ENTPD3 Human, sf9

What is ENTPD3 and what are its key molecular characteristics?

ENTPD3, also known as CD39L3, Ecto-ATP Diphosphohydrolase 3, or NTPDase 3, is an ectonucleotidase that plays a critical role in hydrolyzing extracellular ATP and related nucleotides. When expressed in sf9 baculovirus cells, it forms a single, non-glycosylated polypeptide chain containing 451 amino acids (positions 44-485) with a theoretical molecular mass of 50.7 kDa, though it typically migrates at 50-70 kDa on SDS-PAGE under reducing conditions . The protein contains four apyrase-conserved regions characteristic of NTPases and exhibits a threefold preference for ATP hydrolysis over ADP hydrolysis . When commercially produced, it's often fused to a 6 amino acid His-tag at the C-terminus to facilitate purification through chromatographic techniques .

Why is sf9 insect cell expression system preferred for recombinant ENTPD3 production?

The sf9 baculovirus expression system offers several advantages for ENTPD3 production. This system can produce relatively large amounts of properly folded recombinant proteins with post-translational modifications. Unlike bacterial expression systems, sf9 cells can generate proteins that more closely resemble the native conformation of human ENTPD3 . The baculovirus-insect cell system provides a eukaryotic environment that supports the formation of disulfide bonds and proper protein folding while allowing for higher yields than mammalian cell systems. Additionally, the sf9 system efficiently produces the non-glycosylated form of ENTPD3, which is advantageous for certain structural and functional studies where glycosylation heterogeneity might complicate analysis .

How should researchers store and handle recombinant ENTPD3 to maintain stability?

For optimal stability of ENTPD3 Human (sf9), researchers should store the protein at 4°C if the entire vial will be used within 2-4 weeks. For longer periods, storage at -20°C is recommended. To prevent protein degradation during extended storage, it's advisable to add a carrier protein (0.1% HSA or BSA) . Multiple freeze-thaw cycles should be strictly avoided as they can significantly reduce enzymatic activity and promote protein degradation. ENTPD3 is typically provided in a formulation containing Phosphate Buffered Saline (pH 7.4) with 10% glycerol to enhance stability . When working with the protein, researchers should maintain sterile conditions and consider aliquoting the stock solution to minimize repeated freezing and thawing.

How can researchers verify the enzymatic activity of recombinant ENTPD3?

To assess the enzymatic activity of recombinant ENTPD3, researchers should employ ATP/ADP hydrolysis assays that monitor the conversion of these substrates to their respective products. Given ENTPD3's threefold preference for ATP over ADP hydrolysis , a comparative activity assay with both substrates can verify both function and substrate specificity. Malachite green phosphate detection assays can quantify the released inorganic phosphate. Alternatively, researchers can use coupled enzyme assays that link ATP hydrolysis to a colorimetric or fluorometric readout.

Kinetic parameters including Km and Vmax should be determined under physiologically relevant conditions (pH 7.4, 37°C). When characterizing a new batch of ENTPD3, comparing its activity to previous lots or established standards is essential for experimental consistency. Activity measurements should be performed in the presence of divalent cations (Ca²⁺, Mg²⁺) as these are typically required for optimal ectonucleotidase function.

What are the methodology considerations when using ENTPD3 as a beta cell marker?

ENTPD3 has emerged as a valuable marker for mature beta cells, particularly in stem cell-derived β-cell research . When utilizing ENTPD3 as a beta cell marker, researchers should consider several methodological approaches:

• Immunohistochemistry/Immunofluorescence: Co-staining with ENTPD3 and insulin (C-peptide) antibodies allows identification of mature beta cells. ENTPD3 is readily expressed in C-peptide positive (CPEP+) cells within stem cell-derived beta cell clusters, particularly marking CPEP+ caps .

• Flow Cytometry: Using ENTPD3-specific antibodies in combination with other markers (like insulin-GFP) enables isolation of mature beta cell populations from heterogeneous cell mixtures.

• Single-cell RNA sequencing: For transcriptomic profiling, ENTPD3 expression serves as a reliable indicator of beta cell maturity, as demonstrated in analyses of patients at different stages of Type 1 Diabetes .

• Validation across disease states: Researchers should verify ENTPD3 expression across relevant disease conditions, as it has been shown to be expressed consistently from healthy individuals to those with autoantibodies and recent-onset T1D .

What techniques are employed to develop ENTPD3-specific CAR-Tregs for immunotherapy applications?

The development of ENTPD3-specific Chimeric Antigen Receptor (CAR) regulatory T cells (Tregs) involves several sophisticated methodological approaches:

• Target identification and validation: Researchers must first confirm ENTPD3 expression in target tissues. Histological analysis of pancreatic samples and single-cell RNA sequencing have demonstrated consistent ENTPD3 co-expression with insulin across multiple stages of Type 1 Diabetes .

• CAR design using phage display: Novel phage display approaches have been employed to generate CARs directed against ENTPD3. This method has proven effective for developing scFvs (single-chain variable fragments) that can be incorporated into CAR constructs .

• Functional assessment: After transducing human nTregs with ENTPD3-specific CARs, researchers evaluate activation by measuring upregulation of markers including CD69, CD137, and the Treg-specific marker GARP following ENTPD3 exposure .

• Cross-suppression capability: Testing involves coculture experiments with ENTPD3-expressing B cells, allospecific T effector cells, and ENTPD3 CAR Tregs to demonstrate that ENTPD3 CAR Tregs can suppress T cells with different specificities—a crucial function for controlling autoimmune responses .

• In vivo targeting assessment: Due to challenges with direct in vivo testing, researchers evaluate ENTPD3 CAR T cells' capacity to detect and interact with reaggregated human islets, comparing their performance to other antigen-specific cells like preproinsulin-specific CD8+ T cells .

How does ENTPD3 expression pattern in pancreatic islets inform its use as a therapeutic target?

ENTPD3 exhibits a distinctive expression pattern in pancreatic islets that makes it particularly valuable as a therapeutic target. In-depth histological analysis of pancreatic samples from patients at various stages of Type 1 Diabetes has revealed that ENTPD3 is consistently co-expressed with insulin across multiple disease stages, from autoantibody-positive through stage 3 T1D . This consistent expression throughout disease progression is critical for therapeutic targeting.

Single-cell RNA sequencing data analysis further confirms strong ENTPD3 expression in beta cells across both autoantibody-positive and overt T1D stages. Interestingly, ENTPD3 is also expressed in delta cells, suggesting a broader yet still islet-specific expression pattern . In prediabetic and diabetic NOD mice, ENTPD3 expression is concentrated in islets with minimal expression in surrounding ductal cells, confirming the suitability of this model for testing ENTPD3-targeted therapies .

The somewhat broader expression pattern within islets (beta and delta cells) combined with minimal expression elsewhere creates an advantageous therapeutic targeting profile—allowing for focused immunomodulation at the site of autoimmune attack while minimizing off-target effects.

What are the potential advantages and limitations of using ENTPD3 as a target for anti-tumor therapies?

ENTPD3 has shown promise as a target for cancer immunotherapy, particularly in combination approaches. Research demonstrates that combining anti-ENTPD3 monoclonal antibodies with anti-CD73 antibodies produces significant anti-tumor effects, with one study reporting a Δ T/ΔC value of 44.09 .

Advantages:

• The combination targeting approach addresses multiple enzymatic pathways involved in immunosuppressive adenosine production within the tumor microenvironment.

• By targeting both ENTPD3 and CD73, the therapy may overcome compensatory mechanisms that can develop when targeting single enzymes.

• The relatively restricted expression pattern of ENTPD3 may reduce off-target effects compared to more broadly expressed immunotherapy targets.

Limitations:

• The enzymatic redundancy within the ectonucleotidase family means that other enzymes (like ENTPD1/CD39) may partially compensate for ENTPD3 inhibition.

• Patient stratification may be necessary, as ENTPD3 expression levels likely vary across tumor types and individual patients.

• As with many targeted therapies, resistance mechanisms could emerge through downregulation of ENTPD3 or upregulation of alternative pathways.

Researchers should consider these factors when designing preclinical studies and clinical trials involving ENTPD3-targeted cancer therapies.

How does ENTPD3 function in neuronal tissues compared to pancreatic islets?

In contrast, ENTPD3 appears to have a more specialized and non-redundant role in pancreatic islets. It serves as a marker for mature beta cells and particularly for stem cell-derived β-cells formed by self-aggregation . This differential importance across tissues suggests tissue-specific regulation and function of ENTPD3.

The role of ENTPD3 in regulating extracellular ATP levels likely has context-dependent consequences: in neurons, it may modulate purinergic signaling affecting pain and sensory processes, while in pancreatic islets, it could influence insulin secretion and cellular function through altered purinergic signaling. These distinctions highlight the importance of tissue-specific analysis when investigating ENTPD3 function or developing targeted therapeutics.

What are the common pitfalls in purifying recombinant ENTPD3 from sf9 cells and how can they be addressed?

Purifying recombinant ENTPD3 from sf9 cells presents several technical challenges that researchers should anticipate:

• Protein aggregation: ENTPD3 can form aggregates during purification, particularly when expressed with a His-tag . To minimize this, researchers should optimize lysis conditions (buffer composition, detergent concentration) and consider including low concentrations of glycerol (10%) throughout the purification process .

• Maintaining enzymatic activity: Ectonucleotidases like ENTPD3 require divalent cations for activity. Purification buffers should contain appropriate concentrations of Ca²⁺ or Mg²⁺ to maintain the native conformation and activity.

• Proteolytic degradation: The sf9 system contains endogenous proteases that can degrade the target protein. Adding protease inhibitor cocktails during cell lysis and early purification steps is essential. Additionally, conducting purification at 4°C can minimize proteolytic activity.

• His-tag accessibility: The C-terminal His-tag used in many ENTPD3 constructs may sometimes be partially buried within the protein structure. Including mild denaturants or optimizing tag placement (N-terminal versus C-terminal) can improve purification efficiency.

• Endotoxin contamination: For downstream cell-based assays, especially immunological studies, endotoxin removal is critical. Additional purification steps like Triton X-114 phase separation or specialized endotoxin removal resins should be considered for ENTPD3 preparations intended for sensitive applications like CAR-Treg studies .

How can researchers accurately quantify ENTPD3 expression levels across different tissue samples and disease states?

Accurate quantification of ENTPD3 across tissues and disease states requires a multi-modal approach:

• Immunohistochemistry with digital quantification: As demonstrated in studies of ENTPD3 in pancreatic samples from T1D patients, digital image analysis of immunostained tissues can provide semi-quantitative measures of protein expression . This approach should include proper controls and standardization across staining batches.

• Single-cell RNA sequencing: This technique has been successfully employed to quantify ENTPD3 transcript levels across different cell types in pancreatic tissue at various stages of T1D . The advantage is cell-type specific resolution, though correlation with protein levels should be verified.

• Flow cytometry: Using validated anti-ENTPD3 antibodies, researchers can quantify surface expression levels on various cell populations. This is particularly useful for comparing expression across different cell types or disease states within the same sample.

• Quantitative proteomics: Techniques like targeted mass spectrometry can provide absolute quantification of ENTPD3 protein levels, allowing for more direct comparisons across tissue types and disease conditions than relative methods.

• Enzymatic activity assays: As a functional readout, measuring the nucleotidase activity attributable to ENTPD3 (using specific inhibitors to distinguish from other ectonucleotidases) can provide insight into the functional consequences of expression changes.

When comparing across disease states, researchers should normalize samples appropriately and consider the impact of inflammation, cell stress, and other pathological processes on ENTPD3 expression and function.

What are the key considerations for developing highly specific antibodies against ENTPD3 for research applications?

Developing highly specific antibodies against ENTPD3 requires careful attention to several factors:

• Antigen design: Researchers should select unique regions of ENTPD3 that differ from other ENTPD family members, particularly ENTPD1 (CD39) and ENTPD2, which share structural similarities. Structural analysis can help identify surface-exposed epitopes unique to ENTPD3.

• Validation across multiple systems: Antibodies should be validated using multiple techniques including Western blotting, immunoprecipitation, flow cytometry, and immunohistochemistry. Validation in ENTPD3 knockout models or cells where ENTPD3 is silenced via siRNA is particularly valuable.

• Cross-reactivity testing: Comprehensive testing against other ENTPD family members is essential to ensure specificity. This is particularly important for therapeutic applications like the anti-ENTPD3 monoclonal antibodies used in cancer therapy .

• Epitope mapping: Understanding precisely which region of ENTPD3 is recognized helps predict potential cross-reactivity and functionality. This is especially important for antibodies intended to modulate ENTPD3 activity rather than just detect it.

• Application-specific optimization: Antibodies that work well for one application (e.g., flow cytometry) may perform poorly in others (e.g., immunohistochemistry). Application-specific validation and optimization are therefore critical.

• Species cross-reactivity: For translational research, developing antibodies that recognize both human and mouse ENTPD3 enables more direct comparison between preclinical models and human studies, as demonstrated in research on ENTPD3 expression in diabetes models .

What emerging technologies could enhance ENTPD3-targeted therapeutic approaches?

Several cutting-edge technologies show promise for advancing ENTPD3-targeted therapies:

• Bispecific antibody platforms: Developing bispecific antibodies that simultaneously target ENTPD3 and complementary targets (such as CD73) could enhance therapeutic efficacy beyond what has been observed with antibody combinations .

• CRISPR-engineered CAR-Tregs: Using CRISPR/Cas9 to precisely insert ENTPD3-specific CARs into regulatory T cells could improve the consistency and safety of cellular therapies for autoimmune conditions like T1D .

• Nanobody-based approaches: Developing smaller antibody fragments (nanobodies) against ENTPD3 might improve tissue penetration, particularly in solid tumors for cancer applications or pancreatic islets for diabetes applications.

• Targeted protein degradation: PROTAC (Proteolysis Targeting Chimera) technology could be applied to ENTPD3, allowing for controlled degradation rather than just inhibition of the protein in pathological contexts.

• In vivo editing of ENTPD3: Direct modification of ENTPD3 expression in specific tissues through in vivo gene editing could provide longer-lasting therapeutic effects without requiring repeated administration of biologics.

• Extracellular vesicle delivery systems: Engineering extracellular vesicles to deliver ENTPD3-modulating payloads could enhance targeting to specific tissues while minimizing systemic exposure.

These technologies could address current limitations in specificity, durability, and tissue accessibility of ENTPD3-targeted approaches.

How might the dual expression of ENTPD3 in beta and delta cells influence islet biology and diabetes pathophysiology?

The discovery that ENTPD3 is expressed in both beta and delta cells has significant implications for understanding islet biology and diabetes pathophysiology :

• Paracrine signaling coordination: ENTPD3 may regulate extracellular ATP/ADP levels, potentially coordinating paracrine signaling between different islet cell types. This could influence the synchronized secretion of insulin (from beta cells) and somatostatin (from delta cells).

• Disease progression dynamics: The persistent expression of ENTPD3 in delta cells even in long-standing T1D, when beta cells are largely destroyed, suggests potential alterations in islet microenvironment ATP regulation throughout disease progression .

• Therapeutic targeting considerations: Therapies targeting ENTPD3 for beta cell protection might inadvertently affect delta cell function. This could be either beneficial or detrimental depending on the specific mechanism and could explain some variability in therapeutic responses.

• Biomarker potential: The dual expression pattern suggests that measuring circulating ENTPD3 or anti-ENTPD3 antibodies might reflect combined beta and delta cell mass/stress, potentially providing a more comprehensive biomarker for islet health than beta cell-specific markers alone.

• Evolutionary significance: The shared expression pattern might reflect evolutionary conservation of purinergic signaling mechanisms in hormone-secreting islet cells, pointing to fundamental regulatory pathways worth investigating.

Future research should specifically examine the functional consequences of ENTPD3 activity in delta cells compared to beta cells, and how these distinct roles may converge or diverge in diabetes pathogenesis.