Browse
Submit Data
Databases
API
Help

Dataset Information

33 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Generation and Identification of Peptide-Based Monoclonal Antibodies Against Vacuolar Proton Pyrophosphatase of Toxoplasma gondii.

ABSTRACT: Vacuolar proton pyrophosphatase (V-PPase), an electrogenic proton pump widely distributed in non-mammalian species, is one of the important targets for acidocalcisomes. In this study, a novel method of peptide-based antibody generation was performed to produce monoclonal antibodies (MAbs) against Toxoplasma gondii V-PPase. Three hybridomas were identified and confirmed by ELISA, Western blotting, and immunofluorescence. All of them can react with an 85?kDa band of T. gondii protein in purified acidocalcisomal fraction. The three MAbs were all specific to the synthetic peptide of YTKAADVGADLSGKNEYGMSEDDPRNPAC, corresponding to amino acids at the location of 292aa-320aa of TgVP1 amino acid sequence. These specific MAbs will be valuable tools for further study of T. gondii infection biology, pathogenesis, and host immune response.

SUBMITTER: Tong C

PROVIDER: S-EPMC4492704 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Generation and Identification of Peptide-Based Monoclonal Antibodies Against Vacuolar Proton Pyrophosphatase of Toxoplasma gondii.

Tong Chengbi C Xiao Bin B Cheng Shasha S Li Wei W Liao Xiaoqing X Luo Shuhong S Hao Wenbo W

Monoclonal antibodies in immunodiagnosis and immunotherapy 20150601 3

Vacuolar proton pyrophosphatase (V-PPase), an electrogenic proton pump widely distributed in non-mammalian species, is one of the important targets for acidocalcisomes. In this study, a novel method of peptide-based antibody generation was performed to produce monoclonal antibodies (MAbs) against Toxoplasma gondii V-PPase. Three hybridomas were identified and confirmed by ELISA, Western blotting, and immunofluorescence. All of them can react with an 85 kDa band of T. gondii protein in purified a ...[more]

PMID: 26090597

Similar Datasets

Vacuolar proton pyrophosphatase activity and pyrophosphate (PPi) in Toxoplasma gondii as possible chemotherapeutic targets.

Project description:The addition of PP(i) promoted the acidification of a subcellular compartment in cell homogenates of Toxoplasma gondii tachyzoites, implying the presence of a proton-translocating pyrophosphatase. The proton gradient was collapsed by addition of the K(+)/H(+) antiporter nigericin, and was also inhibited by addition of the PP(i) analogue aminomethylenediphosphonate (AMDP). Both proton transport and PP(i) hydrolysis were dependent upon K(+), but Na(+) caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, NaF and to the thiol reagent N-ethylmaleimide. This activity was unaffected by common inhibitors of phosphohydrolases, except that NaO(3)V (sodium orthovanadate) stimulated the activity by 87%. Immunofluorescence microscopy, using antisera raised against conserved peptide sequences of a plant vacuolar pyrophosphatase, suggested that the pyrophosphatase in T. gondii tachyzoites was located in the plasma membrane and intracellular vacuoles of the parasite. High-field (31)P-NMR spectroscopy showed that PP(i )was more abundant than ATP in tachyzoites. Bisphosphonates (PP(i) analogues), drugs that are used in the treatment of bone diseases, inhibited proton transport and PP(i) hydrolysis in tachyzoite homogenates, and also inhibited intracellular proliferation of tachyzoites in tissue culture cells.

| S-EPMC1221200 | biostudies-other

Production and characterization of monoclonal antibodies against <i>Toxoplasma gondii</i> ROP18 with strain-specific reactivity.

Project description:The rhoptry kinase 18 of Toxoplasma gondii (TgROP18) has been identified as a key virulence factor that allows the parasite to escape from host immune defences and promotes its proliferation in host cells. Although much research is focused on the interaction between host cells and TgROP18, the development of monoclonal antibodies (mAbs) against TgROP18 has not been reported till date. To produce mAbs targeting TgROP18, two hybridomas secreting mAbs against TgROP18, designated as A1 and T2, were generated using cell fusion technology. The subtypes of the A1 and T2 mAbs were identified as IgG3 λ and IgM κ, and peptide scanning revealed that the core sequences of the antigenic epitopes were 180LRAQRRRSELVFE192 and 351NYFLLMMRAEADM363, respectively. The T2 mAb specifically reacted with both T. gondii type I and Chinese I, but not with T. gondii type II, Plasmodium falciparum or Schistosoma japonicum. Finally, the sequences of heavy chain and light chain complementarity-determining regions of T2 were amplified, cloned and characterized, making the modification of the mAb feasible in the future. The development of mAbs against TgROP18 would aid the investigation of the molecular mechanisms underlying the modulation of host cellular functions by TgROP18, and in the development of strategies to diagnose and treat Toxoplasmosis.

| S-EPMC10317636 | biostudies-literature

rROP2(186-533): a novel peptide antigen for detection of IgM antibodies against Toxoplasma gondii.

Project description:Toxoplasma gondii infections are prevalent in a wide range of mammalian hosts including humans. Infection in pregnant women may cause the transmission of parasite to the fetus that makes serious problems. IgM antibodies against Toxoplasma (Toxo-IgM) have been believed to be significant indicators for both recently acquired and congenital toxoplasmosis. So far, however, there has not been any recognized protein of T. gondii that specifically reacts to IgM antibodies. Here, an antigen exclusively for detection of IgM antibodies screened by two-dimensional electrophoresis and mass spectrometry has been reported. The study identified 13 Toxoplasma proteins probed by IgG antibodies and one (rhpotry protein 2 [ROP2]) by IgM antibodies with human sera of Toxo-IgM(-)-IgG(+) and -IgM(+)-IgG(-), respectively, which had been prescreened by Toxo-IgM and -IgG commercial kits from the suspected cases. Following cloning, expression, and purification of the fragment of ROP2(186-533), an enzyme-linked immunosorbent assay with rROP2(186-533) to measure IgM and IgG antibodies was developed. As a result, 100%(48/48) of sera with Toxo-IgM(+)-IgG(-)showed positive Toxo-IgM but none of them (0%) showed positive Toxo-IgG when rROP2(186-533) was used as antigen. Neither Toxo-IgG nor Toxo-IgM antibodies were found when tested with 59 sera of Toxo-IgM(-)-IgG(+). These results indicate that rROP2(186-533) could be used as an antigen that specifically capture Toxo-IgM antibodies and may have a high potential in the serological diagnosis of both acute acquired and congenital toxoplasmosis.

| S-EPMC3250629 | biostudies-literature

A vacuolar-H(+) -pyrophosphatase (TgVP1) is required for microneme secretion, host cell invasion, and extracellular survival of Toxoplasma gondii.

Project description:The vacuolar proton pyrophosphatase (H(+) -PPase) of Toxoplasma gondii (TgVP1), a membrane proton pump, localizes to acidocalcisomes and a novel lysosome-like compartment termed plant-like vacuole (PLV) or vacuolar compartment (VAC). We report the characterization of a T. gondii null mutant for the TgVP1 gene. Propagation of these mutants decreased significantly because of deficient attachment and invasion of host cells, which correlated with deficient microneme secretion. Processing of cathepsin L (CPL) in these mutants was deficient only when the parasites were incubated in the presence of low concentrations of the vacuolar H(+) -ATPase (V-H(+) -ATPase) inhibitor bafilomycin A1 , suggesting that either TgVP1 or the T. gondii?V-H(+) -ATPase (TgVATPase) are sufficient to support CPL processing. The lack of TgVP1 did not affect processing of micronemal proteins, indicating that it does not contribute to proMIC maturations. The TgVP1?null mutants were more sensitive to extracellular conditions and were less virulent in mice. We demonstrate that T. gondii tachyzoites possess regulatory volume decrease capability during hypo-osmotic stress and this ability is impaired in TgVP1?null mutants implicating TgVP1 in osmoregulation. We hypothesize that osmoregulation is needed for host cell invasion and that TgVP1 plays a role during the normal lytic cycle of T. gondii.

| S-EPMC4159726 | biostudies-literature

A plasma membrane-type Ca(2+)-ATPase co-localizes with a vacuolar H(+)-pyrophosphatase to acidocalcisomes of Toxoplasma gondii.

Project description:Ca(2+)-ATPases are likely to play critical roles in the biochemistry of Toxoplasma gondii, since these protozoa are obligate intracellular parasites and the Ca(2+) concentration in their intracellular location is three orders of magnitude lower than in the extracellular medium. Here, we report the cloning and sequencing of a gene encoding a plasma membrane-type Ca(2+)-ATPase (PMCA) of T.gondii (TgA1). The predicted protein (TgA1) exhibits 32-36% identity to vacuolar Ca(2+)-ATPases of Trypanosoma cruzi, Saccharomyces cerevisiae, Entamoeba histolytica and Dictyostelium discoideum. Sequencing of both cDNA and genomic DNA from T.gondii indicated that TgA1 contains two introns near the C-terminus. A hydropathy profile of the protein suggests 10 transmembrane domains. TgA1 suppresses the Ca(2+) hypersensitivity of a mutant of S.cerevisiae that has a defect in vacuolar Ca(2+) accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that TgA1 localizes to the plasma membrane and co-localizes with the vacuolar H(+)-pyrophosphatase to intracellular vacuoles identified morphologically and by X-ray microanalysis as the acidocalcisomes. This vacuolar-type Ca(2+)-ATPase could play an important role in Ca(2+) homeostasis in T.gondii.

| S-EPMC140201 | biostudies-literature

Vacuolar Proton Pyrophosphatase Is Required for High Magnesium Tolerance in Arabidopsis.

Project description:Magnesium (Mg2+) is an essential nutrient in all organisms. However, high levels of Mg2+ in the environment are toxic to plants. In this study, we identified the vacuolar-type H⁺-pyrophosphatase, AVP1, as a critical enzyme for optimal plant growth under high-Mg conditions. The Arabidopsisavp1 mutants displayed severe growth retardation, as compared to the wild-type plants upon excessive Mg2+. Unexpectedly, the avp1 mutant plants retained similar Mg content to wild-type plants under either normal or high Mg conditions, suggesting that AVP1 may not directly contribute to Mg2+ homeostasis in plant cells. Further analyses confirmed that the avp1 mutant plants contained a higher pyrophosphate (PPi) content than wild type, coupled with impaired vacuolar H⁺-pyrophosphatase activity. Interestingly, expression of the Saccharomyces cerevisiae cytosolic inorganic pyrophosphatase1 gene IPP1, which facilitates PPi hydrolysis but not proton translocation into vacuole, rescued the growth defects of avp1 mutants under high-Mg conditions. These results provide evidence that high-Mg sensitivity in avp1 mutants possibly resulted from elevated level of cytosolic PPi. Moreover, genetic analysis indicated that mutation of AVP1 was additive to the defects in mgt6 and cbl2 cbl3 mutants that are previously known to be impaired in Mg2+ homeostasis. Taken together, our results suggest AVP1 is required for cellular PPi homeostasis that in turn contributes to high-Mg tolerance in plant cells.

| S-EPMC6274811 | biostudies-literature

Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment.

Project description:Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide-MHCII complexes.

| S-EPMC4909947 | biostudies-literature

The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii.

Project description:Iron is essential to cells as a cofactor in enzymes of respiration and replication, however without correct storage, iron leads to the formation of dangerous oxygen radicals. In yeast and plants, iron is transported into a membrane-bound vacuole by the vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii. Here, we assess the role of VIT and iron storage in T. gondii. By deleting VIT, we find a slight growth defect in vitro, and iron hypersensitivity, confirming its essential role in parasite iron detoxification, which can be rescued by scavenging of oxygen radicals. We show VIT expression is regulated by iron at transcript and protein levels, and by altering VIT localization. In the absence of VIT, T. gondii responds by altering expression of iron metabolism genes and by increasing antioxidant protein catalase activity. We also show that iron detoxification has an important role both in parasite survival within macrophages and in virulence in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.

| S-EPMC10281983 | biostudies-literature

The Vacuolar Zinc Transporter TgZnT Protects Toxoplasma gondii from Zinc Toxicity.

Project description:Zinc (Zn2+) is the most abundant biological metal ion aside from iron and is an essential element in numerous biological systems, acting as a cofactor for a large number of enzymes and regulatory proteins. Zn2+ must be tightly regulated, as both the deficiency and overabundance of intracellular free Zn2+ are harmful to cells. Zn2+ transporters (ZnTs) play important functions in cells by reducing intracellular Zn2+ levels by transporting the ion out of the cytoplasm. We characterized a Toxoplasma gondii gene (TgGT1_251630, TgZnT), which is annotated as the only ZnT family Zn2+ transporter in T. gondii TgZnT localizes to novel vesicles that fuse with the plant-like vacuole (PLV), an endosome-like organelle. Mutant parasites lacking TgZnT exhibit reduced viability in in vitro assays. This phenotype was exacerbated by increasing zinc concentrations in the extracellular media and was rescued by media with reduced zinc. Heterologous expression of TgZnT in a Zn2+-sensitive Saccharomyces cerevisiae yeast strain partially restored growth in media with higher Zn2+ concentrations. These results suggest that TgZnT transports Zn2+ into the PLV and plays an important role in the Zn2+ tolerance of T. gondii extracellular tachyzoites.IMPORTANCEToxoplasma gondii is an intracellular pathogen of human and animals. T. gondii pathogenesis is associated with its lytic cycle, which involves invasion, replication, egress out of the host cell, and invasion of a new one. T. gondii must be able to tolerate abrupt changes in the composition of the surrounding milieu as it progresses through its lytic cycle. We report the characterization of a Zn2+ transporter of T. gondii (TgZnT) that is important for parasite growth. TgZnT restored Zn2+ tolerance in yeast mutants that were unable to grow in media with high concentrations of Zn2+ We propose that TgZnT plays a role in Zn2+ homeostasis during the T. gondii lytic cycle.

| S-EPMC6531880 | biostudies-literature

Epitope mapping of 11 monoclonal antibodies directed against Neisserial adesin A by peptide microarray

Project description:The aim of the study was to determine the epitope targeted by four different HumAbs and the cross-reactivity to linear peptide epitopes of 5 different Neisserial adesin A (NadA) variants. the HumAbs were diluted at 1:200 and incubated on a custom PepStar Peptide Microarray platform printed with 348 different peptides.

2018-03-07 | GSE98881 | GEO

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