RESUMO
Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.
Assuntos
Proteínas do Olho/química , Proteínas de Ligação ao Retinol/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/química , Bovinos , Microscopia Crioeletrônica , Proteínas do Olho/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação ao Retinol/imunologia , Imagem Individual de MoléculaRESUMO
The goal of this study was to investigate the glycosylation profile of native immunoglobulin (Ig)G present in serum immune complexes in patients with rheumatoid arthritis (RA). To accomplish this, lectin binding assays, detecting the accessibility of glycans present on IgG-containing immune complexes by biotinylated lectins, were employed. Lectins capturing fucosyl residues (AAL), fucosylated tri-mannose N-glycan core sites (LCA), terminal sialic acid residues (SNA) and O-glycosidically linked galactose/N-acetylgalactosamine (GalNac-L) were used. Patients with recent-onset RA at baseline and after 3-year follow-up were investigated. We found that native IgG was complexed significantly more often with IgM, C1q, C3c and C-reactive protein (CRP) in RA patients, suggesting alterations of the native structure of IgG. The total accessibility of fucose residues on captured immune complexes to the respective lectin was significantly higher in patients with RA. Moreover, fucose accessibility on IgG-containing immune complexes correlated positively with the levels of antibodies to cyclic citrullinated peptides (anti-CCP). We also observed a significantly higher accessibility to sialic acid residues and galactose/GalNAc glyco-epitopes in native complexed IgG of patients with RA at baseline. While sialic acid accessibility increased during treatment, the accessibility of galactose/GalNAc decreased. Hence, successful treatment of RA was associated with an increase in the SNA/GalNAc-L ratio. Interestingly, the SNA/GalNAc-L ratio in particular rises after glucocorticoid treatment. In summary, this study shows the exposure of glycans in native complexed IgG of patients with early RA, revealing particular glycosylation patterns and its changes following pharmaceutical treatment.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Artrite Reumatoide/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Polissacarídeos/química , Polissacarídeos/imunologia , Adulto , Idoso , Complexo Antígeno-Anticorpo/química , Artrite Reumatoide/terapia , Proteína C-Reativa/imunologia , Proteína C-Reativa/metabolismo , Complemento C1q/imunologia , Complemento C1q/metabolismo , Complemento C3c/imunologia , Complemento C3c/metabolismo , Feminino , Fucose/metabolismo , Galactose/metabolismo , Glicosilação , Humanos , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Lectinas/metabolismo , Masculino , Pessoa de Meia-Idade , Polissacarídeos/metabolismo , Sambucus nigra , Ácidos Siálicos/metabolismoRESUMO
Therapeutic monoclonal antibodies (mAbs) represent a milestone in pharmacological development. Their superiority is based on the combination of high specificity, low toxicity, and long half-life that characterizes biologics. If biologics have Achilles' heel, it is their potential immunogenicity. To better understand the impact of the size of immune complexes of mAbs on anti-drug antibody (ADA) dependent adverse reactions in Macaca fascicularis, we developed an efficient high-throughput size exclusion chromatography- (SEC-) based methodology that enables analysis of the size, size distribution, and ratio of free and ADA-complexed mAb in serum allowing for assessment of formation and clearance of circulating ADA-mAb immune complexes (CIC).
Assuntos
Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Cromatografia em Gel/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Isoanticorpos/imunologia , Isoanticorpos/metabolismo , Macaca fascicularis , Ligação Proteica , Coloração e Rotulagem , Toxicologia/métodosRESUMO
Copper(I)-catalyzed azide-alkyne cycloaddition, or CuAAC click chemistry, is an efficient method for bioconjugation aiming at chemical and biological applications. Herein, we demonstrate how the CuAAC method can provide novel phospholipid-protein conjugates with a high potential for the diagnostics and therapy of autoimmune conditions. In doing this, we, for the first time, covalently bind via 1,2,3-triazole linker biologically complementary molecules, namely phosphoethanol amine with human ß2-glycoprotein I and prothrombin. The resulting phospholipid-protein conjugates show high binding affinity and specificity for the autoimmune antibodies against autoimmune complexes. Thus, the development of this work might become a milestone in further diagnostics and therapy of autoimmune diseases that involve the production of autoantibodies against the aforementioned phospholipids and proteins, such as antiphospholipid syndrome and systemic lupus erythematosus.
Assuntos
Antígenos/química , Autoanticorpos/química , Etanolaminas/química , Imunoconjugados/química , Protrombina/química , beta 2-Glicoproteína I/química , Alcinos/química , Complexo Antígeno-Anticorpo/química , Antígenos/imunologia , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/imunologia , Azidas/química , Química Click , Reação de Cicloadição , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Ligação Proteica , Soluções , Triazóis/químicaRESUMO
The quantitative dependences of the immune complement-dependent lysis of a monodisperse suspension of 200-nm liposomes whose membrane was sensitized by monovalent hapten (2,4-DNP-epsilon-caproyl-DPPE) or a polyvalent antigen (LPS from F. tularensis) on the initial concentration of specific antibodies (IgG) to hapten and the antigen have been investigated. The quantity of antibodies binding sites on the surface of liposome was evaluated. The difference between the complement-dependent immune lysis of poly- and monodisperse suspensions of liposomes was shown. The experimental results are well described by the direct binding model.
Assuntos
Complexo Antígeno-Anticorpo/química , Proteínas do Sistema Complemento/química , Lipossomas Unilamelares/química , Lipossomas Unilamelares/imunologia , Anticorpos Monoclonais/química , Colesterol/química , Francisella tularensis/química , Haptenos/química , Imunoglobulina G/química , Lecitinas/química , Lipopolissacarídeos/química , Organofosfatos/química , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/imunologiaRESUMO
CdTe nanowires made by self-organization of CdTe nanoparticles in aqueous media were separately conjugated with complementary biological connectors, such as antigen-antibody and biotin-streptavidin. Transmission electron microscopy images and Forster resonance energy transfer measurements in nanowire superstructures with different diameters indicate that biological affinity of the attached proteins results in the formation of crossbar and end-to-side connections between the nanowires. A prototype of a logical circuit made from a triangular arrangement of the nanowires spontaneously assembled on a Si substrate was examined by conducting atomic force microscopy. While diode-like behavior was observed in the sides of the triangle, the nanowire junction points were found to be nonconductive. It was attributed to high tunneling barrier created by protein molecules wedged between the nanowires. Suggestions are made how to reduce it or use the insulating gap between the nanowires as a framework for single-electron devices.
Assuntos
Compostos de Cádmio/química , Cristalização/métodos , Eletroquímica/métodos , Nanotubos/química , Nanotubos/ultraestrutura , Proteínas/química , Proteínas/ultraestrutura , Telúrio/química , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/ultraestrutura , Materiais Revestidos Biocompatíveis/química , Condutividade Elétrica , Eletroquímica/instrumentação , Eletrônica , Teste de Materiais , Nanotecnologia/métodos , SemicondutoresRESUMO
The influence of laser therapy on the course of Parkinson's disease (PD) was studied in 70 patients. This influence appeared adaptogenic both in the group with elevated and low MAO B and Cu/Zn SOD activity. Laser therapy resulted in reduction of neurological deficit, normalization of the activity of MAO B, Cu/Zn-SOD and immune indices. There was a correlation between humoral immunity and activity of the antioxidant enzymes (SOD, catalase). This justifies pathogenetically the use of laser therapy in PD.
Assuntos
Terapia com Luz de Baixa Intensidade , Doença de Parkinson/imunologia , Doença de Parkinson/radioterapia , Idoso , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/química , Catalase/sangue , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Peso Molecular , Monoaminoxidase/sangue , Doença de Parkinson/sangue , Doença de Parkinson/fisiopatologia , Valor Preditivo dos Testes , Valores de Referência , Superóxido Dismutase/sangueRESUMO
Antienzyme polyclonal antibodies against stem bromelain were raised in male albino rabbits and the Fab' monomers isolated from the IgG of the immune sera. Incubation of bromelain with the Fab' resulted in binding and gel filtration of the resulting complex suggested a 1:1 stoichiometry. Complexing with the Fab' resulted in significant stabilization of bromelain against thermal inactivation and alkaline pH.
Assuntos
Bromelaínas/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Animais , Complexo Antígeno-Anticorpo/química , Bromelaínas/administração & dosagem , Estabilidade Enzimática/imunologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Masculino , CoelhosRESUMO
Much research is now focused on a potential vaccine for Alzheimer's disease (AD). Current studies involve administering the amyloid beta peptide (Abeta) in Freund's complete adjuvant, which cannot be used in humans. Our studies show that the immune complex of Abeta is taken up by a receptor-mediated process at the blood-brain barrier (BBB). The success of immunization for AD, therefore, may be critically dependent on circulating Abeta levels which are lower in AD patients compared to AD transgenic mice. Moreover, we have found that modifying the antibody with polyamine increases its BBB permeability and may provide a better approach to passive immunization for Alzheimer's disease.
Assuntos
Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/farmacocinética , Barreira Hematoencefálica/efeitos dos fármacos , Proteínas de Transporte/efeitos dos fármacos , Imunização/métodos , Fragmentos de Peptídeos/farmacocinética , Receptores de Peptídeos/efeitos dos fármacos , Vacinas/farmacocinética , Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/farmacologia , Barreira Hematoencefálica/imunologia , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Encéfalo/metabolismo , Proteínas de Transporte/imunologia , Relação Dose-Resposta a Droga , Humanos , Radioisótopos do Iodo/farmacocinética , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Poliaminas/química , Poliaminas/farmacocinética , Receptores de Peptídeos/imunologia , Vacinas/síntese químicaRESUMO
Monoclonal antibody NC6.8 is specific for the superpotent sweetener, N-(p-cyanophenyl)-N'-(diphenylmethyl)-guanidiniumacetic++ + acid. The three-dimensional structure of the complex shows the close proximity of complementary charged residues on the antibody and groups of the hapten. As a result, association is dependent on the pH, dielectric, and ionic strength of the medium. Continuum electrostatics methods are used to calculate the pH-dependent association energetics of NC6.8 with the superpotent sweetener. In addition to providing a titration profile, the calculations quantitatively assess the relative influence of charged groups on the energetics of association. Models of site directed mutants are constructed to probe the influence of each charged interface residue on the pH-dependent energetics of association. Examination of electrostatic contribution to free energy of association in mutant complexes, where the key acidic residues on the antibody are neutralized, shows that charge complementarity at the combining site is an important requirement for hapten binding. Also, based on the pKa values of several combining site tyrosine residues, aromatic pi-stacking and van der Waal's contacts between the antibody and hapten contribute to the specificity of the complex.