RESUMO
The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.
Assuntos
Acetilcolinesterase/metabolismo , Acetilcolinesterase/química , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Domínio Catalítico , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Clonagem Molecular , Regiões Determinantes de Complementaridade , Ativação Enzimática , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrP(Sc) accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 microg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.
Assuntos
Colostro/química , Leite/química , Proteínas PrPSc/análise , Scrapie/metabolismo , Scrapie/transmissão , Animais , Química Encefálica , Feminino , Humanos , Glândulas Mamárias Animais/química , Camundongos , Camundongos Transgênicos , Proteínas PrPSc/patogenicidade , Gravidez , Carneiro Doméstico , Distribuição TecidualRESUMO
We compared the biochemical properties of a wild type recombinant normal human cellular prion protein, rPrP(c), with a recombinant mutant human prion protein that has three additional octapeptide repeats, rPrP(8OR). Monoclonal antibodies that are specific for the N terminus of rPrP(c) react much better with rPrP(8OR) than rPrP(c), suggesting that the N terminus of rPrP(8OR) is more exposed and hence more available for antibody binding. The N terminus of PrP(c) contains a glycosaminoglycan binding motif. Accordingly, rPrP(8OR) also binds more glycosaminoglycan than rPrP(c). In addition, the divalent cation copper modulates the conformations of rPrP(c) and rPrP(8OR) differently. When compared with rPrP(c), rPrP(8OR) is also more susceptible to oxidative damage. Furthermore, the abnormalities associated with rPrP(8OR) are recapitulated, but even more profoundly, in another insertion mutant, which has five extra octapeptide repeats, rPrP(10OR). Therefore, insertion mutants appear to share common features, and the degree of abnormality is proportional to the number of insertions. Any of these anomalies may contribute to the pathogenesis of inherited human prion disease.
Assuntos
Mutação , Oxigênio/química , Príons/química , Motivos de Aminoácidos , Anticorpos Monoclonais/química , Cálcio/metabolismo , Carbono/química , Cátions , Cobre/química , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos , Glicosaminoglicanos/química , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Genéticos , Mutagênese Sítio-Dirigida , Oxigênio/metabolismo , Peptídeos/química , Proteínas PrPC/química , Príons/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
d-amino acids are present in some peptides from amphibian skin. These residues are derived from the corresponding L-amino acids present in the respective precursors. From skin secretions of Bombinae, we have isolated an enzyme that catalyzes the isomerization of an L-Ile in position 2 of a model peptide to D-allo-Ile. In the course of this reaction, which proceeds without the addition of a cofactor, radioactivity from tritiated water is incorporated into the second position of the product. The amino acid sequence of this isomerase could be deduced from cloned cDNA and genomic DNA. After expression of this cDNA in oocytes of Xenopus laevis, isomerase activity could be detected. Polypeptides related to the frog skin enzyme are present in several vertebrate species, including humans.
Assuntos
Isomerases de Aminoácido/metabolismo , Aminoácidos/biossíntese , Aminoácidos/química , Anuros/metabolismo , Pele/enzimologia , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/isolamento & purificação , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Animais , Anuros/genética , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Feminino , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oócitos/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Estereoisomerismo , Xenopus laevisRESUMO
The use of anti-PrP antibodies represents one of the most promising strategies for the treatment of prion diseases. In the present study, we screened various anti-PrP antibodies with the aim of identifying those that would block PrP(Sc) replication in prion-infected cell culture. Two antibodies, SAF34 recognizing the flexible octarepeats region on HuPrP protein, and SAF61 directed against PrP amino acid residues (144-152), not only inhibited PrP(Sc) formation in prion-infected neuroblastoma cells but also decreased the PrP(C) levels in non-infected N2a cells. In addition, treatment with both SAF34 and SAF61 antibodies decreased PrP(C) and PrP(Sc) levels in the cells synergistically. In the presence of both antibodies, our results showed that the mode of action which leads to the disappearance of PrP(Sc) in cells is directly coupled to PrP(C) degradation by reducing the half-life of the PrP(C) protein.
Assuntos
Anticorpos Monoclonais/farmacologia , Proteínas PrPC/imunologia , Proteínas PrPC/metabolismo , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/imunologia , Doenças Priônicas/imunologia , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Humanos , Camundongos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Doenças Priônicas/terapiaRESUMO
The concept of specific immune treatment against allergic diseases requires the development of antibodies capable of specifically neutralizing anti-allergen antibodies. The aim of the present study was to investigate whether a novel approach, consisting in raising anti-idiotypic blocking antibodies through peptide immunization, could be envisaged in the field of allergy. Using allergy to cow's milk as a model, we prepared polyclonal antibodies against a peptide that is complementary (i.e. hydropathically opposed) to a major epitope of bovine beta-lactoglobulin (BLG), one of the main allergens of bovine milk. Anti-complementary peptide antibodies were found to neutralize in vitro both well-characterized anti-BLG monoclonal antibodies from mice sensitized to BLG and anti-BLG IgE from two patients suffering from milk allergy. These results suggest a new strategy for the functional inhibition of specific disease-associated IgE that may be applicable to the specific treatment of various allergic disorders.