PLPBP Antibody

What is PLPBP and why is it significant in research?

PLPBP (formerly known as PROSC) is an evolutionarily conserved protein that binds pyridoxal 5′-phosphate (PLP), the active form of vitamin B6. Its significance stems from its association with vitamin B6-dependent epilepsy, where mutations in the PLPBP gene cause severe neurological symptoms . The protein is present in both cytosolic and mitochondrial compartments and appears to play roles beyond simple PLP binding . Research interest has increased since the discovery that PLPBP deficiency markedly alters cellular PLP and PL (pyridoxal) concentrations, affecting amino acid homeostasis .

What validated applications exist for PLPBP antibodies in cellular studies?

PLPBP antibodies have been validated for multiple experimental applications including Western blotting, immunohistochemistry, and flow cytometry . These techniques allow researchers to:

• Detect native PLPBP in various human cell types and tissues

• Evaluate subcellular localization (confirming dual cytosolic and mitochondrial presence)

• Quantify expression levels in different experimental conditions

• Track alterations in PLPBP levels during pathological states

For optimal results, researchers should verify that their selected antibody has been validated for their specific application and cellular model, as some antibodies may perform better in certain techniques than others .

How can PLPBP antibodies help characterize normal versus pathological protein states?

PLPBP antibodies are valuable tools for distinguishing between normal and disease-associated variants of the protein. In vitamin B6-dependent epilepsy, mutations such as p.Leu175Pro, p.Arg241Gln, and p.Pro87Leu affect protein folding, stability, and cofactor binding . Antibodies can help researchers:

• Compare wild-type versus mutant PLPBP expression levels

• Assess protein stability differences through pulse-chase experiments

• Visualize altered subcellular localization patterns

• Detect conformational changes that might affect epitope accessibility

• Monitor oligomerization states (PLPBP exists predominantly as dimers with a minor monomeric fraction)

This approach has revealed that disease-causing mutations significantly impact PLPBP's structure-function relationship, with some mutations causing gross misfolding (like p.Leu175Pro) while others affect solubility or stability without obvious structural changes .

How can researchers investigate PLPBP's unexpected dimeric structure?

Recent research has uncovered that human PLPBP exists primarily in a dimeric state rather than the previously assumed monomeric form . To investigate this unexpected oligomerization:

• Chemical cross-linking coupled with mass spectrometry can be employed using cell-permeable cross-linkers like DSSO

• Size-exclusion chromatography can separate monomeric and dimeric forms for further analysis

• Analytical ultracentrifugation provides complementary data on oligomeric states

• UV/vis spectroscopy can confirm PLP binding in both monomeric and dimeric species by detecting characteristic internal aldimine peaks (λmax ≈ 336 nm and λmax ≈ 425 nm)

The dimeric assembly significantly affects cofactor accessibility, as demonstrated by studies with d-cycloserine (DCS). The internal aldimine of monomeric PLPBP is almost completely lost upon incubation with 10 mM DCS, while the dimeric form retains its aldimine even at 50 mM DCS , suggesting functional differences between oligomeric states.

What strategies can be employed to map PLPBP's protein interaction network using antibodies?

To elucidate PLPBP's cellular function through its interaction network:

• Employ co-immunoprecipitation (co-IP) with PLPBP antibodies combined with cross-linking agents like DSSO

• Analyze pull-down complexes using mass spectrometry to identify interaction partners

• Validate key interactions through reciprocal co-IP or proximity ligation assays

• Perform gene ontology (GO) analysis to identify functional clusters among interacting proteins

Studies using this approach have revealed that approximately 85% of PLPBP interactors in HEK293 cells are cytosolic proteins, with a significant enrichment of cytoskeleton- and cell division-associated proteins (about 22%) . Interestingly, contrary to previous hypotheses, PLP-dependent enzymes are not overrepresented among PLPBP interactors, suggesting the protein is not primarily involved in cofactor delivery to apo-enzymes .

How can PLPBP antibodies help investigate the protein's role in vitamin B6-dependent epilepsy?

To study PLPBP's involvement in vitamin B6-dependent epilepsy:

• Generate cellular models expressing epilepsy-causing PLPBP variants through transfection

• Use antibodies to compare mutant versus wild-type expression, stability, and localization

• Conduct proteomic analyses in PLPBP knockout and rescued cell lines

• Investigate changes in key metabolic pathways affected by PLPBP mutations

Research has shown that PLPBP knockout cells display significant proteomic changes, including upregulation of cytoskeleton- and cell division-associated proteins . Additionally, two PLP-dependent enzymes important for H₂S synthesis (CTH and CBS) are downregulated, potentially linking PLPBP deficiency to pathological mechanisms affecting muscle and nervous system function .

What are the optimal sample preparation techniques for PLPBP antibody applications?

For successful PLPBP detection across different experimental platforms:

When preparing samples for PLP-binding studies, it's crucial to consider that PLPBP binds PLP through a Schiff-base linkage at Lys47, which can be confirmed through LC-MS/MS analysis after reduction with NaBH₄ .

How can researchers accurately quantify PLPBP expression levels in different tissues?

For precise quantification of PLPBP:

• Establish calibration curves using recombinant PLPBP at known concentrations

• Include appropriate housekeeping protein controls for normalization

• Consider the oligomeric state of PLPBP, as dimeric and monomeric forms may exhibit different antibody accessibility

• For absolute quantification, use techniques like AQUA (Absolute QUAntification) with isotope-labeled peptide standards

It's worth noting that PLPBP expression levels in yeast and HeLa cells are approximately 10-fold higher than the median protein copy number , which should be considered when interpreting quantification results in different experimental systems.

What controls should be included when using PLPBP antibodies in research?

To ensure experimental validity:

For knockout validation, CRISPR/Cas9-generated PLPBP-deficient cells show distinct phenotypes, including proteomic changes involving cytoskeleton proteins and PLP-dependent enzymes , which can serve as functional validation of antibody specificity.

How can researchers address potential cross-reactivity with other PLP-binding proteins?

PLPBP is one of many cellular proteins that bind PLP. To ensure specificity:

• Validate antibody specificity against recombinant PLPBP and related proteins

• Use epitope mapping to confirm the antibody targets unique regions of PLPBP

• Employ PLPBP knockout cells as negative controls to confirm signal absence

• Consider competition assays with recombinant protein to verify signal displacement

While PLPBP shares the common feature of PLP binding with many enzymes, its unique TIM-barrel-like structure provides distinct epitopes that can be targeted by specific antibodies.

What strategies can help detect both monomeric and dimeric forms of PLPBP?

To successfully analyze both oligomeric states:

• Use native gel electrophoresis or BN-PAGE (Blue Native PAGE) instead of standard SDS-PAGE

• Apply gentle sample preparation conditions that preserve protein-protein interactions

• Consider chemical cross-linking to stabilize dimeric forms before analysis

• Use size-exclusion chromatography to separate oligomeric states prior to antibody-based detection

Research has shown that the dimeric form of PLPBP exhibits different cofactor accessibility compared to the monomeric form, with the dimer potentially serving as a PLP storage mechanism . This functional difference may be critical when interpreting experimental results.

How can researchers investigate PLPBP's role in cellular PLP homeostasis?

To study PLPBP's proposed function as a PLP storage protein:

• Use antibodies to track PLPBP expression under different vitamin B6 concentrations

• Combine immunoprecipitation with metabolite analysis to measure bound versus free PLP

• Implement pulse-chase experiments with labeled vitamin B6 to monitor turnover rates

• Study the effects of PLPBP knockdown/knockout on cellular PLP levels and metabolism

Research indicates that PLPBP deficiency increases free cellular PLP levels, which may subsequently be detoxified by reduction to PNP (pyridoxine 5'-phosphate) due to PLP's reactive aldehyde group . This mechanism could explain the high PNP levels observed in PLPBP knockout cells and patient fibroblasts .

How can PLPBP antibodies contribute to understanding the protein's role in cytoskeleton organization?

To investigate PLPBP's unexpected connection to cytoskeletal regulation:

• Use antibodies for co-localization studies with cytoskeletal proteins identified in interaction networks

• Perform proximity ligation assays to confirm direct interactions in situ

• Compare cytoskeletal organization in wild-type versus PLPBP-deficient cells using immunofluorescence

• Implement live-cell imaging with fluorescently tagged PLPBP antibody fragments to track dynamic interactions

Interaction studies have identified associations between PLPBP and components of the γ-tubulin ring complex (TUBGCP2–4), proteins involved in centriole and spindle formation (CEP78, CEP97, CEP350, CP110), and subunits of the F-actin capping complex (CAPZA1–A2, CAPZB) , suggesting a structural or regulatory role beyond PLP metabolism.

What methodologies can help determine if PLPBP has enzymatic activity?

Although primarily characterized as a binding protein, to investigate potential enzymatic functions:

• Immunopurify PLPBP using specific antibodies for in vitro activity assays

• Compare wild-type and catalytic site mutants (such as the PLP-binding Lys47) for functional differences

• Develop activity-based protein profiling approaches specific to potential PLPBP catalytic mechanisms

• Combine structural information with metabolomic analyses in PLPBP-modulated systems

While current evidence primarily supports a non-enzymatic role for PLPBP , its evolutionary conservation and structural features warrant investigation of potential catalytic activities that may have been overlooked.

How can researchers investigate tissue-specific functions of PLPBP using antibodies?

To explore PLPBP's tissue-specific roles, particularly in the context of neurological and muscular pathologies:

• Employ immunohistochemistry with validated PLPBP antibodies across different tissue types

• Analyze expression patterns in tissues affected by vitamin B6-dependent epilepsy

• Investigate co-expression with tissue-specific PLP-dependent enzymes

• Study developmental expression patterns in models of PLPBP-associated disorders

Experimental evidence suggests that PLPBP may have tissue-specific functions beyond PLP binding, particularly in muscle integrity and nervous system function . These specialized roles may explain why PLPBP mutations primarily manifest as neurological symptoms despite the protein's ubiquitous expression.