RESUMO
Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogendeuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups. This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis. Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD. The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.
Assuntos
Mapeamento de Epitopos , Radical Hidroxila , Pegadas de Proteínas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Mapeamento de Epitopos/métodos , Pegadas de Proteínas/métodos , SARS-CoV-2/imunologia , SARS-CoV-2/química , Radical Hidroxila/química , Humanos , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Ligação Proteica , COVID-19/virologia , COVID-19/imunologia , Sítios de Ligação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/química , Espectrometria de Massas/métodos , Domínios ProteicosRESUMO
Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases termed gingipains, and in this study we have used the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naive camel nanobody library and used phage display to select one nanobody toward RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for RgpB given that it did not bind to the homologous gingipain HRgpA. This indicated the presence of a binding epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted soluble RgpB. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection and that the presented nanobody-based assay could supplement existing methods for P. gingivalis detection.
Assuntos
Adesinas Bacterianas/análise , Anticorpos Antibacterianos/imunologia , Infecções por Bacteroidaceae/diagnóstico , Cisteína Endopeptidases/análise , Porphyromonas gingivalis/isolamento & purificação , Anticorpos de Cadeia Única/imunologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Sequência de Aminoácidos , Infecções por Bacteroidaceae/microbiologia , Biomarcadores/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases Gingipaínas , Humanos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Porphyromonas gingivalis/enzimologia , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saliva/microbiologiaRESUMO
Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by degradation of complement system proteins and by inactivation of the antimicrobial peptide LL-37 by proteolytic cleavage. This makes karilysin a highly interesting virulence factor to study in the framework of drug development and diagnostics. However, to date the presence of karilysin in clinical samples has not been demonstrated due to the lack of specific probes. In the present work, a high titer and stable affinity-purified avian IgY antibody against karilysin was developed. By surface plasmon resonance imaging the IgY affinity was found to be in the low nanomolar range. The antibody could be used to detect karilysin in saliva samples by immuno-blotting and was specific when tested towards human MMP-3. Furthermore, an avian IgY-based immunoassay was developed, which demonstrated low intra- and interday assay variability (CV's below 10%). Application of the immunoassay on a well-characterized set of saliva samples from adolescents with or without signs of periodontitis showed that it was possible to detect karilysin in saliva. A significant difference in karilysin concentration was found between saliva from participants with signs of periodontitis and saliva from healthy controls (pâ¯=â¯.0024). The median of karilysin levels among periodontitis cases was 957â¯pg/ml (IQR, 499-2132â¯pg/ml) and the median for controls was 569â¯pg/ml (IQR, 210-1343â¯pg/ml). Collectively our data confirm the presence of karilysin in clinical samples. The described IgY-based immunoassay may prove useful as part of protein-based biomarker screenings in the clinic or in point-of care settings.
Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/fisiologia , Ensaio de Imunoadsorção Enzimática , Infecções por Bactérias Gram-Negativas/diagnóstico , Imunoglobulinas/imunologia , Metaloproteinases da Matriz/imunologia , Periodontite/diagnóstico , Saliva/microbiologia , Tannerella forsythia/imunologia , Fatores de Virulência/imunologia , Adolescente , Especificidade de Anticorpos , Proteínas de Bactérias/imunologia , Estudos de Casos e Controles , Feminino , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Masculino , Periodontite/microbiologia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Tannerella forsythia/patogenicidade , VirulênciaRESUMO
Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a vital role in extracellular conversion of inactive plasminogen into catalytically active plasmin. Activated plasmin facilitates the release of several proteolytic enzymes, which control processes like pericellular proteolysis and remodeling of ECM. uPA and the receptor uPAR, are overexpressed in a number of malignant tumours and uPA/uPAR play major roles in adhesion, migration, invasion and metastasis of cancer cells. Elevated levels of uPA have been reported as a risk biomarker for disease relapse, increased cancer malignancy and poor survival prognosis. For these reasons uPA is considered an important target for anticancer drug therapy. In this study we isolated two camel single domain antibodies (nanobodies) from a naïve library by phage display. The nanobody sequences were sequence-optimized for Escherichia coli expression, cloned into the pET22-B(+) vector system, expressed in BL-21 cells and purified from the periplasmic fraction by IMAC. ELISA tests demonstrated that the purified nanobodies were specific for uPA when tested towards other trypsin-like serine proteases. The apparent affinities of the nanobodies were determined by competitive ELISA to 80 nM and 522 nM, respectively. The best binder did not inhibit uPA (nAb-C3), however the lowest affinity binder (nAb-C8) was able to inhibit the uPA-mediated cleavage of the substrate S-2444. The results validate the naïve library as a resource for retrieval of relevant lead molecules and the novel uPA-nanobodies can be useful pharmacological tools to study uPA structure-function relationships.