Recombinant Macaca fascicularis Hypoxanthine-guanine phosphoribosyltransferase (HPRT1)

What is the biological function of Hypoxanthine-guanine phosphoribosyltransferase in Macaca fascicularis?

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme in the purine salvage pathway, catalyzing the conversion of hypoxanthine to inosine-5′-monophosphate (IMP) and guanine to guanosine-5′-monophosphate (GMP) using phosphoribosyl pyrophosphate (PRPP) as the phosphoribosyl donor . This enzymatic reaction represents a critical metabolic shortcut that allows cells to recycle purine bases rather than synthesizing them de novo, which is energetically more demanding. In Macaca fascicularis, as in other primates, HPRT1 is particularly important in tissues with high rates of nucleic acid turnover and limited capacity for de novo purine synthesis, such as neural tissues.

The enzyme's mechanism involves replacing the 1-pyrophosphate group in phosphoribosyl pyrophosphate with the corresponding free nucleobase (either hypoxanthine or guanine) . This salvage pathway becomes especially crucial under conditions of metabolic stress or when de novo purine synthesis is compromised. Studies indicate that while HPRT1 is not universally essential across all organisms, its function is particularly important in higher mammals, including Macaca fascicularis, where its activity helps maintain cellular nucleotide pools necessary for DNA replication, RNA synthesis, and various signaling pathways.

How does Macaca fascicularis HPRT1 compare structurally to human HPRT1?

Macaca fascicularis (cynomolgus monkey) HPRT1 shares approximately 99% amino acid sequence identity with human HPRT1, making it an excellent model for human studies. This remarkable conservation reflects the essential nature of this enzyme in primate purine metabolism. The high sequence similarity translates to nearly identical three-dimensional structures, with conservation of all catalytically important residues including those involved in substrate binding and catalysis.

Key structural features including the active site architecture, PRPP binding domain, and purine base binding pocket are virtually indistinguishable between the two species. X-ray crystallography models suggest that both enzymes form homotetramers as their biologically active quaternary structure, with each monomer containing a core domain with a five-stranded parallel β-sheet surrounded by α-helices. The primary differences between Macaca fascicularis and human HPRT1 are found in non-catalytic surface residues that do not significantly affect enzymatic function or substrate specificity. This high degree of conservation makes Macaca fascicularis HPRT1 an ideal surrogate for human HPRT1 in preclinical studies, particularly for developing therapeutics targeting purine metabolism disorders.

What are the optimal conditions for enzymatic activity of recombinant Macaca fascicularis HPRT1?

Recombinant Macaca fascicularis HPRT1 demonstrates optimal enzymatic activity under specific biochemical conditions that closely resemble human HPRT1 requirements. For maximum activity, the enzyme requires a slightly alkaline environment with optimal pH ranging between 7.4-7.8 when using Tris-HCl buffer systems. Temperature optimization studies show peak activity at 37°C, which corresponds to the physiological body temperature of Macaca fascicularis.

Divalent metal ions, particularly Mg²⁺, are absolutely essential cofactors for HPRT1 activity, with optimal concentration typically between 5-10 mM MgCl₂ . The Mg²⁺ ions coordinate with the phosphate groups of PRPP and stabilize the transition state during catalysis. In the absence of magnesium, enzymatic activity is negligible. Kinetic studies indicate Macaca fascicularis HPRT1 follows Michaelis-Menten kinetics with Km values of approximately 3.2 μM for hypoxanthine and 1.9 μM for guanine, demonstrating slightly higher affinity for guanine as substrate. The enzyme exhibits relatively fast turnover rates with kcat values around 50 s⁻¹ under optimal conditions.

The enzyme maintains stability and activity in the presence of reducing agents such as DTT (1-5 mM) or TCEP (0.5-2 mM), which help prevent oxidation of cysteine residues. Furthermore, the addition of glycerol (5-10%) enhances stability without compromising enzymatic activity during extended experimental procedures.

What expression systems are most effective for producing recombinant Macaca fascicularis HPRT1?

Several expression systems have been successfully employed for recombinant Macaca fascicularis HPRT1 production, each offering distinct advantages depending on research requirements. The most widely used systems include:

Bacterial expression in E. coli represents the most cost-effective and high-yield approach. Optimal results are typically achieved using pET vector systems in BL21(DE3) or Rosetta strains, which compensate for codon bias. Expression protocols typically employ induction with 0.5-1.0 mM IPTG at OD600 0.6-0.8, followed by overnight expression at reduced temperatures (18-25°C) to promote proper folding. This system can yield 10-15 mg/L of culture, though some researchers report inclusion body formation at high expression levels, necessitating refolding procedures.

Mammalian expression systems using HEK293T or CHO cells provide optimal post-translational modifications and folding but at higher cost and lower yields (typically 2-5 mg/L). These systems are particularly valuable when studying interactions with other primate proteins or when post-translational modifications are suspected to affect function. Transfection efficiency and expression time (typically 48-72 hours) significantly impact yield and quality.

Insect cell expression using baculovirus vectors in Sf9 or Hi5 cells offers a balanced approach with moderate yields (5-10 mg/L) and proper folding. This system proves particularly advantageous for structural biology applications requiring highly homogeneous protein preparations. The choice between expression systems should be guided by specific research objectives, required protein quantity, and downstream applications.

What purification strategy provides the highest yield and purity of recombinant Macaca fascicularis HPRT1?

A robust purification strategy for recombinant Macaca fascicularis HPRT1 typically employs a multi-step chromatographic approach. The most effective protocol begins with affinity chromatography using either His-tagged HPRT1 (Ni-NTA or IMAC with imidazole gradient elution, 20-250 mM) or GST-tagged HPRT1 (Glutathione-Sepharose with reduced glutathione elution, 10-20 mM). This initial step provides 80-90% purity with recovery rates of 70-80% from the crude lysate.

Following affinity purification, an optional but recommended step involves tag removal using proteolytic cleavage with TEV, PreScission, or thrombin protease, followed by reverse affinity chromatography to remove both the protease and cleaved tag. The next step employs ion exchange chromatography, with Q-Sepharose at pH 8.0 (HPRT1 has a pI of approximately 6.2) and NaCl gradient elution (0-500 mM), which effectively separates HPRT1 from similarly sized contaminants and nucleic acid remnants.

How can enzymatic activity of purified Macaca fascicularis HPRT1 be reliably measured?

Several validated methods exist for measuring HPRT1 enzymatic activity, each with specific advantages depending on research objectives. The most commonly employed techniques include:

The spectrophotometric coupled assay represents a popular continuous monitoring approach, where HPRT1 activity is coupled to IMP/GMP production followed by NAD+ reduction to NADH via IMP/GMP dehydrogenase. This reaction creates a measurable increase in absorbance at 340 nm that directly correlates with HPRT1 activity. The assay typically contains purified HPRT1 (1-10 μg/ml), substrate (50-100 μM hypoxanthine or guanine), PRPP (1 mM), MgCl₂ (5 mM), IMP/GMP dehydrogenase (2-5 units), and NAD+ (0.5 mM) in 50 mM Tris-HCl buffer (pH 7.4). This method offers moderate sensitivity (0.5-5 nmol/min/mg protein) and is readily adaptable to microplate format for high-throughput screening.

Radiochemical assays provide superior sensitivity (0.05-0.5 nmol/min/mg protein) by tracking the conversion of radiolabeled substrates ([¹⁴C]-hypoxanthine or [³H]-guanine) to nucleotide products. After the reaction, products are separated from substrates using thin-layer chromatography or selective precipitation, followed by scintillation counting. While more labor-intensive, this method experiences minimal interference from sample components and is particularly valuable for precise kinetic studies.

HPLC-based assays offer direct quantification of nucleotide products with high specificity. The reaction mixture containing HPRT1, substrates, and PRPP is incubated at 37°C for 10-30 minutes before being stopped with acid or heat treatment. Products are then separated and quantified by HPLC with UV detection at 254 nm. This method provides a detection sensitivity of 0.1-1 nmol/min/mg protein and is particularly useful for complex samples or when analyzing multiple reaction products simultaneously.

How can recombinant Macaca fascicularis HPRT1 be used for inhibitor screening and development?

Recombinant Macaca fascicularis HPRT1 serves as an excellent platform for inhibitor screening due to its high similarity to human HPRT1 while providing a non-human primate model system for preclinical development. A comprehensive inhibitor screening workflow generally involves multiple phases with increasing stringency:

Primary screening typically employs high-throughput enzymatic assays in 384-well microplate format, where HPRT1 activity is measured in the presence of compound libraries (typically at 10-50 μM concentration). The spectrophotometric coupled assay described earlier is particularly suitable due to its adaptability to automation. Alternative approaches include fragment-based screening using thermal shift assays (differential scanning fluorimetry) to identify compounds that alter protein thermal stability upon binding. Virtual screening based on available crystal structures can also identify potential inhibitors for prioritized testing.

Secondary validation methods focus on promising hits from primary screens, analyzing dose-response relationships to determine IC₅₀ values and establishing structure-activity relationships (SAR). Kinetic analysis reveals inhibition mechanisms (competitive, non-competitive, uncompetitive) and provides Ki values. These studies specifically probe whether compounds compete with hypoxanthine/guanine binding, PRPP binding, or function through alternative mechanisms such as allosteric inhibition.

Cellular validation studies transition from biochemical to biological systems, typically employing Macaca fascicularis cell lines (e.g., primary fibroblasts or lymphoblasts) to confirm target engagement. Metabolomic profiling using LC-MS/MS can verify cellular effects on purine nucleotide pools, while comparative analysis with human cell lines helps predict translational potential. The conserved nature of HPRT1 across primates makes Macaca fascicularis an excellent surrogate for human systems in early development phases .

What experimental approaches are most effective for studying the structural biology of Macaca fascicularis HPRT1?

Structural biology investigations of Macaca fascicularis HPRT1 employ various complementary techniques to elucidate its three-dimensional organization and dynamic properties. X-ray crystallography remains the gold standard for high-resolution structural determination, requiring highly pure (>95%) and homogeneous protein preparations. Successful crystallization typically employs vapor diffusion methods (hanging or sitting drop) with protein concentrations of 10-15 mg/mL in buffers containing 20 mM HEPES pH 7.5, 150 mM NaCl, and 1-2 mM DTT. Co-crystallization with substrate analogs or inhibitors stabilizes the protein conformation and provides insights into binding interactions.

Nuclear Magnetic Resonance (NMR) spectroscopy complements crystallography by providing information about protein dynamics in solution. This approach requires isotopically labeled protein (¹⁵N, ¹³C) produced in minimal media with labeled precursors. While the relatively large size of tetrameric HPRT1 (~110 kDa) presents challenges for traditional NMR studies, selective labeling strategies and modern TROSY-based experiments can overcome size limitations for targeted structural investigations of specific regions.

Cryo-electron microscopy (cryo-EM) offers an alternative approach that avoids crystallization requirements. Though the relatively small size of HPRT1 presents resolution challenges for traditional single-particle cryo-EM, recent advances in detector technology and image processing have extended resolution capabilities for smaller proteins. Sample preparation typically involves 3-5 mg/mL protein applied to glow-discharged grids, followed by vitrification under optimized blotting conditions.

Computational approaches including homology modeling and molecular dynamics simulations provide complementary insights, particularly for comparative studies between species or for investigating conformational changes during catalysis. These in silico approaches benefit from the high sequence conservation between Macaca fascicularis and human HPRT1, allowing accurate model generation even in the absence of direct experimental structures.

How can recombinant Macaca fascicularis HPRT1 contribute to understanding Lesch-Nyhan syndrome and other HPRT1-related disorders?

Lesch-Nyhan syndrome is a severe neurological disorder caused by mutations in the human HPRT1 gene, resulting in purine metabolism dysregulation. Recombinant Macaca fascicularis HPRT1 serves as an invaluable tool for investigating this and related disorders due to the close evolutionary relationship between macaques and humans. Several experimental approaches leverage this recombinant protein to advance our understanding of disease mechanisms.

Site-directed mutagenesis of recombinant Macaca fascicularis HPRT1 can replicate human disease-associated mutations, creating a platform for detailed biochemical characterization. Enzymatic assays comparing wild-type and mutant proteins reveal how specific mutations affect catalytic efficiency, substrate binding, protein stability, and quaternary structure formation. Thermal denaturation studies and limited proteolysis experiments provide insights into structural destabilization caused by pathogenic mutations. These biochemical studies help classify mutations based on their molecular consequences (catalytic defects versus structural instability), which correlates with clinical severity.

Cell-based models utilizing CRISPR/Cas9 gene editing of Macaca fascicularis fibroblasts or induced pluripotent stem cells (iPSCs) create physiologically relevant disease models. These cellular systems allow investigation of downstream consequences of HPRT1 deficiency, including altered purine metabolism, nucleotide imbalances, and global transcriptional changes . The advantage of Macaca fascicularis-derived cellular models over rodent systems lies in their closer genetic and physiological similarity to humans, particularly regarding neural development and function, which are prominently affected in Lesch-Nyhan syndrome.

Therapeutic development applications include screening for pharmacological chaperones that stabilize mutant HPRT1 proteins with folding defects, evaluation of gene therapy approaches, and development of enzyme replacement strategies. The high similarity between Macaca fascicularis and human HPRT1 ensures that therapeutic approaches successful in macaque systems have enhanced translational potential for human applications.

What strategies can address low activity or stability issues with recombinant Macaca fascicularis HPRT1?

Researchers frequently encounter stability and activity challenges when working with recombinant HPRT1. These issues stem from multiple factors, each requiring specific troubleshooting approaches. Protein misfolding represents a common problem, particularly in bacterial expression systems, manifesting as aggregation in size exclusion chromatography or aberrant thermal denaturation profiles. Effective solutions include expression at reduced temperatures (16-18°C), inclusion of folding enhancers in culture media (glycerol 5-10%, L-arginine 50-100 mM), and co-expression with chaperone systems such as GroEL/ES or DnaK/DnaJ/GrpE.

Post-translational modifications can significantly impact enzyme functionality. When expressing in bacterial systems that lack mammalian PTM capability, alternative expression in insect or mammalian cells may restore activity. Mass spectrometry analysis can identify unexpected modifications that might affect catalytic function. Metal cofactor depletion during purification frequently causes activity loss, as HPRT1 requires Mg²⁺ for catalysis. This can be addressed by supplementing purification buffers with MgCl₂ (1-5 mM) and avoiding strong chelators like EDTA in purification buffers .

Cysteine oxidation presents another common issue affecting activity. HPRT1 contains catalytically important cysteine residues that, when oxidized, compromise function. Maintaining reducing conditions throughout purification using freshly prepared DTT (1-5 mM) or the more stable TCEP (0.5-2 mM) effectively prevents this problem. For long-term storage, optimized buffer conditions include 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 10% glycerol, and 2 mM DTT. Storing the protein in small aliquots at -80°C prevents repeated freeze-thaw cycles that accelerate denaturation.

How can potential contamination with host cell HPRT1 be controlled during recombinant Macaca fascicularis HPRT1 purification?

Contamination with host cell HPRT1 represents a significant concern when purifying recombinant Macaca fascicularis HPRT1, particularly when subsequent experiments require high specificity. This contamination can lead to misleading results in inhibitor screening, kinetic analyses, or structural studies. Several approaches effectively address this challenge depending on the expression system and experimental requirements.

Affinity tag selection offers the first line of defense against host contamination. When expressing in E. coli, which possesses its own HPRT (hpt gene product), using a large affinity tag like GST (26 kDa) rather than a smaller His-tag creates a significant size difference between recombinant and native enzyme, facilitating separation during purification. Furthermore, incorporating an additional orthogonal tag (dual-tagging with His and FLAG, for example) allows sequential affinity steps that dramatically reduce host enzyme carryover.

Expression in HPRT1-deficient host strains provides an elegant solution that eliminates contamination concerns at the source. Several commercially available E. coli strains with hpt gene knockouts have been developed for this purpose. For mammalian expression, HPRT1-deficient cell lines derived from Lesch-Nyhan patients or CRISPR-engineered knockout lines serve as excellent hosts for recombinant production. These deficient backgrounds ensure that any HPRT1 activity detected originates exclusively from the recombinant protein.

Immunological techniques using antibodies specific to Macaca fascicularis HPRT1 that do not cross-react with the host HPRT1 can be employed for immunoaffinity purification or for detecting contamination in western blot validation. Specific activity assays comparing the purified enzyme preparation to known standards of pure Macaca fascicularis HPRT1 can quantitatively assess contamination levels and validate preparation purity.

What quality control measures should be implemented when working with recombinant Macaca fascicularis HPRT1?

Rigorous quality control is essential when working with recombinant Macaca fascicularis HPRT1 to ensure experimental reproducibility and reliability. A comprehensive quality control workflow should assess multiple protein characteristics including purity, identity, integrity, activity, and homogeneity.

Purity assessment represents the foundation of quality control, typically employing SDS-PAGE with Coomassie staining (detection limit ~100 ng) or silver staining (detection limit ~1-10 ng) to visualize contaminants. Image analysis software quantifies purity percentage, with >95% purity desired for most applications. Complementary techniques such as capillary electrophoresis or analytical HPLC provide higher resolution for detecting closely related contaminants. Western blotting with anti-HPRT1 antibodies confirms protein identity while revealing potential degradation products or truncations.

Activity assays measure functional quality using the methodologies described earlier. Specific activity (μmol/min/mg) serves as a key metric, with batch-to-batch consistency indicating reliable protein quality. Thermal shift assays (differential scanning fluorimetry) assess protein folding and stability by measuring the melting temperature (Tm). Well-folded Macaca fascicularis HPRT1 typically exhibits a sharp, single transition with Tm around 52-55°C, while the presence of multiple transitions suggests heterogeneity or partial unfolding.

Oligomeric state analysis through size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) verifies quaternary structure formation. Native HPRT1 forms homotetramers (~110 kDa), and deviation from this state may indicate improper folding or aggregation. Mass spectrometry provides the ultimate verification of protein integrity, confirming molecular weight and potentially revealing post-translational modifications or unexpected chemical alterations that may impact function.

What statistical approaches are most appropriate for analyzing inhibition studies with recombinant Macaca fascicularis HPRT1?

For single-point inhibition screening, where compounds are tested at a fixed concentration, Z-factor analysis provides a statistical measure of assay quality. A Z-factor between 0.5-1.0 indicates an excellent assay suitable for high-throughput screening. Percent inhibition values should be normalized against appropriate positive controls (complete inhibition) and negative controls (vehicle only). When screening large compound libraries, multiple comparison corrections such as Benjamini-Hochberg false discovery rate (FDR) control prevent type I errors resulting from multiple hypothesis testing.

Dose-response experiments require nonlinear regression analysis, typically using a four-parameter logistic model to determine IC₅₀ values. This model accounts for the sigmoidal shape of most inhibition curves while allowing for variable Hill slopes and asymmetric responses. Confidence intervals for IC₅₀ values provide essential information about estimate precision, with narrower intervals indicating higher reliability. Model comparison approaches using Akaike Information Criterion (AIC) help select the most appropriate model when alternative inhibition mechanisms are considered.

Enzyme kinetic studies examining inhibition mechanisms require more sophisticated statistical treatment. Global fitting approaches simultaneously analyze multiple datasets (varying substrate and inhibitor concentrations) to distinguish between competitive, non-competitive, uncompetitive, or mixed inhibition models. F-tests compare nested models to determine whether more complex models (with additional parameters) provide statistically significant improvements in fit quality. Dixon and Cornish-Bowden plots offer graphical methods for inhibition mechanism determination, complementing computational approaches.

How should species differences be analyzed when comparing Macaca fascicularis HPRT1 with human HPRT1?

Comparative analysis between Macaca fascicularis and human HPRT1 provides valuable insights for translational research, but requires methodical approaches to identify meaningful differences while acknowledging experimental limitations. A comprehensive comparative strategy addresses sequence, structural, functional, and cellular dimensions of enzyme biology.

Structural comparisons utilize available crystal structures or homology models to visualize three-dimensional consequences of sequence differences. Superposition of structures quantifies root-mean-square deviation (RMSD) as a measure of structural divergence, with significant differences typically defined as RMSD >1Å. Molecular dynamics simulations explore conformational flexibility differences that may not be apparent in static structures, particularly regarding substrate access channels or induced-fit mechanisms.

Functional comparisons require side-by-side biochemical characterization under identical experimental conditions. Statistical approaches like extra sum-of-squares F-test determine whether differences in kinetic parameters (Km, kcat, kcat/Km) between species are statistically significant. Similarly, thermal stability comparisons using differential scanning fluorimetry with statistical validation through t-tests or ANOVA identify meaningful differences in protein robustness.

Pharmacological comparisons examining inhibitor potency across species employ correlation analysis to quantify the relationship between human and Macaca fascicularis HPRT1 IC₅₀ values across multiple compounds. Bland-Altman plots visualize systematic differences in inhibitor sensitivity, while regression analysis with 95% prediction intervals estimates the expected range of human values based on macaque data.

What approaches can identify post-translational modifications in recombinant Macaca fascicularis HPRT1?

Post-translational modifications (PTMs) can significantly influence enzyme function, stability, and interactions, making their identification crucial for comprehensive protein characterization. Multiple complementary analytical approaches effectively detect and characterize PTMs in recombinant Macaca fascicularis HPRT1, each with specific advantages.

Mass spectrometry-based proteomics represents the gold standard for PTM identification. Bottom-up proteomics employing enzymatic digestion (typically with trypsin) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identifies site-specific modifications through mass shifts in modified peptides. Electron transfer dissociation (ETD) or electron capture dissociation (ECD) fragmentation techniques preserve labile modifications better than conventional collision-induced dissociation (CID). For comprehensive analysis, enrichment strategies targeting specific modifications enhance detection sensitivity. These include phosphopeptide enrichment using titanium dioxide or IMAC, glycopeptide enrichment using lectin affinity, and ubiquitination enrichment using ubiquitin remnant antibodies.

Top-down proteomics analyzing intact protein by high-resolution mass spectrometry provides a complementary approach that preserves the relationship between multiple modifications on the same protein molecule. This technique reveals modification stoichiometry and identifies proteoforms with distinct modification patterns. Native mass spectrometry further extends this approach to examine how modifications affect quaternary structure formation.

Specialized biochemical assays target specific modifications with high sensitivity. Pro-Q Diamond staining detects phosphorylation on SDS-PAGE gels, while periodic acid-Schiff staining reveals glycosylation. Western blotting using modification-specific antibodies (anti-phospho, anti-acetyl, anti-SUMO) offers another targeted approach. Enzymatic treatments that remove specific modifications (phosphatases, deglycosylases, deubiquitinases) followed by mobility shift analysis on SDS-PAGE provide functional confirmation of modification presence.

Site-directed mutagenesis of potential modification sites to non-modifiable residues (e.g., Ser/Thr to Ala for phosphorylation, Lys to Arg for acetylation/ubiquitination) combined with functional analysis reveals the biological significance of identified modifications. This approach is particularly valuable for understanding how PTMs affect enzymatic activity, protein-protein interactions, or stability.