Recombinant Xenopus laevis Hyaluronan synthase 3 (has3)

What is Xenopus laevis hyaluronan synthase 3 (has3) and how does it function?

Hyaluronan synthase 3 (has3) in Xenopus laevis is a member of the glycosyltransferase (GT) family-2 enzymes responsible for synthesizing hyaluronan (HA), an essential matrix polysaccharide in vertebrates. Has3 functions by sequentially adding monosaccharide units from uridine diphosphate-activated (UDP) donors to form the disaccharide repeats of HA polysaccharide with the structure [-3-GlcNAc-1-β-4-GlcA-1-β-] . The enzyme catalyzes the addition of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) in an alternating pattern, releasing UDP as a second reaction product. Like other hyaluronan synthases, has3 contains multiple transmembrane domains and is lipid-dependent, suggesting it creates an intraprotein HAS-lipid pore through which growing HA chains can be translocated across cell membranes .

How is has3 characterized in the Xenopus model system?

Has3 in Xenopus laevis (also referred to as xhas3) is characterized as part of the chitin synthase/hyaluronan synthase family of glycosyltransferases . The gene information is documented in Xenbase (the Xenopus model organism knowledgebase), which serves as a comprehensive resource for Xenopus-related genomic and biological research data . Has3 plays significant roles in embryonic development, making it particularly valuable in the Xenopus model system which has been extensively used for developmental and cell biology research . The Xenopus model offers large eggs and embryos with rapid external development, making it ideal for studying developmental processes involving has3 .

What expression systems are optimal for producing recombinant Xenopus laevis has3?

Based on established protocols for related proteins, bacterial expression systems (particularly E. coli) provide a cost-effective and relatively simple approach for recombinant has3 production . For has3 and related hyaluronan synthases, the following expression systems have proven effective:

• E. coli expression systems: Suitable for producing partial or full-length has3 protein with N-terminal tags (such as His-tags) for purification purposes . This approach has been successfully used for has-rs protein production.

• Insect cell expression systems: For more complex proteins requiring eukaryotic post-translational modifications. This system has been employed for expression of other Xenopus proteins requiring proper folding and functionality .

• Saccharomyces cerevisiae: Particularly useful for hyaluronan synthases as demonstrated with Xenopus laevis HAS1, as yeast lacks endogenous hyaluronan and UDP-GlcA, allowing for clean in vitro initiation of HA biosynthesis without background interference .

The choice between these systems should be guided by the intended application and whether enzymatic activity needs to be preserved.

What are the critical factors for purification of recombinant has3 while maintaining structural integrity?

Successful purification of recombinant has3 while preserving its structural integrity requires careful consideration of several factors:

• Affinity tag selection: N-terminal His-tags are commonly used for purification of hyaluronan synthases and related proteins from Xenopus laevis . This approach allows for efficient capture using Ni-NTA affinity chromatography.

• Buffer composition: Tris or HEPES-based buffers with pH 7.5-8.0 are typically used for hyaluronan synthases, often supplemented with:

  • 5-10% glycerol to enhance protein stability

  • 150-300 mM NaCl for ionic strength

  • Protease inhibitors during initial lysis steps

• Elution and storage conditions:

  • Gradual imidazole gradient (20-500 mM) for elution

  • Storage in buffers containing 5-50% glycerol at -20°C/-80°C

  • Aliquoting to avoid repeated freeze-thaw cycles, which can significantly reduce enzymatic activity

• Membrane protein considerations: As has3 contains transmembrane domains, inclusion of appropriate detergents or lipids during purification may be necessary to maintain proper folding and function if the full transmembrane structure is needed .

What assays are available to measure recombinant has3 enzymatic activity?

Several complementary methods can be employed to assess the enzymatic activity of recombinant has3:

• Radiochemical incorporation assays: Measuring the incorporation of radiolabeled UDP-sugars (UDP-[14C]GlcA or UDP-[3H]GlcNAc) into high molecular weight hyaluronan. This approach allows quantification of synthesis rates under various conditions.

• Mass spectrometry analysis: Characterization of reaction products to confirm proper disaccharide formation and chain elongation patterns. This has been particularly useful in identifying novel products like chitin-UDP oligomers made by hyaluronan synthases .

• Size exclusion chromatography: Analyzing the molecular weight distribution of synthesized hyaluronan to assess processivity and chain length control by the enzyme.

• Fluorescence-based assays: Using fluorescently labeled substrates or products to monitor enzyme activity in real-time, potentially in combination with single-molecule imaging approaches similar to those used for other glycosyltransferases .

• Native PAGE analysis: Used to study protein-protein interactions and complex formation, which may be relevant for understanding regulatory mechanisms of has3 activity .

How can researchers differentiate between reducing-end and non-reducing-end mechanisms of has3 activity?

Distinguishing between these mechanisms requires specialized approaches:

• Pulse-chase labeling experiments: By initiating synthesis with one labeled substrate followed by chase with unlabeled substrate, then analyzing the location of the label in the final product using exoglycosidase digestion. This approach has confirmed that Class I hyaluronan synthases (including those from Xenopus) elongate HA at the reducing end .

• Mass spectrometry analysis of early reaction products: Identifying the initial oligosaccharides formed and the sequential addition pattern. This has been essential in demonstrating that some hyaluronan synthases initially create chitin oligomers before switching to HA synthesis .

• Heterologous expression in systems lacking UDP-GlcA: Expression in Saccharomyces cerevisiae allows clean initiation of HA biosynthesis in vitro without background interference, making mechanistic studies more definitive .

• Single-molecule imaging techniques: These can be adapted to visualize the growing HA chain and determine the directionality of elongation, similar to approaches used for other processive enzymes .

How does the membrane topology of has3 influence its function in hyaluronan biosynthesis and translocation?

The membrane topology of has3 is critical to its dual function in hyaluronan synthesis and translocation:

• Transmembrane domain organization: Has3, like other Class I hyaluronan synthases, contains multiple transmembrane domains that are proposed to form an intraprotein pore. This pore structure is believed to facilitate the continuous translocation of growing HA chains across the cell membrane to the extracellular space .

• Pendulum model hypothesis: This model suggests a mechanism for HA translocation where the growing HA-UDP chain moves through an alternating pendulum-like motion through the membrane pore without requiring direct ATP hydrolysis. Instead, the energy from the glycosidic bond formation may drive the translocation process .

• Lipid dependencies: The activity of hyaluronan synthases is lipid-dependent, suggesting specific lipid interactions may be necessary for proper pore formation or enzyme function. These interactions could be studied using reconstituted liposome systems with purified recombinant has3 .

• Experimental approaches: The membrane topology can be investigated using techniques such as:

  • Cysteine scanning mutagenesis combined with accessibility assays

  • Fluorescence resonance energy transfer (FRET) to map proximities between domains

  • Cryo-electron microscopy for structural characterization of the membrane-embedded enzyme

How is has3 expression and function regulated during Xenopus embryonic development?

Understanding the developmental regulation of has3 requires integration of multiple experimental approaches:

• Stage-specific expression analysis: Using the Normal Table of Xenopus development (available on Xenbase) as a reference for precise staging, researchers can analyze has3 expression patterns across developmental timepoints . This can be performed using techniques such as:

  • RT-qPCR for quantitative expression analysis

  • In situ hybridization to visualize spatial expression patterns

  • RNA-seq for genome-wide expression profiling

• Functional studies: The role of has3 during development can be investigated through:

  • Morpholino-mediated knockdown

  • CRISPR/Cas9 gene editing to generate has3 mutations

  • Overexpression studies using microinjection of has3 mRNA

• Hyaluronan detection and localization: Visualizing the product of has3 activity during development using:

  • Hyaluronan-binding proteins coupled to fluorescent reporters

  • Immunohistochemistry with antibodies specific to hyaluronan

  • Metabolic labeling approaches to track newly synthesized hyaluronan

• Integration with Xenbase resources: The Xenopus landmarks table and developmental stage illustrations can help researchers precisely correlate has3 expression/function with specific developmental events .

What are the optimal storage conditions for preserving recombinant has3 activity?

Based on protocols for related hyaluronan synthases and has-rs protein, the following storage conditions are recommended:

• Short-term storage (up to one week): Store working aliquots at 4°C to maintain activity while avoiding freeze-thaw cycles .

• Long-term storage (months to years):

  • Store at -20°C/-80°C in buffer containing 5-50% glycerol (with 50% being optimal for maximum stability)

  • Aliquot into small volumes to avoid repeated freeze-thaw cycles

  • Use Tris/PBS-based buffer, pH 8.0 with 6% trehalose as an additional stabilizing agent

• Reconstitution from lyophilized form:

  • Briefly centrifuge vials prior to opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% before aliquoting and storing at -20°C/-80°C

• Stability considerations:

  • Shelf life for liquid form is typically 6 months at -20°C/-80°C

  • Shelf life for lyophilized form is approximately 12 months at -20°C/-80°C

  • Activity should be verified after long-term storage before use in critical experiments

How does has3 compare to other has family members in Xenopus laevis?

Research into the comparative biology of has family members reveals important functional and evolutionary insights:

• Sequence and structural comparisons: Has3 belongs to the same glycosyltransferase family (GT-2) as has1 and has2, sharing conserved catalytic domains but with distinct sequence regions that may confer specific functional properties . Comparisons of has-family proteins can provide insights into the evolution of glycosyltransferase mechanisms.

• Expression patterns: The three has enzymes (has1, has2, and has3) likely show differential expression patterns during development and across tissues, suggesting specialized roles in hyaluronan production. Xenbase resources can be utilized to compare expression data across different developmental stages .

• Functional specialization: Studies in other vertebrates suggest has3 typically produces shorter HA chains than has1 and has2. Similar patterns may exist in Xenopus, with each enzyme having distinct roles in producing HA of different molecular weights for specific developmental or physiological functions .

• Evolutionary conservation: Comparative analysis between Xenopus laevis (allotetraploid) and Xenopus tropicalis (diploid) has genes can provide insights into sub-functionalization following genome duplication events. This is particularly relevant given that X. laevis underwent a hybridization event 17-18 MYA resulting in two subgenomes (L and S chromosomes) .

What is the potential relationship between has3 activity and chitin oligomer synthesis?

Recent research has revealed intriguing connections between hyaluronan synthases and chitin synthesis:

• Dual enzymatic activity: Studies have shown that hyaluronan synthases from Xenopus laevis (specifically XlHAS1) can synthesize chitin oligomers in addition to hyaluronan . This suggests has3 may possess similar dual functionality, with the ability to produce (GlcNAc-β1,4)n oligomers under certain conditions.

• Initiation mechanism hypotheses: The synthesis of chitin-UDP oligomers by hyaluronan synthases supports the reducing end mechanism for sugar addition and suggests a possible role for chitin oligomers as self-primers for HA biosynthesis . This could mean that has3 initially creates short chitin oligomers before transitioning to alternating GlcNAc-GlcA addition for hyaluronan synthesis.

• Evolutionary implications: This dual functionality may reflect the evolutionary history of these enzymes, potentially indicating that modern hyaluronan synthases evolved from ancestral chitin synthases. Comparative studies across species could provide further insights into this evolutionary relationship .

• Methodological approaches: Researchers can investigate this relationship using:

  • Mass spectrometry to identify chitin oligomers produced by recombinant has3

  • Mutation studies to identify residues critical for substrate selectivity

  • Comparative analysis of reaction kinetics with different UDP-sugar substrates

What genomic and bioinformatic resources are available for Xenopus laevis has3 research?

Several specialized resources support research on Xenopus has3:

• Xenbase (https://www.xenbase.org/): The primary Xenopus model organism knowledgebase integrating diverse genomic and biological data . Relevant has3 resources include:

  • Gene expression data across developmental stages

  • Anatomical expression maps

  • Genome browser integration

  • Literature curation specific to has3

• Normal Table of Xenopus Development: A comprehensive resource with 133 high-quality illustrations from fertilization to metamorphosis, available on Xenbase for precise staging of developmental processes . This resource includes:

  • Detailed morphological features at each stage

  • Landmarks table for quick stage identification

  • Open-access images available under creative commons license

• Xenopus Stock Centers: Five established stock centers provide access to various Xenopus strains for research, including inbred J strain with sequenced genome (particularly useful for genetic studies of has3) .

• Bioinformatic tools:

  • JBrowse genome browser for visualizing has3 genomic context

  • BLAST tools optimized for Xenopus sequences

  • Gene expression data from GEO and SRA integrated with Xenbase

How can CRISPR/Cas9 technology be optimized for studying has3 function in Xenopus?

CRISPR/Cas9 approaches in Xenopus offer powerful tools for has3 functional studies:

• Guide RNA design considerations for Xenopus has3:

  • Account for the allotetraploid nature of X. laevis when designing guide RNAs

  • Target conserved regions between L and S homeologs if both need to be modified

  • Use Xenbase genome browser to identify suitable target sites with minimal off-target potential

• Delivery methods:

  • Microinjection of Cas9 protein and guide RNAs into fertilized eggs

  • Targeted injection into specific blastomeres for tissue-specific effects

  • Use of doxycycline-inducible Cas9 for temporal control of editing

• Phenotypic analysis approaches:

  • Utilize the Normal Table of Xenopus development for precise staging and phenotype characterization

  • Apply hyaluronan-specific staining methods to assess changes in HA production and distribution

  • Use the Xenopus Phenotype Ontology for standardized phenotype reporting

• Validation strategies:

  • T7 endonuclease assays for mutation detection

  • Direct sequencing of PCR products spanning the target site

  • Functional rescue experiments by co-injection of wild-type has3 mRNA

By applying these resources and techniques, researchers can conduct comprehensive investigations into the structure, function, and developmental roles of Xenopus laevis has3, contributing to our understanding of hyaluronan biology across vertebrate species.