Recombinant Chicken Hyaluronan synthase 3 (HAS3)

What is Recombinant Chicken Hyaluronan synthase 3 (HAS3)?

Recombinant Chicken Hyaluronan synthase 3 (HAS3) is a laboratory-produced form of the naturally occurring enzyme responsible for synthesizing hyaluronan, a critical component of vertebrate extracellular and cell-associated matrices. This recombinant protein is derived from Gallus gallus (chicken) and can be expressed in systems like E. coli for research purposes. The enzyme belongs to the HAS family with the EC number 2.4.1.212 and has alternative nomenclature including hyaluronate synthase 3, hyaluronic acid synthase 3, CHAS3, and HA synthase 3 . As a recombinant protein, it offers researchers a controlled way to study hyaluronan synthesis mechanisms outside of complex cellular systems while maintaining the functional characteristics of the native enzyme.

How does HAS3 differ from other hyaluronan synthases (HAS1 and HAS2)?

Hyaluronan synthases exist as three homologous isoforms (HAS1, HAS2, and HAS3) that exhibit distinct differences in several key aspects:

• Tissue Distribution: Each HAS isoform shows unique expression patterns across different tissues, with HAS3 having its own characteristic distribution profile .

• Enzymatic Characteristics: HAS3 typically produces shorter hyaluronan chains compared to HAS1 and HAS2. It generally exhibits higher intrinsic activity than the other isoforms, allowing it to synthesize hyaluronan more rapidly under similar conditions .

• Regulatory Mechanisms: The three synthases respond differently to various cellular signals and growth factors. HAS3 shows distinct patterns of transcriptional regulation and post-translational modification compared to HAS1 and HAS2 .

• Functional Roles: The differences in enzymatic properties and regulation suggest distinct biological roles for each synthase in processes such as embryogenesis, inflammation, wound healing, and tumor progression .

What is the role of HAS3 in hyaluronan synthesis?

HAS3 plays a critical role in the biosynthesis of hyaluronan, a high molecular weight polysaccharide crucial for tissue structure and function. The enzyme catalyzes the polymerization of UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc) to form the repeating disaccharide units of hyaluronan . This synthesis process is fundamental for maintaining the structural integrity of tissues like skin, cartilage, eyes, and joints .

Beyond structural roles, HAS3-synthesized hyaluronan contributes to dynamic cellular processes including:

• Embryonic Development: Supporting cellular migration and tissue patterning

• Inflammatory Responses: Mediating immune cell recruitment and activation

• Wound Healing: Facilitating cellular proliferation and migration

• Cancer Metastasis: Potentially supporting tumor cell invasion and migration

Research suggests that the chain length and production rate of hyaluronan by HAS3 may have specific functional implications in these biological contexts.

What are the optimal storage conditions for recombinant chicken HAS3?

Optimal storage of recombinant chicken HAS3 is critical to maintaining its stability and enzymatic activity. Based on manufacturer recommendations and research protocols, the following storage guidelines should be observed:

• Temperature: Store at -20°C to -80°C for long-term preservation. The shelf life of lyophilized recombinant HAS3 is approximately 12 months at these temperatures, while liquid formulations typically maintain stability for about 6 months .

• Working Aliquots: For ongoing experiments, working aliquots can be stored at 4°C for up to one week. Repeated freezing and thawing should be strictly avoided as this significantly reduces enzyme activity .

• Reconstitution: Prior to use, briefly centrifuge vials to bring contents to the bottom. Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For optimal stability, add glycerol to a final concentration of 5-50% (with 50% being commonly recommended) before aliquoting for long-term storage .

• Handling: Minimize exposure to room temperature and avoid contamination of stocks to prevent degradation and maintain purity levels (typically >85% as assessed by SDS-PAGE) .

How is HAS3 activity regulated in cells?

HAS3 activity is regulated through multiple mechanisms that enable precise control of hyaluronan production in response to cellular needs:

• Transcriptional Regulation: Expression of HAS3 genes varies based on tissue type and is responsive to growth factors and cytokines, allowing tissue-specific and context-dependent control of hyaluronan synthesis .

• Post-translational Modifications: Phosphorylation represents a key regulatory mechanism for HAS3 activity. Research demonstrates that HAS3 undergoes serine phosphorylation, which can be significantly enhanced by various cellular effectors .

• cAMP-Dependent Regulation: The membrane-permeable analogue of cAMP, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), has been shown to enhance HAS3 phosphorylation substantially. In unstimulated cells, the stoichiometry of FLAG-HAS3 phosphorylation was approximately 0.11, increasing to as much as 0.32 in cells stimulated with CPT-cAMP .

• Other Signal Transduction Pathways: Additional effectors, including phorbol esters (activators of protein kinase C) and epidermal growth factor (EGF), have also been observed to enhance HAS3 phosphorylation, suggesting multiple signaling pathways converge to regulate this enzyme .

How does phosphorylation affect the function of HAS3?

Phosphorylation of HAS3 appears to serve as a critical regulatory switch for enzyme activity, though the relationship between phosphorylation state and functional outcomes is complex:

• Activity Modulation: Phosphorylation likely alters the catalytic efficiency of HAS3, potentially by inducing conformational changes that affect substrate binding or the rate of chain elongation. Research indicates that changes in phosphorylation state correlate with alterations in hyaluronan production rates .

• Stoichiometry Significance: Studies have measured the stoichiometry of HAS3 phosphorylation, finding it to be approximately 0.11 in unstimulated cells, increasing to as much as 0.32 in cells stimulated with cAMP analogues. This significant change (~3-fold increase) suggests a direct relationship between phosphorylation levels and enzymatic activity .

• Serine-Specific Phosphorylation: Research has identified serine residues as the primary phosphorylation sites on HAS3. The specific serine residues involved and their positions within the protein structure likely determine the functional consequences of phosphorylation .

• Signaling Integration: The phosphorylation state of HAS3 may serve as an integration point for multiple cellular signaling pathways, allowing hyaluronan production to respond dynamically to diverse physiological stimuli and cellular conditions .

To fully characterize these relationships, researchers should employ both site-directed mutagenesis to modify potential phosphorylation sites and kinase/phosphatase inhibitors to manipulate phosphorylation states while monitoring changes in enzyme activity.

What methods can be used to study HAS3 phosphorylation in vitro?

Investigating HAS3 phosphorylation requires specialized techniques that can detect and quantify this post-translational modification with high sensitivity and specificity:

• Radioisotope Labeling: The incorporation of [32P]Pi into recombinant HAS3 represents a powerful approach for detecting phosphorylation events. This technique has successfully demonstrated serine phosphorylation of HAS3 in COS-7 cells and revealed enhancement by various effectors, particularly cAMP analogues .

• Epitope Tagging Strategies: Utilizing epitope tags (such as FLAG) facilitates protein tracking and purification. For example, a FLAG (DYKDDDDK) epitope-tagged version of human HAS3 has been effectively used to study phosphorylation . The experimental workflow typically includes:

  • Transfection of expression systems with tagged constructs

  • Radiolabeling with [32P]Pi

  • Extraction and affinity purification using anti-FLAG antibodies

  • Analysis via SDS-PAGE and autoradiography

• Stoichiometry Determination: Employing reference proteins with known phosphorylation sites (such as modified EGFP) enables quantitative assessment of phosphorylation stoichiometry. This approach has revealed that FLAG-HAS3 phosphorylation increases from approximately 0.11 in unstimulated cells to 0.32 in cells stimulated with 8-(4-chlorophenylthio)-cAMP .

• Protein Extraction Optimization: Due to HAS3's membrane association, effective extraction requires careful detergent selection. Research indicates that while non-ionic or zwitterionic detergents like CHAPS may be insufficient, SDS solubilization followed by dilution with buffers containing non-ionic detergents enables successful affinity purification .

• Phosphorylation Site Mapping: Mass spectrometry combined with phosphopeptide enrichment methods can identify specific phosphorylated residues within HAS3, providing insight into structure-function relationships.

How can one distinguish between the enzymatic activities of different HAS isoforms?

Differentiating the enzymatic activities of HAS1, HAS2, and HAS3 requires methodological approaches that account for their distinct biochemical properties:

• Product Characterization Analysis: The three HAS isoforms produce hyaluronan chains of different lengths and at different rates. Analyzing the size distribution of synthesized hyaluronan using methods such as gel filtration chromatography or multi-angle light scattering can help distinguish between isoforms. HAS3 typically produces shorter chains than HAS1 and HAS2 .

• Kinetic Analyses: Determining the kinetic parameters (Km, Vmax) for each isoform using purified recombinant proteins can reveal distinct substrate affinities and catalytic efficiencies. These experiments require carefully controlled conditions and highly purified enzyme preparations.

• Isoform-Specific Inhibitors: While not widely available for all HAS isoforms, the development and use of isoform-specific inhibitors or antibodies could help distinguish their activities in complex systems.

• Chain Elongation Direction Studies: Research suggests differences in the mechanism of chain elongation between native and recombinant synthases. Methods to study the direction of chain elongation include:

  • Differential labeling of reducing terminal and non-reducing terminal domains using radiolabeled substrates like UDP-[³H]GlcNAc and UDP-[¹⁴C]GlcA

  • Digestion with exoglycosidases such as β-glucuronidase and β-N-acetylglucosaminidase

  • Monitoring changes in isotope ratios during digestion to determine the direction of chain growth

• Recombinant Expression Systems: Using heterologous expression of individual HAS isoforms in systems lacking endogenous hyaluronan synthesis capacity allows for isolated study of each enzyme's properties without interference.

What experimental approaches can be used to study the direction of chain elongation in HAS3-mediated hyaluronan synthesis?

Understanding the direction of chain elongation in HAS3-mediated hyaluronan synthesis requires sophisticated experimental designs that can track the addition of new monosaccharides:

• Differential Isotopic Labeling: This approach involves using distinct radioisotope-labeled UDP-sugar precursors to mark different regions of the growing chain. For example:

  • Incubating membranes containing HAS3 with UDP-[³H]GlcNAc and UDP-[¹⁴C]GlcA creates differentially labeled reducing terminal and non-reducing terminal domains

  • The resulting ³H/¹⁴C ratio in the synthesized hyaluronan provides information about the directionality of chain growth

• Exoglycosidase Digestion Assays:

  • Treatment of the labeled hyaluronan with exoglycosidases (β-glucuronidase and β-N-acetylglucosaminidase) that specifically cleave from the non-reducing end

  • Monitoring changes in the isotope ratio during digestion reveals whether synthesis occurs at the reducing or non-reducing end

  • Research with native eukaryotic synthases indicates extension at the reducing end, while some recombinant synthases appear to extend at the non-reducing end

• Competition Studies:

  • If synthesis occurs at the reducing end, UDP-hyaluronan terminus should compete with substrates for binding to the active site

  • Experiments demonstrating that increasing substrate concentrations enhance hyaluronan release from the synthase support the reducing-end model of chain elongation

• Substrate Binding Affinity Analysis:

  • Determining the binding affinity of the synthase for UDP-hyaluronan chains

  • Comparing native and recombinant enzyme binding properties to explain differences in chain elongation direction

These methodologies have revealed that native eukaryotic hyaluronan synthases, including HAS3, likely extend chains at the reducing end, while some recombinant systems may show different behavior.

How do post-translational modifications alter HAS3 activity and what techniques are useful for investigating these modifications?

Post-translational modifications (PTMs) of HAS3, particularly phosphorylation, appear to play critical roles in regulating enzyme activity. Investigating these modifications requires specialized methodologies:

• Phosphorylation Site Identification:

  • Phosphoproteomics: Mass spectrometry-based approaches can identify specific phosphorylated residues within HAS3. This typically involves tryptic digestion of purified HAS3, phosphopeptide enrichment, and LC-MS/MS analysis.

  • Site-Directed Mutagenesis: Replacing potential phosphorylation sites (serine residues) with alanine (phospho-null) or aspartate/glutamate (phospho-mimetic) allows assessment of site-specific phosphorylation effects on enzyme activity.

• Quantification of Phosphorylation Levels:

  • Stoichiometry Determination: Using reference proteins with known phosphorylation levels enables estimation of HAS3 phosphorylation stoichiometry. This approach has shown that FLAG-HAS3 phosphorylation increases from approximately 0.11 in unstimulated cells to 0.32 in cells stimulated with cAMP analogues .

  • Phospho-specific Antibodies: Development of antibodies recognizing specific phosphorylated residues in HAS3 would facilitate monitoring of phosphorylation states under various conditions.

• Signaling Pathway Analysis:

  • Kinase/Phosphatase Inhibitors: Treatment with specific inhibitors can help identify the signaling pathways regulating HAS3 phosphorylation. Research has shown enhancement of HAS3 phosphorylation by effectors including CPT-cAMP, PMA, and EGF, suggesting involvement of PKA, PKC, and receptor tyrosine kinase pathways, respectively .

  • Phosphatase Treatment: Incubation of purified phosphorylated HAS3 with phosphatases (e.g., bacterial alkaline phosphatase) can confirm the identity of modifications and their reversibility.

• Correlation of Modifications with Activity:

  • In vitro Enzyme Assays: Comparing the activity of differentially modified HAS3 preparations can establish relationships between specific PTMs and catalytic properties.

  • Structure-Function Analysis: Combining structural modeling with experimental data on modified residues can reveal how PTMs affect enzyme conformation and function.

These approaches collectively provide insights into how PTMs like phosphorylation dynamically regulate HAS3 activity in response to cellular signals.

What are the challenges in designing experiments to study HAS3 function in different tissue contexts?

Investigating HAS3 function across diverse tissue contexts presents several methodological challenges that researchers must address through careful experimental design:

• Tissue-Specific Expression Patterns:

  • HAS3 shows variable expression across different tissues, requiring precise characterization of endogenous levels before experimental manipulation .

  • Techniques such as RNA-seq, qPCR, and immunohistochemistry with isoform-specific antibodies are essential for mapping expression patterns.

  • Researchers must consider that tissue-specific co-expression of different HAS isoforms may lead to functional redundancy or complementation.

• Microenvironment Influences:

  • The tissue microenvironment, including pH, ion concentrations, and available substrates, can significantly affect HAS3 activity.

  • In vitro systems may not accurately recapitulate these conditions, necessitating careful adjustment of experimental parameters.

  • Co-culture systems incorporating relevant cell types may better represent in vivo conditions.

• Substrate Availability Variations:

  • UDP-GlcA and UDP-GlcNAc concentrations may vary between tissues, affecting HAS3 activity.

  • Metabolic labeling experiments should account for potential differences in precursor uptake and incorporation rates across tissue types.

• Regulatory Network Differences:

  • Signaling pathways regulating HAS3 phosphorylation (e.g., cAMP-dependent pathways) may function differently across tissues .

  • Tissue-specific transcription factors and epigenetic modifications may alter HAS3 gene expression in ways unique to each context.

  • Systematic analysis of HAS3 responses to stimuli across different cell types can help map these regulatory networks.

• Functional Outcome Assessment:

  • The biological consequences of HAS3 activity may differ between tissues (e.g., structural roles in cartilage versus signaling roles in inflammation).

  • Appropriate functional readouts must be selected based on tissue-specific hyaluronan roles.

  • Both gain-of-function (overexpression) and loss-of-function (knockdown/knockout) approaches are valuable for comprehensive functional assessment.

Addressing these challenges requires integrated approaches combining molecular, cellular, and tissue-level analyses to fully understand the context-dependent functions of HAS3.

How can one troubleshoot issues with recombinant HAS3 expression and purification?

Expressing and purifying functional recombinant HAS3 presents several technical challenges that researchers commonly encounter. Here are methodological approaches to address these issues:

• Expression System Selection:

  • E. coli Systems: While commonly used for recombinant chicken HAS3 production , bacterial systems may lack appropriate post-translational modifications and membrane environments.

  • Eukaryotic Systems: COS-7 cells have been successfully used for FLAG-tagged human HAS3 expression , providing a more native environment for proper folding and modification.

  • Troubleshooting: If expression levels are low, consider codon optimization, use of stronger promoters, or inducible expression systems to reduce potential toxicity.

• Solubilization Strategies:

  • Detergent Selection: Research has shown that non-ionic or zwitterionic detergents like CHAPS may be insufficient for HAS3 solubilization. SDS has been used successfully, with subsequent dilution in buffers containing non-ionic detergents for affinity purification .

  • Protocol Optimization: Detergent concentration, buffer composition, temperature, and incubation time should be systematically optimized for maximal recovery of active protein.

• Affinity Purification Approaches:

  • Tag Selection: FLAG tags have been successfully employed for HAS3 purification using anti-FLAG M2 monoclonal antibody agarose .

  • Binding and Elution Conditions: Ensure complete binding by confirming the absence of target protein in flow-through fractions. Specific elution with FLAG peptide can improve purity compared to direct elution with SDS-containing buffers .

  • Contaminant Reduction: Silver staining of purified fractions helps assess purity. Be aware that Ig heavy and light chains from affinity resins may appear as contaminants at approximately 25 and 50 kDa .

• Activity Preservation:

  • Storage Optimization: Store purified recombinant HAS3 at -20°C to -80°C with 5-50% glycerol to maintain activity. Avoid repeated freeze-thaw cycles .

  • Activity Assays: Develop sensitive assays to confirm that purified protein retains enzymatic activity, such as measuring incorporation of radiolabeled UDP-sugars into hyaluronan.

• Protein Verification:

  • Western Blotting: Confirm identity and integrity using appropriate antibodies against HAS3 or epitope tags.

  • Mass Spectrometry: Peptide mapping can verify protein identity and identify any post-translational modifications or proteolytic degradation.

By systematically addressing these aspects, researchers can improve the yield and quality of recombinant HAS3 preparations for subsequent functional studies.

What are the methodological considerations for comparing HAS3 from different species?

Comparative analysis of HAS3 from different species (such as chicken and human) requires careful methodological considerations to ensure valid interpretations:

• Sequence Homology Analysis:

  • Perform comprehensive sequence alignments to identify conserved domains, active sites, and potential phosphorylation sites.

  • Phylogenetic analysis can reveal evolutionary relationships and help predict functional conservation or divergence.

  • Special attention should be given to catalytic domains and regions involved in post-translational modifications.

• Expression System Standardization:

  • Use identical expression systems (e.g., E. coli or COS-7 cells ) for all species variants to eliminate system-dependent variations.

  • Ensure consistent vector design, tag placement, and induction conditions to minimize non-species-related differences.

  • Verify that expression levels are comparable to avoid artifacts from concentration differences.

• Biochemical Characterization:

  • Compare enzymatic parameters (Km, Vmax, substrate preference) under identical reaction conditions.

  • Assess product characteristics (chain length, rate of synthesis) using standardized analytical methods.

  • Evaluate sensitivity to inhibitors or activators to identify potential species-specific regulatory mechanisms.

• Phosphorylation Analysis:

  • Compare phosphorylation patterns using identical methodologies, such as [32P]Pi radiolabeling .

  • Determine whether the same effectors (e.g., CPT-cAMP) enhance phosphorylation across species variants.

  • Quantify phosphorylation stoichiometry under standardized conditions to identify species differences in regulation.

• Structural Studies:

  • Consider how sequence differences might affect protein structure and function.

  • Techniques such as circular dichroism, limited proteolysis, or if feasible, X-ray crystallography or cryo-EM can reveal structural similarities and differences.

  • Computational modeling can predict how sequence variations might affect substrate binding or catalytic mechanism.

• Functional Complementation:

  • Test whether HAS3 from one species can functionally replace that of another in cellular or in vivo models.

  • This approach can reveal biologically relevant functional conservation or divergence beyond biochemical characteristics.

These methodological considerations ensure that observed differences between species variants of HAS3 reflect genuine biological distinctions rather than experimental artifacts.