Recombinant Mouse Cytosolic carboxypeptidase 2 (Agbl2), partial

What is Recombinant Mouse Cytosolic carboxypeptidase 2 (Agbl2) and what are its primary functions?

Recombinant Mouse Cytosolic carboxypeptidase 2 (Agbl2) is a protein enzyme that belongs to the cytosolic carboxypeptidase family. Its primary functions include:

• Mediating deglutamylation of tubulin and non-tubulin target proteins

• Catalyzing the removal of polyglutamate side chains on the gamma-carboxyl group of glutamate residues within the C-terminal tail of tubulin protein

• Cleaving tubulin long-side-chains

• Catalyzing the removal of polyglutamate residues from the carboxy-terminus of non-tubulin proteins

These enzymatic activities play crucial roles in microtubule dynamics and cytoskeletal organization. The deglutamylation process is a post-translational modification that affects protein function and interaction networks, influencing various cellular processes including cell division, intracellular transport, and morphogenesis.

What expression systems are available for producing Recombinant Mouse Agbl2?

Recombinant Mouse Agbl2 can be produced using several expression systems, each with distinct advantages for different research applications:

The biotinylated version is produced using E. coli biotin ligase (BirA), which covalently attaches biotin to the 15 amino acid AviTag peptide with high specificity . This provides a convenient tool for detection, immobilization, and pull-down experiments.

What are the optimal storage and reconstitution conditions for Recombinant Mouse Agbl2?

For optimal stability and activity of Recombinant Mouse Agbl2:

• Initial handling: Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitution: Use deionized sterile water to reconstitute the lyophilized powder to a concentration of 0.1-1.0 mg/mL

• Long-term storage: Add 5-50% glycerol (final concentration) and aliquot for storage at -20°C/-80°C

• Quality control: Verify protein purity (>85% by SDS-PAGE) before experimental use

Following these procedures minimizes freeze-thaw cycles and prevents protein degradation, ensuring consistent experimental results. The addition of glycerol prevents ice crystal formation during freezing, which can denature proteins.

How does Agbl2 contribute to cancer progression mechanisms?

Agbl2 has been identified as a critical oncogenic factor in hepatocellular carcinoma (HCC), with multiple mechanisms contributing to cancer progression:

• Overexpression pattern: AGBL2 is frequently overexpressed in HCC tissues and cell lines compared to normal tissues

• Clinical correlations: High expression of AGBL2 positively associates with:

  • Increased tumor size

  • Tumor multiplicity

  • Advanced clinical stage (p < 0.05)

  • Poor prognosis (independent prognostic factor)

• Cellular mechanisms:

  • Enhances HCC cell survival and proliferation in vitro

  • Promotes tumor growth in vivo

  • Inhibits apoptosis by enhancing IRGM-regulated autophagy

  • Up-regulates TPX2 expression and Aurora A activity

These findings suggest that AGBL2 functions through dual pathways: modulating autophagy to prevent cell death and activating Aurora A-dependent cell cycle progression. The correlation between AGBL2 expression and clinical features makes it a potential biomarker for prognosis and therapeutic targeting in HCC.

What methodologies are most effective for studying Agbl2's enzymatic activity?

To effectively measure and characterize Agbl2's deglutamylation activity, researchers should consider the following methodological approaches:

• Substrate preparation:

  • Purified tubulin with polyglutamylated C-terminal tails

  • Synthetic peptides containing polyglutamate chains

  • Fluorescently labeled substrates for real-time activity monitoring

• Activity assays:

  • Mass spectrometry to detect changes in substrate mass after deglutamylation

  • HPLC analysis of released glutamate residues

  • Antibody-based detection using glutamylation-specific antibodies (Western blot)

  • Fluorescence polarization assays with labeled substrates

• Controls and validations:

  • Use of catalytically inactive Agbl2 mutants (negative control)

  • Comparative analysis with other AGBL family members

  • Inhibitor studies (competitive and non-competitive)

  • Kinetic parameter determination (Km, Vmax, kcat)

• In-cell verification:

  • Immunofluorescence microscopy to visualize tubulin modification status

  • Co-immunoprecipitation to capture enzyme-substrate complexes

  • Live-cell imaging with fluorescent tubulin to monitor dynamics

These approaches provide complementary data on Agbl2's catalytic mechanism, substrate specificity, and cellular function, enabling comprehensive characterization of this enzyme's activity in both purified systems and cellular contexts.

How does co-expression of RARRES1 and AGBL2 impact renal cell carcinoma prognosis?

The co-expression patterns of RARRES1 (a carboxypeptidase inhibitor) and AGBL2 (its substrate carboxypeptidase) define distinct prognostic groups in renal cell carcinoma (RCC):

Multivariate analysis confirmed that the combination of RARRES1 cytoplasmic/negative and AGBL2 positive/negative immunostaining is a significant independent risk factor for tumor progression, indicating 11–15 times higher risk of cancer relapse compared to membranous RARRES1 expression .

These expression patterns significantly correlated with tumor size, grade, necrosis, T stadium, and stage (p < 0.001). The vast majority of AGBL2-positive cases occurred in tumors with RARRES1 membrane positivity. Of 454 conventional RCC with membranous RARRES1 expression, 110 tumors showed AGBL2-positive staining as well .

This co-expression pattern analysis provides a powerful prognostic tool for identifying patients who may benefit from more intensive surveillance and adjuvant therapy following surgery.

What is known about Agbl2's role in cellular signaling pathways beyond tubulin modification?

Beyond its canonical role in tubulin deglutamylation, Agbl2 engages in multiple signaling pathways:

• Autophagy regulation:

  • Enhances IRGM-regulated autophagy

  • This function contributes to apoptosis inhibition in cancer cells

  • May modulate autophagic flux through post-translational modifications of autophagy-related proteins

• Aurora A kinase pathway:

  • Up-regulates TPX2 expression

  • Enhances Aurora A activity

  • Promotes cell proliferation through cell cycle regulation

  • Potentially influences centrosome maturation and spindle assembly

• Potential developmental pathways:

  • A maternally inherited heterozygous change in AGBL2 was identified in a patient with:

    • Heart abnormalities

    • Global developmental delay

    • Brain MRI abnormality

    • Seizures (infantile spasms)

  • This suggests possible roles in neuronal development and cardiac function

These non-canonical functions expand our understanding of Agbl2 as a multifunctional protein beyond cytoskeletal regulation. Research is ongoing to determine whether the AGBL2 genetic variant is causative for the developmental phenotypes observed in the clinical case.

What experimental design considerations are important when investigating Agbl2's role in tumor growth in vivo?

When designing in vivo experiments to study Agbl2's role in tumor growth:

• Model selection:

  • Xenograft models using cell lines with Agbl2 overexpression or knockdown

  • Orthotopic implantation for organ-specific microenvironment effects

  • Patient-derived xenografts for clinical relevance

  • Genetically engineered mouse models with conditional Agbl2 expression

• Experimental controls:

  • Use of catalytically inactive Agbl2 mutants to distinguish enzymatic from scaffolding functions

  • Rescue experiments with wild-type Agbl2 in knockdown models

  • Paired analysis of primary tumors and metastatic lesions

• Assessment parameters:

  • Tumor volume measurements (caliper and imaging)

  • Survival analysis

  • Metastasis evaluation (histology, molecular markers)

  • Immunohistochemistry for proliferation (Ki-67), apoptosis (cleaved caspase-3), and autophagy markers (LC3, p62)

  • Analysis of tubulin modification status in tumor tissues

• Mechanistic investigations:

  • Ex vivo analysis of signaling pathway activation (Aurora A, TPX2)

  • Autophagy flux assessment in freshly isolated tumor cells

  • Pharmacological intervention studies targeting Agbl2-dependent pathways

  • Analysis of tumor microenvironment interactions

• Therapeutic implications:

  • Testing potential Agbl2 inhibitors in established tumors

  • Combination therapy approaches (with autophagy inhibitors, Aurora kinase inhibitors)

  • Assessment of resistance mechanisms

These design considerations ensure robust evaluation of Agbl2's contributions to tumor growth while providing mechanistic insights that may guide therapeutic development.

How can researchers validate the specificity and activity of purchased Recombinant Mouse Agbl2?

To validate recombinant Agbl2 before experimental use:

• Purity assessment:

  • SDS-PAGE analysis (expect >85% purity)

  • Western blot with specific anti-Agbl2 antibodies

  • Mass spectrometry for precise molecular weight confirmation

• Activity verification:

  • In vitro deglutamylation assay using polyglutamylated peptides

  • Comparison with established activity standards

  • Dose-dependent activity measurements

• Functional validation:

  • Complementation of Agbl2-depleted cell extracts

  • Rescue experiments in Agbl2 knockdown cells

  • Co-immunoprecipitation with known binding partners

• Troubleshooting inactive protein:

  • Check protein folding (circular dichroism spectroscopy)

  • Verify absence of aggregation (dynamic light scattering)

  • Test different buffer conditions for optimal activity

  • Evaluate potential inhibitors in the preparation

Systematic validation ensures experimental reproducibility and prevents misleading results from working with inactive or non-specific protein preparations.

What are the methodological considerations for investigating Agbl2's impact on autophagy?

To effectively study Agbl2's influence on autophagy:

• Autophagy flux measurement:

  • LC3-I to LC3-II conversion with and without lysosomal inhibitors

  • p62/SQSTM1 degradation kinetics

  • Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) to distinguish autophagosomes from autolysosomes

  • Long-lived protein degradation assays

• IRGM-Agbl2 interaction analysis:

  • Co-immunoprecipitation with appropriate controls

  • Proximity ligation assay for in situ interaction detection

  • FRET/BRET for dynamic interaction studies

  • Domain mapping to identify critical interaction regions

• Experimental manipulation approaches:

  • Genetic approaches: Agbl2 overexpression, knockdown/knockout

  • Pharmacological: Autophagy inducers (rapamycin, starvation) and inhibitors (chloroquine, bafilomycin A1)

  • Stress conditions: Nutrient deprivation, hypoxia, oxidative stress

• Readouts for functional consequences:

  • Cell survival assays following stress conditions

  • Apoptosis measurement (Annexin V/PI staining, caspase activation)

  • Cell cycle analysis to correlate with proliferation effects

  • Morphological assessment of autophagosome formation (electron microscopy)

Since Agbl2 enhances IRGM-regulated autophagy to inhibit apoptosis in HCC cells , these approaches will help elucidate the mechanistic details of this relationship and identify potential intervention points for therapeutic development.

What are promising avenues for developing Agbl2-targeted therapeutics?

Based on the oncogenic roles of Agbl2, several therapeutic development approaches show promise:

• Direct enzyme inhibition strategies:

  • Small molecule inhibitors targeting the catalytic domain

  • Peptide-based competitive inhibitors mimicking substrate recognition sites

  • Allosteric modulators affecting enzyme conformation

  • Structure-based drug design utilizing crystallographic data

• Pathway intervention approaches:

  • Dual targeting of Agbl2 and Aurora A signaling

  • Combination with autophagy inhibitors to enhance apoptosis sensitivity

  • IRGM-Agbl2 interaction disruptors

  • TPX2 expression modulators

• Translational considerations:

  • Biomarker development for patient stratification

  • Rational drug combinations based on expression patterns

  • Predictive models for treatment response

  • Resistance mechanism anticipation

• Delivery innovations:

  • Cancer-specific targeting strategies

  • Nucleic acid-based therapeutics (siRNA, antisense oligonucleotides)

  • Antibody-drug conjugates recognizing tumor cells with high Agbl2 expression

The strong association between Agbl2 overexpression and poor prognosis in HCC , along with its role in RCC progression when co-expressed with certain RARRES1 patterns , supports its validity as a therapeutic target. The dual role in autophagy modulation and Aurora A activation provides multiple intervention points that could be exploited for comprehensive pathway inhibition.

What is the potential significance of Agbl2 in neurodevelopmental disorders?

The identification of an AGBL2 genetic variant in a patient with neurological symptoms opens new research directions:

• Genotype-phenotype correlations:

  • Systematic assessment of AGBL2 variants in patients with:

    • Seizure disorders (particularly infantile spasms)

    • Global developmental delay

    • Brain structural abnormalities

    • Cardiac developmental issues

• Neurobiological mechanisms:

  • Role of tubulin post-translational modifications in:

    • Neuronal migration and differentiation

    • Axon guidance and synaptogenesis

    • Microtubule dynamics during brain development

    • Excitatory/inhibitory balance in neural circuits

• Model systems for investigation:

  • Patient-derived iPSCs differentiated into neurons

  • Agbl2 knockout/knockin mouse models

  • CRISPR-engineered variants in zebrafish

  • Brain organoids to study 3D developmental processes

• Translational implications:

  • Early molecular diagnosis

  • Development of precision therapies targeting affected pathways

  • Potential biomarkers for treatment response

  • Prevention strategies for high-risk pregnancies

The Undiagnosed Diseases Network's ongoing research into this maternally inherited heterozygous change in AGBL2 may reveal new functions for this enzyme in neurodevelopment, distinct from its better-characterized roles in cancer progression.