Browse
Submit Data
Databases
API
Help

Dataset Information

30 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Epitope mapping of an anti-diacylglycerol kinase delta monoclonal antibody DdMab-1.

ABSTRACT: Diacylglycerol kinase ? (DGK?) is a type II DGK, which catalyzes diacylglycerol phosphorylation to produce phosphatidic acid. DGK? is expressed in several types of tissues and organs including the stomach, testis, bone marrow, and lymph node. Here, we established an anti-human DGK? (hDGK?) mAb, DdMab-1 (mouse IgG2a, kappa), which is useful for Western blot analysis. We also introduced deletion or point mutations to hDGK?, and performed western blotting to determine the binding epitope of DdMab-1. DdMab-1 reacted with the dN670 mutant, but not with the dN680 mutant, indicating that the N-terminus of the DdMab-1 epitope is mainly located between amino acids 670 and 680 of the protein. Further analysis using point mutants demonstrated that R675A, R678A, K679A, and K682A mutants were not detected, and V680A was only weakly detected by DdMab-1, indicating that Arg675, Arg678, Lys679, Val680 and Lys682 are important for binding of DdMab-1 to hDGK?.

SUBMITTER: Sano M

PROVIDER: S-EPMC7481522 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Epitope mapping of an anti-diacylglycerol kinase delta monoclonal antibody DdMab-1.

Sano Masato M Asano Teizo T Kaneko Mika K MK Kato Yukinari Y

Biochemistry and biophysics reports 20200902

Diacylglycerol kinase δ (DGKδ) is a type II DGK, which catalyzes diacylglycerol phosphorylation to produce phosphatidic acid. DGKδ is expressed in several types of tissues and organs including the stomach, testis, bone marrow, and lymph node. Here, we established an anti-human DGKδ (hDGKδ) mAb, DdMab-1 (mouse IgG<sub>2a</sub>, kappa), which is useful for Western blot analysis. We also introduced deletion or point mutations to hDGKδ, and performed western blotting to determine the binding epitope ...[more]

PMID: 32944659

Similar Datasets

Epitope mapping of anti-mouse podoplanin monoclonal antibody PMab-1.

Project description:Mouse podoplanin (mPDPN) is a type I transmembrane sialoglycoprotein, which is expressed on lymphatic endothelial cells, podocytes of the kidney, and type I alveolar cells of the lung. mPDPN is known as a platelet aggregation-inducing factor and possesses four platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 in the N-terminus and PLAG4 in the middle of the mPDPN protein. mPDPN overexpression in cancers has been reportedly associated with hematogenous metastasis through interaction with the C-type lectin-like receptor 2 of platelets. We previously reported a rat anti-mPDPN monoclonal antibody clone PMab-1, which was developed by immunizing the PLAG2 and PLAG3 domains of mPDPN. PMab-1 is very useful in flow cytometry, western blot, and immunohistochemical analyses to detect both normal cells and cancers. However, the binding epitope of PMab-1 remains to be clarified. In the present study, flow cytometry, enzyme-linked immunosorbent assay, and immunohistochemical analyses were utilized to investigate the epitope of PMab-1. The results revealed that the critical epitope of PMab-1 is Asp39 and Met41 of mPDPN. These findings can be applied to the production of more functional anti-mPDPN monoclonal antibodies.

| S-EPMC6039309 | biostudies-literature

Epitope mapping of an anti-alpha thalassemia/mental retardation syndrome X-linked monoclonal antibody AMab-6.

Project description:The alpha-thalassemia/mental-retardation-syndrome-X-linked (ATRX) gene is located on the q arm of the X chromosome. ATRX gene mutations were first discovered in pancreatic neuroendocrine tumors, and subsequently in other cancer subtypes, including gliomas. Molecular subgrouping of gliomas has been more important than conventional histological classifications. Mutations in the isocitrate dehydrogenase (IDH), telomerase reverse transcriptase (TERT) promoter, and ATRX and the codeletion of chromosomes 1p/19q are used as biomarkers for diagnosing the subtypes of diffuse gliomas. We recently developed a sensitive monoclonal antibody (mAb) AMab-6 against ATRX by immunizing mice with recombinant human ATRX. AMab-6 can help to detect ATRX mutations via Western blotting and immunohistochemical analyses. In this study, we characterized the binding epitope of AMab-6 using enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunohistochemical analysis, and found that Gln2368 of ATRX is critical for AMab-6 binding to ATRX. Our findings could be applied to the production of more functional anti-ATRX mAbs.

| S-EPMC6068083 | biostudies-literature

Epitope mapping of a monoclonal antibody directed against Neisserial Heparin Binding Antigen

Project description:This SuperSeries is composed of the SubSeries listed below.

2016-05-27 | GSE81379 | GEO

Diacylglycerol kinase delta promotes lipogenesis.

Project description:We have studied the relationship between diacylglycerol kinase delta (DGK?) and lipogenesis. There is a marked increase in the expression of DGK? during the differentiation of 3T3-L1 cells to adipocytes, as well as in the synthesis of neutral and polar lipids. When 3T3-L1 undifferentiated fibroblasts are transfected to express DGK?, there is increased triglyceride synthesis without differentiation to adipocytes. Hence, expression of DGK? promotes lipogenesis. Lipid synthesis is decreased in DGK? knockout mouse embryo fibroblasts, especially for lipids with shorter acyl chains and limited unsaturation. This reduction occurs for both neutral and polar lipids. These findings suggest reduced de novo lipid synthesis. This is confirmed by measuring the incorporation of glycerol into polar and neutral lipids, which is higher in the wild type cells than in the DGK? knockouts. In comparison, there was no change in lipid synthesis in DGK? knockout mouse embryo fibroblasts. We also demonstrate that the DGK? knockout cells had a lower expression of acetyl-CoA carboxylase and fatty acid synthase as well as a lower degree of activation by phosphorylation of ATP citrate lyase. These three enzymes are involved in the synthesis of long chain fatty acids. Our results demonstrate that DGK? markedly increases lipid synthesis, at least in part as a result of promoting the de novo synthesis of fatty acids.

| S-EPMC3864584 | biostudies-literature

Epitope mapping of a monoclonal antibody directed against Neisserial Heparin Binding Antigen by protein microarray

Project description:The aim of the study was to determine the epitope targeted by 31E10/E7 mouse monoclonal antibody (mAb). mAb 31E10/E7 was diluted at 1:2000 and incubated on a non-commercial Protein Microarray platform printed with NHBA specific recombinant protein fragments and full length NHBA of different variants.

2016-05-27 | GSE81378 | GEO

Epitope mapping of a monoclonal antibody directed against Neisserial Heparin Binding Antigen by peptide microarray

Project description:The aim of the study was to determine the epitope targeted by 31E10/E7 mouse monoclonal antibody (mAb) and the cross-reactivity to linear peptide epitopes of 10 different Neisserial Heparin Binding Antigen (NHBA) variants. mAb 31E10/E7 was diluted at 1:200 and incubated on a custom PepStar Peptide Microarray platform printed with 560 different peptides in three independent experiments.

2016-05-27 | GSE81377 | GEO

Epitope mapping for the monoclonal antibody that inhibits intramolecular electron transfer in flavocytochrome b2.

Project description:Flavocytochrome b(2) (yeast L-lactate dehydrogenase) carries one FMN and one protohaem IX on each of its four subunits. The prosthetic groups are bound to separate domains, the haem domain (residues 1-99) and the flavin domain (residues 100-485), which interact for electron transfer between lactate-reduced FMN and haem b(2); in vivo, the latter reduces cytochrome c. In the crystal structure, one haem domain out of two is mobile. Previously we have described a monoclonal antibody, raised against the tetramer, that only recognizes the native haem domain and prevents electron transfer between flavin and haem, while having no effect on flavin reduction by the substrate [Miles, Lederer and Lê (1998) Biochemistry 37, 3440-3448]. In order to understand the structural basis of the uncoupling between the domains, we proceeded to site-directed mutagenesis, so as to map the epitope on the surface of the haem domain. We analysed the effects of 14 mutations at 12 different positions, located mostly in the domain interface or at its edge; we also analysed the effect of replacing protohaem IX with its dimethyl ester. We used as criteria the antibody-mediated inhibition of cytochrome c reduction by flavocytochrome b(2), competitive ELISA tests and surface plasmon resonance. We have thus defined a minimal epitope surface on the haem domain; it encompasses positions 63, 64, 65, 67, 69 and 70 and one or both haem propionates. When the haem and flavin domains are docked for electron transfer, the 65, 67 and 70 side chains, as well as the haem propionates, are excluded from solvent. The present results thus indicate that, when bound, the antibody acts as a wedge between the domains and constitutes a physical barrier to electron transfer.

| S-EPMC1223457 | biostudies-other

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134.

Project description:The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377-RGDSFTHTPP-386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

| S-EPMC5986659 | biostudies-literature

Epitope Mapping by NMR of a Novel Anti-A? Antibody (STAB-MAb).

Project description:Alzheimer´s Disease (AD) is one of the most common neurodegenerative disorders worldwide. Excess of ?-amyloid (A?), a peptide with a high propensity to misfold and self-aggregate, is believed to be the major contributor to the observed neuronal degeneration and cognitive decline in AD. Here, we characterize the epitope of a novel anti-A? monoclonal antibody, the STAB-MAb, which has previously demonstrated picomolar affinities for both monomers (KD?=?80 pM) and fibrils (KD?=?130 pM) of A?(1-42) and has shown therapeutic efficacy in preclinical mouse models of AD. Our findings reveal a widespread epitope that embraces several key A? residues that have been previously described as important in the A? fibrillation process. Of note, STAB-MAb exhibits a stronger affinity for the N-terminus of A? and stabilizes an ?-helix conformation in the central to N-terminal region of the peptide, in addition to disrupting a characteristic salt-bridge of a hairpin structure present in fibrils. The NMR derived epitope supports the observed results from ThT-monitored fluorescence and electron microscopy experiments, in which STAB-MAb was shown to inhibit the formation of aggregates and promote disruption of pre-formed fibrils. In combination with the published in vitro and in vivo assays, our study highlights STAB-MAb as a rare and versatile antibody with analytical, diagnostic and therapeutic efficacy.

| S-EPMC6706428 | biostudies-literature

Epitope mapping of anti-drug antibodies to a clinical candidate bispecific antibody.

Project description:Anti-drug antibodies (ADA) can limit the efficacy and safety of therapeutic antibodies. However, determining the exact nature of ADA interactions with the target drug via epitope mapping is challenging due to the polyclonal nature of the IgG response. Here, we demonstrate successful proof-of-concept for the application of hydroxyl radical footprinting (HRF)-mass spectrometry for epitope mapping of ADAs obtained from goats that were administered a knob-into-hole bispecific antibody (BsAb1). Subsequently, we performed epitope mapping of ADAs obtained from cynomolgus (cyno) monkeys that were administered BsAb1 as we described in a recently published paper. Herein, we provide the first data to demonstrate the feasibility of using HRF for ADA epitope mapping, and show that both goat and cyno-derived ADAs specifically target the complementary-determining regions in both arms of BsAb1, suggesting that the ADA epitopes on BsAb1 may be species-independent.

| S-EPMC8794239 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data