RESUMO
Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.
Assuntos
Doença de Alzheimer/metabolismo , Anticorpos/imunologia , Epitopos/imunologia , Proteínas tau/imunologia , Doença de Alzheimer/genética , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/genética , Encéfalo/metabolismo , Galinhas , Epitopos/química , Epitopos/genética , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fosforilação , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.
Assuntos
Membrana Celular/metabolismo , Lipoproteínas LDL/metabolismo , Modelos Moleculares , Proteínas Recombinantes de Fusão/farmacologia , Receptores Depuradores Classe E/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/química , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Reagentes de Ligações Cruzadas/química , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Receptores Depuradores Classe E/antagonistas & inibidores , Receptores Depuradores Classe E/genéticaRESUMO
Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.