MPPED2 Antibody

What is MPPED2 and what is its primary function in cellular processes?

MPPED2 (Metallophosphoesterase Domain Containing 2) is a protein that functions as a metallophosphodiesterase, cleaving 3',5'-cyclic phosphate nucleosides into 5'-phosphate nucleosides. It regulates the levels of cyclic second messengers including cAMP and cGMP and their degradation rates . MPPED2 exhibits low phosphodiesteric activity against cAMP and cGMP due to an amino acid replacement (glycine replacing histidine) at position 252 in the highly conserved catalytic site . This unique substitution allows the active site to retain AMP or GMP with strong affinity but reduces its catalytic efficiency.

MPPED2 is highly expressed in fetal and adult brain tissue and plays roles in neurodevelopment . Recent studies indicate MPPED2 functions as a tumor suppressor in multiple cancer types.

What types of MPPED2 antibodies are currently available for research applications?

Currently available MPPED2 antibodies can be categorized based on several characteristics:

For example, catalog number ABIN6750479 is a rabbit polyclonal antibody targeting the N-terminal region with reactivity to human, mouse, rat, cow, horse, rabbit, pig, and chicken MPPED2 . Similarly, catalog number 13270-1-AP is a rabbit polyclonal antibody validated for WB, IHC, and IF/ICC applications primarily on human samples .

How does MPPED2 expression vary across different tissues and what implications does this have for antibody selection?

MPPED2 shows tissue-specific expression patterns that researchers should consider when selecting antibodies:

• Highest expression occurs in brain tissue, making it an ideal positive control

• Expression is downregulated in multiple cancer types, including glioblastoma, cervical cancer, thyroid neoplasia, and breast cancer

• Expression levels correlate with brain development stages, with high expression in fetal brain

These expression patterns have methodological implications:

• Brain tissue should be used as a positive control when validating antibodies

• For cancer studies, paired normal-tumor samples are essential for comparative analysis

• When working with low-expressing tissues, more sensitive detection methods may be required

• The observed molecular weight is consistently reported as approximately 33 kDa

What are the optimal conditions for Western blot detection of MPPED2?

Based on validated protocols from multiple antibody suppliers, optimal Western blot conditions for MPPED2 detection include:

For troubleshooting weak signals:

• Increase protein loading for tissues with low MPPED2 expression

• Extend primary antibody incubation to overnight at 4°C

• Use enhanced chemiluminescence detection systems with longer exposure times

• Consider concentrating protein samples when working with cell lines showing low expression

How should samples be prepared for optimal immunohistochemical detection of MPPED2?

For successful IHC detection of MPPED2, the following protocol parameters have been validated:

For brain tissue specifically, studies have shown strong MPPED2 immunoreactivity in normal brain samples, which progressively decreases in low-grade gliomas and is minimally detected in glioblastoma tissues . This pattern makes brain tissue an excellent model for validating antibody specificity and optimizing staining protocols.

What controls should be included when using MPPED2 antibodies in experimental protocols?

To ensure reliable and reproducible results with MPPED2 antibodies, incorporate these essential controls:

Example: In glioblastoma studies, researchers validated MPPED2 antibody specificity by comparing expression patterns in normal brain tissue (high expression), low-grade gliomas (moderate expression), and glioblastomas (low expression), correlating protein detection with mRNA expression data from databases like TCGA and Gravendeel .

How is MPPED2 expression altered in different cancer types and what methodologies best detect these changes?

MPPED2 expression is consistently downregulated across multiple cancer types:

In glioblastoma specifically, MPPED2 downregulation:

• Correlates with tumor grade (lowest in grade IV tumors)

• Shows a negative correlation with the aggressive mesenchymal subtype

• Is associated with promoter hypermethylation (r = -0.4495, p < 0.0028)

• Correlates positively with patient survival

For reliable detection of these expression changes, multi-modal approaches are recommended:

• Start with mRNA expression analysis (qRT-PCR or RNA-seq)

• Confirm at protein level with Western blotting (quantitative)

• Visualize cellular and tissue distribution changes with IHC or IF

• Correlate with methylation status of the MPPED2 promoter region

What functional studies demonstrate MPPED2's role as a tumor suppressor and how can researchers replicate these approaches?

Multiple functional studies support MPPED2's tumor suppressor role:

To replicate these functional studies, researchers should:

• Generate stable or transient MPPED2-overexpressing cell lines using appropriate expression vectors

• Confirm overexpression by both qRT-PCR and Western blotting

• Perform comparative functional assays (proliferation, migration, drug sensitivity)

• Analyze molecular pathway alterations through Western blotting for key signaling components

• Include appropriate controls (empty vector transfected cells)

The documented inhibitory effects on PI3K/AKT and NF-kB pathways may be related to MPPED2's role in regulating cAMP levels, as subtle changes in cAMP have been shown to modulate the phosphorylation of both p65 and PI3K/AKT .

How can MPPED2 be studied in relation to p16INK4A expression in cervical carcinoma?

Recent research has revealed an interesting relationship between MPPED2 and p16INK4A in cervical carcinoma:

To study this relationship, researchers can:

• Use a tissue cohort of cervical samples with known HPV status (high-risk HPV positive, low-risk HPV positive, and HPV negative)

• Perform serial section IHC for both MPPED2 and p16INK4A

• Apply semi-quantitative scoring methods as described by Kaur et al.:

  • Grade 0-3 based on staining intensity (no staining to strong)

  • Estimate percentage of positive cells (0-100%)

  • Calculate total score by combining intensity and percentage (maximum 8)

• Correlate expression patterns with clinical parameters and HPV status

• Perform in vitro studies using HPV-positive and HPV-negative cervical cancer cell lines to examine the functional relationship

How does MPPED2 polymorphism rs2065418 affect systemic inflammation and what methodologies are used to study this association?

The rs2065418 polymorphism in the MPPED2 gene has been associated with altered systemic inflammation and clinical outcomes:

Research methodologies to study this association include:

• Genotyping trauma patients using real-time PCR to identify rs2065418 variants

• Stratifying patients based on injury severity (e.g., ISS ≥ 25)

• Collecting and analyzing clinical data including:

  • Hospital length of stay

  • Multiple Organ Dysfunction Scores (MODScores)

  • Duration of mechanical ventilation

  • Plasma creatinine levels over time

• Measuring plasma cytokine levels to assess inflammatory profiles

• Statistical analysis to correlate genotype with clinical and inflammatory outcomes

Research has found that a certain injury severity threshold must be exceeded to observe the greater impact of rs2065418 TT genotype on outcomes, suggesting context-dependent effects of this polymorphism .

What techniques are available for studying MPPED2's phosphodiesterase activity and substrate specificity?

As a metallophosphoesterase, MPPED2's enzymatic activity can be studied using these approaches:

Researchers should note that MPPED2 exhibits relatively low catalytic efficiency compared to other phosphodiesterases due to the glycine substitution at position 252, which enables strong substrate binding but reduces turnover rate . Studies in Drosophila have confirmed that the ortholog (dMpped) hydrolyzes phosphodiester substrates including cAMP and cGMP in a metal-dependent manner, suggesting evolutionary conservation of this function .

How can evolutionary conservation of MPPED2 be leveraged in research using model organisms?

MPPED2 is highly conserved across species, offering opportunities for comparative research:

Research approaches leveraging evolutionary conservation:

• Cross-species rescue experiments (e.g., expressing human MPPED2 in dMpped knockout flies)

• Comparative expression analysis across developmental stages in different organisms

• Structural biology approaches to identify conserved functional domains

• Study of simpler organisms to elucidate basic MPPED2 functions before moving to more complex models

In Drosophila, knockout of dMpped resulted in reduced lifespan, elevated cAMP and cGMP levels in the brain, misregulation of immune pathways, and defective olfactory perception. Importantly, neuronal expression of mammalian MPPED2 restored normal lifespan in dMpped knockout flies, demonstrating functional conservation .

How should researchers address inconsistent MPPED2 detection across different antibodies and experimental systems?

When facing inconsistent MPPED2 detection, consider these troubleshooting approaches:

Validation protocol recommendation:

• Start with well-characterized samples (e.g., brain tissue positive control)

• Compare multiple antibodies targeting different epitopes

• Verify correlation between protein and mRNA expression levels

• Include MPPED2-overexpressing cells as additional positive controls

• Optimize protocol for each specific antibody and application

What are the key considerations when designing experiments to study epigenetic regulation of MPPED2?

MPPED2 downregulation in various cancers has been linked to promoter hypermethylation:

To investigate epigenetic regulation of MPPED2:

• Analyze promoter methylation status using:

  • Bisulfite sequencing (gold standard for detailed methylation patterns)

  • Methylation-specific PCR (MSP) for targeted analysis

  • Pyrosequencing for quantitative methylation assessment

• Correlate methylation with expression levels (qRT-PCR, Western blot)

• Perform demethylation experiments:

  • Treat cells with 5-aza-2'-deoxycytidine (DNA methyltransferase inhibitor)

  • Measure MPPED2 re-expression following treatment

• Chromatin immunoprecipitation (ChIP) to assess histone modifications

• Analyze TCGA and other public datasets for correlations between methylation and expression across cancer types

The significant inverse correlation between MPPED2 expression and its promoter methylation in GBM samples (p < 0.0028) provides strong evidence that hypermethylation is a key mechanism of MPPED2 silencing in cancer.

How can researchers effectively study MPPED2's role in modulating sensitivity to chemotherapeutic agents?

Building on findings that MPPED2 restoration enhances temozolomide sensitivity in glioblastoma:

Methodological recommendations:

• Generate stable cell lines with inducible MPPED2 expression to control expression timing and levels

• Perform dose-response curves with multiple chemotherapeutic agents

• Analyze both short-term (24-72h) and long-term (colony formation) effects

• Investigate mechanisms through which MPPED2 enhances drug sensitivity:

  • Changes in drug efflux/influx

  • DNA damage response modifications

  • Alterations in apoptotic threshold

  • Effects on PI3K/AKT and NF-kB survival pathways

• Validate findings across multiple cell lines representing different cancer subtypes

The observed sensitization to temozolomide through MPPED2 overexpression in glioblastoma cells provides a foundation for investigating similar effects with other chemotherapeutic agents and in other cancer types .