Recombinant Chicken Low molecular weight phosphotyrosine protein phosphatase (ACP1)

What is the basic structural and functional characterization of Chicken Low molecular weight phosphotyrosine protein phosphatase (ACP1)?

Chicken Low molecular weight phosphotyrosine protein phosphatase (ACP1) is a member of the phosphotyrosine protein phosphatase family with a UniProt identification number Q5ZKG5. The mature protein has a sequence spanning amino acid positions 2-158, with its full amino acid sequence being AAGEVKSVLFVCLGNICRSPIAEAVFRKLVTDEKVENKWRIDSAATSTEIGNPPDYRGQTCMKKHGITMNHIARQVTKDDFQTFDYILCMDESNLRDLKRKSNQVKDCKAKIELLGAYDPQKQLIIEDPYYGNEKDFETVYEQCVRCCKAFLEKPH . Functionally, this enzyme catalyzes the hydrolysis of protein tyrosine phosphate to produce protein tyrosine and orthophosphate, while also hydrolyzing orthophosphoric monoesters to alcohol and orthophosphate . The enzyme displays dual functionality as both an acid phosphatase (EC 3.1.3.2) and a protein tyrosine phosphatase (EC 3.1.3.48) .

How does the expression system affect the properties of recombinant Chicken ACP1?

The expression system significantly influences the post-translational modifications, folding, and ultimately the functional characteristics of recombinant Chicken ACP1. When expressed in mammalian cell systems as indicated in the product specifications, the protein typically maintains proper folding and post-translational modifications that more closely resemble the native chicken protein . In contrast, when expressed in bacterial systems such as E. coli (as is common with some recombinant proteins), the protein may lack these modifications, potentially affecting its activity and stability. For research applications requiring functionally active enzyme, mammalian expression systems are generally preferred despite their higher cost and complexity. The purity of the recombinant protein (>85% by SDS-PAGE) indicates a relatively high-quality preparation suitable for most research applications .

What are the optimal storage and handling conditions for maintaining Chicken ACP1 activity?

Maintaining optimal activity of recombinant Chicken ACP1 requires specific storage and handling protocols. The shelf life is significantly affected by storage conditions, buffer composition, temperature, and the inherent stability of the protein. For liquid preparations, storage at -20°C to -80°C provides a shelf life of approximately 6 months, while lyophilized forms can maintain stability for up to 12 months at the same temperature range . Repeated freeze-thaw cycles should be strictly avoided as they promote protein denaturation and activity loss. For ongoing experiments, working aliquots can be stored at 4°C for up to one week to minimize freeze-thaw damage . When reconstituting lyophilized protein, brief centrifugation before opening is recommended to ensure all material is collected at the bottom of the vial . Buffer systems containing stabilizers such as glycerol (5-50%) are commonly used to maintain protein integrity during storage .

What enzymatic assay methodologies are most appropriate for measuring Chicken ACP1 activity and kinetics?

The enzymatic activity of Chicken ACP1 can be assessed through multiple complementary approaches. The most common spectrophotometric assay utilizes p-nitrophenyl phosphate (pNPP) as a substrate, which upon dephosphorylation yields the colored product p-nitrophenol that can be measured at 405 nm. For more physiologically relevant assessments, researchers should consider using phosphotyrosine-containing peptides or proteins as substrates, with quantification of released phosphate using malachite green or similar colorimetric methods. For detailed kinetic analysis, researchers should determine the Michaelis-Menten parameters (Km, Vmax, kcat) using varying substrate concentrations under controlled temperature and pH conditions, ideally at pH 5.0-6.0 to match the enzyme's acidic pH optimum .

For inhibition studies, orthovanadate and phenylarsine oxide are commonly used inhibitors of protein tyrosine phosphatases including ACP1. When designing enzyme kinetic experiments, it's essential to consider the chicken enzyme may have different substrate preferences compared to its human counterpart, which typically acts on tyrosine phosphorylated proteins, low-molecular-weight aryl phosphates, and natural and synthetic acyl phosphates .

How can researchers effectively incorporate protein-protein interaction studies to identify Chicken ACP1 binding partners?

To identify and characterize the interactome of Chicken ACP1, researchers should employ multiple complementary approaches. Co-immunoprecipitation (Co-IP) using antibodies against ACP1 or potential binding partners represents a fundamental method, though the availability of chicken-specific antibodies may be limited. For a more comprehensive approach, proximity-based labeling methods such as BioID or APEX can be employed, where ACP1 is fused to a biotin ligase or peroxidase, allowing biotinylation of proximal proteins for subsequent purification and identification.

Yeast two-hybrid screening offers another powerful system for detecting binary interactions, though it may miss interactions dependent on post-translational modifications. For higher confidence results, researchers should validate interactions using reciprocal Co-IP experiments and provide supporting evidence through co-localization studies using confocal microscopy. Based on studies of human LMWPTP, researchers should specifically investigate interactions with focal adhesion kinase and paxillin, as these interactions have been implicated in migration and adhesion processes in human cell models .

What approaches are recommended for studying the role of Chicken ACP1 in cellular signaling pathways?

Investigating Chicken ACP1's role in signaling cascades requires strategic manipulation of its expression and activity. CRISPR-Cas9 gene editing in chicken cell lines provides a powerful method for generating complete knockouts, while RNA interference (siRNA or shRNA) allows for transient or stable knockdown. Overexpression studies using plasmid transfection with wild-type and catalytically inactive mutants (typically involving mutation of the catalytic cysteine residue) can further elucidate function.

For signaling pathway analysis, researchers should employ phosphorylation-specific antibodies to monitor changes in key signaling proteins following ACP1 manipulation. Western blotting for phosphorylated focal adhesion kinase and paxillin is particularly recommended based on human studies showing LMWPTP's role in regulating these proteins . Phosphoproteomic approaches using mass spectrometry offer unbiased identification of substrates and affected pathways. Real-time monitoring of phosphorylation dynamics can be achieved using FRET-based phosphorylation biosensors in live cells. When interpreting results, researchers should consider that the chicken enzyme may have evolved different substrate specificities compared to mammalian orthologs, particularly in light of the co-evolutionary processes observed in chicken immune system molecules .

What structural features distinguish Chicken ACP1 from its mammalian orthologs, and how do these differences impact function?

Chicken ACP1 shares key structural elements with mammalian orthologs while exhibiting distinct features that may confer unique functional properties. The enzyme belongs to the low molecular weight phosphotyrosine protein phosphatase family, with a core structure containing the characteristic phosphotyrosine protein phosphatase domain. Sequence analysis reveals that Chicken ACP1 contains the critical catalytic motif (C-X5-R) found in all protein tyrosine phosphatases, which forms the phosphate-binding loop essential for catalysis .

Comparative structural analysis between chicken and human ACP1 reveals approximately 70-80% sequence identity, with divergences primarily in surface-exposed regions that may influence substrate recognition and protein-protein interactions. These differences might explain variations in substrate specificity between species. Unlike the human enzyme, which acts on tyrosine phosphorylated proteins, low-MW aryl phosphates, and acyl phosphates, the chicken enzyme may have evolved distinct substrate preferences related to avian-specific signaling pathways . Crystallographic studies of Chicken ACP1 would be valuable for elucidating these structural nuances, particularly given the unexpected features discovered in other chicken immune-related proteins compared to their mammalian counterparts .

How can site-directed mutagenesis be employed to investigate the catalytic mechanism of Chicken ACP1?

Site-directed mutagenesis represents a powerful approach for dissecting the catalytic mechanism and substrate specificity of Chicken ACP1. The catalytic cysteine residue (likely Cys12 based on alignment with human ACP1) should be the primary mutagenesis target, with substitution to serine (C12S) expected to generate a catalytically inactive enzyme that can serve as a negative control in activity assays and potentially function as a substrate trap. The conserved arginine in the phosphate-binding loop is another critical residue whose mutation would significantly impair catalysis while maintaining substrate binding.

Additionally, researchers should target residues in substrate-binding regions to investigate determinants of specificity. Based on the human enzyme's preference for acidic substrates, mutagenesis of positively charged residues in potential substrate-binding pockets could reveal important species-specific differences. When designing mutagenesis experiments, researchers should consider:

What are the challenges in crystallizing Chicken ACP1, and what alternative structural biology approaches might be valuable?

Obtaining high-resolution crystal structures of Chicken ACP1 presents several challenges that researchers should anticipate. The protein's relatively small size (~18 kDa) may result in limited crystal contacts, while potential conformational flexibility in substrate-binding regions can hamper crystallization. Additionally, the presence of multiple isoforms due to alternative splicing could introduce heterogeneity in protein preparations .

To overcome these challenges, researchers should:

• Perform extensive crystallization screening with varied precipitants, pH, temperatures, and additives

• Consider crystallizing the enzyme in complex with substrate analogs or inhibitors to stabilize specific conformations

• Explore surface entropy reduction mutations to promote crystal contacts

If crystallization proves challenging, alternative structural approaches include:

• Small-angle X-ray scattering (SAXS) for low-resolution envelope determination in solution

• Nuclear magnetic resonance (NMR) spectroscopy, which is feasible given the protein's small size

• Cryo-electron microscopy for larger complexes of ACP1 with binding partners

• Hydrogen-deuterium exchange mass spectrometry to probe conformational dynamics and ligand-binding regions

These complementary approaches can provide valuable structural insights even in the absence of crystallographic data.

How does Chicken ACP1 compare to mammalian orthologs in terms of genetic polymorphism and splice variants?

The genetic diversity of Chicken ACP1 presents intriguing evolutionary implications compared to its mammalian counterparts. While human ACP1 is characterized by three common alleles that segregate at the corresponding locus giving rise to six phenotypes (with each allele encoding two electrophoretically different isozymes, Bf and Bs, produced in allele-specific ratios) , the extent of allelic diversity in chicken populations has not been as thoroughly documented. The chicken gene, like its human ortholog, produces multiple alternatively spliced transcript variants encoding distinct isoforms .

This genetic variation may reflect species-specific adaptation, particularly considering the co-evolutionary patterns observed in the chicken immune system. The unexpected features discovered in chicken MHC molecules compared to mammals suggest that phosphatases like ACP1, which participate in immune signaling pathways, might similarly exhibit species-specific functional adaptations . Researchers investigating chicken ACP1 polymorphisms should consider:

• Sequencing the gene from diverse chicken breeds to characterize allelic diversity

• Analyzing expression patterns of splice variants in different tissues

• Comparing enzymatic properties of allelic variants and isoforms

• Examining potential co-evolution with interacting proteins, particularly those involved in immune function

What might the evolutionary conservation of ACP1 across species reveal about its fundamental biological roles?

The evolutionary conservation of ACP1 across diverse species provides valuable insights into its fundamental biological significance. Phylogenetic analysis reveals that ACP1 orthologs are present throughout vertebrates, suggesting ancient origins and conserved functions in core cellular processes. Despite this conservation, species-specific adaptations in sequence and structure likely reflect specialized roles in different organisms.

Comparative analysis between chicken and human ACP1 might reveal:

• Conserved catalytic residues and structural motifs essential for phosphatase activity

• Divergent substrate-binding regions that could indicate species-specific adaptations

• Differential regulatory mechanisms controlling expression and activity

• Lineage-specific protein-protein interaction domains

The human LMWPTP has been implicated in oncogenic signaling in prostate cancer, with overexpression correlating with earlier disease recurrence and reduced patient survival . This raises intriguing questions about whether chicken ACP1 might play similar roles in avian tumor biology, or whether its functions have diverged through evolution. Researchers should consider that while core enzymatic mechanisms may be conserved, the integration of ACP1 into cellular signaling networks might differ significantly between species.

What are the optimal cell culture models for studying Chicken ACP1 function in vitro?

Selecting appropriate cell culture models is crucial for investigating Chicken ACP1 function. Primary chicken embryonic fibroblasts (CEFs) represent an excellent physiological model system, as they maintain the native signaling environment in which the enzyme normally functions. Established chicken cell lines such as DT40 (B cell line) or HD11 (macrophage-like line) offer the advantages of easier maintenance and genetic manipulation. When studying tissue-specific functions, researchers should consider primary cells from relevant tissues such as immune cells, given the potential involvement of phosphatases in immune signaling pathways suggested by studies of chicken MHC molecules .

For heterologous expression studies, both avian and mammalian cell lines (such as CHO or HEK293) can be employed, though researchers should remain mindful that signaling pathways and interaction partners may differ from the native context. When designing in vitro experiments, consider:

• Comparing results across multiple cell types to distinguish cell type-specific from general functions

• Establishing stable cell lines with inducible expression systems for controlled experimental conditions

• Implementing CRISPR-Cas9 gene editing to generate knockout cell lines for loss-of-function studies

• Using fluorescently tagged variants to track subcellular localization, provided the tag doesn't interfere with function

How can researchers effectively study the role of Chicken ACP1 in immune signaling pathways?

Given the unexpected features discovered in chicken MHC molecules and their potential implications for immune system function , investigating Chicken ACP1's role in immune signaling represents a particularly promising research direction. Researchers should employ a multi-faceted approach that includes:

• Ex vivo lymphocyte activation studies: Isolate primary chicken T and B lymphocytes and assess how ACP1 inhibition or knockdown affects activation markers, cytokine production, and proliferation following receptor stimulation.

• Phosphoproteomic analysis: Compare the phosphoproteome of immune cells with normal versus manipulated ACP1 expression/activity to identify potential substrates and affected pathways.

• Transgenic approaches: Generate chicken models with tissue-specific ACP1 knockdown or overexpression in immune compartments to assess in vivo immune function.

• Infection models: Challenge cell cultures or animal models with relevant pathogens to determine if ACP1 modulation affects immune response efficacy.

When interpreting results, researchers should consider the potential co-evolution between ACP1 and other immune signaling components, as suggested by studies showing co-evolution between polymorphic TAP and tapasin genes with class I genes in chickens .

What are the common pitfalls in recombinant Chicken ACP1 expression and purification, and how can they be addressed?

Recombinant expression and purification of Chicken ACP1 present several technical challenges that researchers should anticipate. The expression system chosen significantly impacts protein quality—while E. coli systems offer high yield and simplicity, mammalian cell expression (as used in some commercial preparations) likely provides superior folding and post-translational modifications . Common expression challenges include:

• Low solubility: Address by optimizing induction conditions (temperature, inducer concentration, duration) or using solubility-enhancing fusion tags (MBP, SUMO).

• Protein instability: Incorporate stabilizing buffer components (glycerol, reducing agents) and maintain cold temperatures throughout purification.

• Catalytic inactive preparations: Ensure reducing conditions are maintained to protect the catalytic cysteine from oxidation.

• Contaminating phosphatases: Include phosphatase inhibitors selective for other phosphatase classes during purification.

For optimal purification results, a multi-step approach is recommended:

The final purity should exceed 85% by SDS-PAGE for most applications, with higher purity (>90%) recommended for structural studies .

What quality control measures should be implemented when working with recombinant Chicken ACP1?

Comprehensive quality control is essential for ensuring reliable experimental results with recombinant Chicken ACP1. Researchers should implement the following validation steps:

• Purity assessment: SDS-PAGE with Coomassie staining should demonstrate >85% purity , with more sensitive silver staining to detect minor contaminants.

• Identity confirmation: Western blotting with specific antibodies and/or mass spectrometry peptide mapping to verify protein identity.

• Activity assay: Enzymatic activity using p-nitrophenyl phosphate or physiologically relevant substrates should be measured, with specific activity (units/mg) compared to established benchmarks.

• Endotoxin testing: Particularly for preparations intended for immune cell experiments, as endotoxin contamination can confound results.

• Thermal shift assay: To assess protein stability and proper folding.

• Dynamic light scattering: To verify monodispersity and absence of aggregation.

Batch-to-batch consistency is crucial for longitudinal studies, requiring careful documentation of specific activity and other quality parameters. For preparations showing suboptimal activity, troubleshooting should focus on potential oxidation of the catalytic cysteine, presence of inhibitory contaminants, or protein misfolding.

What are the promising unexplored aspects of Chicken ACP1 that warrant further investigation?

Several unexplored aspects of Chicken ACP1 present compelling opportunities for future research:

• Substrate specificity profiling: Comprehensive characterization of physiological substrates using phosphoproteomic approaches would provide valuable insights into pathway involvement. This is particularly relevant given the differences in sequence between chicken and mammalian orthologs that may confer unique substrate preferences.

• Role in avian-specific signaling: Investigation of potential specialized functions in birds, particularly in light of the unexpected features discovered in chicken MHC molecules and other immune components .

• Tissue-specific expression patterns: Detailed analysis of expression across different tissues and developmental stages to elucidate specialized functions.

• Potential roles in avian diseases: Given the human ortholog's implication in cancer progression , investigation of similar roles in avian tumor biology could reveal conserved oncogenic mechanisms.

• Regulatory mechanisms: Characterization of post-translational modifications and regulatory interactions that control ACP1 activity in avian systems.

• High-resolution structure determination: Crystal or cryo-EM structures would provide valuable insights into species-specific structural features.

These research directions would significantly advance our understanding of this enzyme's biological functions and potential applications in comparative biology and veterinary medicine.

How might new technological advances be applied to deepen our understanding of Chicken ACP1 biology?

Emerging technologies offer promising approaches to advance Chicken ACP1 research:

• CRISPR-Cas9 gene editing: Generation of precise genomic modifications in chicken cell lines and potentially in vivo models using newly developed avian-specific systems.

• Single-cell phosphoproteomics: Examination of ACP1-dependent signaling events with cellular resolution to understand heterogeneous responses.

• Proximity labeling proteomics: BioID or APEX2 fusions to map the spatiotemporal interactome of ACP1 in living cells.

• Cryo-electron microscopy: Structural determination of ACP1 complexes with interaction partners at near-atomic resolution.

• AlphaFold and related AI structure prediction tools: Computational modeling of chicken ACP1 structure and protein-protein interactions to guide experimental design.

• Organoid systems: Development of chicken-derived organoids to study ACP1 function in more physiologically relevant three-dimensional tissue models.

• Nanobodies and intrabodies: Development of highly specific inhibitors for acute inhibition with spatial and temporal precision.

These technological approaches, combined with classical biochemical and cellular methods, promise to provide unprecedented insights into the functions and mechanisms of this important signaling enzyme.