinity. Kinetics studies revealed that this is accomplished through an increase in the forward rate constant of the IgG-Fc gamma RI interaction.
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Inmunoglobulina G/metabolismo , Receptores de IgE/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Inmunoglobulina G/genética , Cinética , Ratones , Datos de Secuencia Molecular , Mutación Puntual , Células Tumorales CultivadasRESUMEN
The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.
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Antígenos Bacterianos/inmunología , Brucella/inmunología , Pared Celular/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Polisacáridos Bacterianos/inmunología , Especificidad de Anticuerpos , Brucella abortus/inmunología , Brucella melitensis/inmunología , Secuencia de Carbohidratos , Simulación por Computador , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Manosa/análogos & derivados , Manosa/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Difracción de Rayos XRESUMEN
Specific functional group modification of an antibody adsorbed to microtitre plates has been used to probe the binding site residues that determine antigen specificity. Chemical modification of adsorbed protein in tandem with enzyme immunoassay (termed CMAP-EIA) consumes only modest amounts of antibody, while allowing a variety of reagents to be rapidly screened in situ. Modification of tyrosine and arginine residues with 1-fluoro-2,4-dinitrobenzene, and p-hydroxyphenylglyoxal resulted in reduced binding of polysaccharide antigen from Yersinia enterocolitica O-polysaccharide to its homologous monoclonal antibody, YsT9-1. Modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide under various conditions indicated that carboxylate groups may also be involved. Parallel experiments with diethylpyrocarbonate and acetic anhydride were used to rule out the involvement of histidine and lysine residues respectively. In all cases, binding of an anti-idiotypic antibody, AJ5, could only be reduced at concentrations of modifying reagent substantially higher than those required to reduce polysaccharide antigen binding to YsT9-1. The results are discussed with regard to the structure of the combining site of YsT9-1 as determined by X ray crystallography and by modelling, and the role of particular residues in complex formation with antigen and in the idiotope.
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Anticuerpos Antibacterianos/inmunología , Sitios de Unión de Anticuerpos/inmunología , Polisacáridos Bacterianos/inmunología , Yersinia enterocolitica/inmunología , Anhídridos Acéticos , Reacciones Antígeno-Anticuerpo/efectos de los fármacos , Sitios de Unión de Anticuerpos/efectos de los fármacos , Carbodiimidas , Dietil Pirocarbonato , Dinitrofluorobenceno , Relación Dosis-Respuesta Inmunológica , Concentración de Iones de Hidrógeno , Técnicas para Inmunoenzimas , Modelos Moleculares , Antígenos O , Fenilglioxal/análogos & derivados , Difracción de Rayos XRESUMEN
Twelve plant lectins from the Papilionoideae subfamily were selected to represent a range of carbohydrate specificities, and their sequences were aligned. Two variability indices were applied to the aligned sequences and the results were analysed using the three-dimensional structures of concanavalin A and the pea lectin. The areas of greatest variability were located in the carbohydrate-binding site region, forming a perimeter around a well-conserved core. These residues are inferred to be specificity determining, in the manner of antibodies, and the most variable position corresponded to Tyr100 in concanavalin A, a known ligand contact residue. In addition to the five peptide loops known to form the binding site from crystallographic studies, a sixth segment with variable residues was located in the binding-site region, and this may contribute to oligosaccharide specificity. In their overall composition, the lectin sites resemble those of the sugar-transport proteins rather than antibodies. The prospects for modelling lectin binding sites by the methods used for antibodies were also assessed.
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Fabaceae/química , Lectinas/química , Plantas Medicinales , Secuencia de Aminoácidos , Sitios de Unión , Metabolismo de los Hidratos de Carbono , Concanavalina A/química , Concanavalina A/genética , Concanavalina A/metabolismo , Variación Genética , Lectinas/genética , Lectinas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Lectinas de Plantas , Alineación de Secuencia , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Competitive labeling of melittin over a range of concentrations in the presence and absence of liposomes provides a series of "snapshots" of the chemical reactivities of melittin's intrinsic nucleophiles. Distinct trends in apparent reactivities were observed for the Gly-1 alpha-amino group and the epsilon-amino groups of Lys-7 and Lys-21 and -23, over a range of concentrations, providing evidence for different forms of associated melittin in solution. The monomer-tetramer transition can be followed, in accord with structural details derived from X-ray crystallography. The reactivity behavior of the alpha-amino group of Gly-1 and the epsilon-amino groups of Lys-21 and Lys-23 suggests these groups undergo similar perturbations in their microenvironments during the monomer-tetramer transition in free solution. Similar changes in reactivity behavior occur upon association of melittin monomers with bilayer-forming lipids. Together, these findings suggest that the local environments of the N- and C-terminal segments have similar physicochemical properties in both the solution tetramer and the lipid-associated complex. The concentration dependence of the chemical properties of melittin is correlated with surface accessibility calculations which are used to provide a framework for interpretation. Aspects of several previously proposed models of membrane lysis can be accounted for by concentration-dependent properties of melittin.
Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Meliteno/química , Secuencia de Aminoácidos , Unión Competitiva , Fenómenos Químicos , Química , Dinitrofluorobenceno/metabolismo , Liposomas/metabolismo , Lisina/química , Sustancias Macromoleculares , Meliteno/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Mapeo Peptídico , SolucionesRESUMEN
A molecular model of the binding site of an anti-carbohydrate antibody (YsT9.1) has been developed using computer-assisted modeling techniques and molecular dynamics calculations. Sequence homologies among YsT9.1 and the Fv regions of McPC603, J539 and human Bence--Jones protein REI, all of which have solved crystal structures, provided the basis for the modeling. The groove-type combining site model had a topography which was complementary to low energy conformers of the polysaccharide, a Brucella O-antigen, and the site could be almost completely filled by a pentasaccharide epitope in either of two docking modes. Putative interactions between this epitope and the antibody are consistent with the known structural requirements for binding and lead to the design of oligosaccharide inhibitors that probe the veracity of the modeled docked complex. Ultimately both the Fv model and the docked complex will be compared with independent crystal structures of YsT9.1 Fab with and without pentasaccharide inhibitor, currently at the stage of refinement.