Recombinant Human Carbohydrate sulfotransferase 3 (CHST3), partial

What is CHST3 and what is its primary biological function?

CHST3 encodes the chondroitin 6-O-sulfotransferase-1 (C6ST-1) enzyme, which belongs to the carbohydrate sulfotransferase family. This enzyme catalyzes a critical modifying step in chondroitin sulfate (CS) synthesis by transferring sulfate groups to the C-6 position of the N-acetylgalactosamine residue of chondroitin. This sulfation process is essential for proper extracellular matrix formation, particularly in connective tissues .

The enzyme displays specificity for multiple substrates including chondroitin, various CS variants, keratan sulfate, and sialyl lactosamine oligosaccharides, with major specificity for chondroitin. CHST3 plays a crucial role in skeletal development, as evidenced by the severe skeletal dysplasias observed in patients with CHST3 mutations .

What structural elements are essential for recombinant CHST3 function?

The CHST3 gene consists of three exons spanning approximately 49.2 kb of genomic sequence with an unusually large 3′ UTR of 5,115 bp. The open reading frame of 1,437 bp codes for a 479 amino acid protein . Critical to its function is the 3′-phosphoadenosine 5′-phosphosulfate (PAPS) binding site, which contains a highly conserved arginine at position 304. This residue is essential for the structure of the cosubstrate binding site, as demonstrated by functional studies showing that mutations at this position (such as R304Q) can completely abolish C6ST-1 activity .

The enzyme is anchored in the Golgi apparatus via its transmembrane domain, positioning it appropriately for the post-translational modification of chondroitin during proteoglycan synthesis .

What experimental approaches can be used to assess CHST3 enzyme activity?

Based on published methodologies, CHST3 enzyme activity can be assessed through sulfotransferase assays using:

• Sample preparation: Cell lysates (from patient fibroblasts or transfected cells) or recombinant enzymes

• Reaction components:

  • Polymer chondroitin as substrate (acceptor)

  • [³⁵S]PAPS as cosubstrate (sulfate donor)

  • Appropriate buffer conditions

• Analytical procedures:

  • Gel filtration to isolate reaction products

  • Liquid scintillation counting for quantification

  • Chondroitinase ABC digestion (5 milliunits) to determine specific sulfation positions

  • HPLC analysis of resulting unsaturated disaccharides

For specific position determination, C6ST activity can be quantified based on [³⁵S]sulfate incorporation into ΔHexA-GalNAc(6S), where ΔHexA and 6S represent unsaturated hexuronic acid and 6-O-sulfate, respectively .

How can recombinant CHST3 be engineered for optimized expression and solubility?

Recombinant CHST3 expression can be optimized using a strategy that addresses the transmembrane domain constraint. As demonstrated in published research, a soluble form of CHST3 can be engineered by:

• Replacing the first NH₂-terminal 48 amino acids (which include the transmembrane domain) with a cleavable insulin signal sequence.

• Fusing this construct to a protein A IgG-binding domain to facilitate purification.

• Expressing the construct in COS-1 cells, allowing secretion of the soluble enzyme into the medium.

• Purifying the recombinant protein using IgG-Sepharose beads, effectively separating it from endogenous C6ST .

This approach yields functional enzyme suitable for in vitro activity assays and structural studies while avoiding the solubility issues associated with the transmembrane domain.

What are the disease associations of CHST3 mutations and how can recombinant CHST3 be used to study these conditions?

CHST3 mutations are associated with a specific form of skeleton dysplasia known as Spondyloepiphyseal dysplasia with congenital joint dislocations (SED) . Clinical manifestations include:

• Short stature

• Kyphoscoliosis

• Joint dislocations

• Clubfoot

• Heart valve anomalies

• Progressive bilateral mixed hearing loss

As of 2015, 30 disease-associated mutations in CHST3 had been found in 45 patients globally, including familial cases from Oman, Tanzania, and Pakistan .

Recombinant CHST3 provides an excellent platform for:

• Functional characterization of patient-specific mutations

• Structure-function relationship studies

• High-throughput screening for therapeutic compounds

• Development of enzyme replacement therapy approaches

Molecular modeling of CHST3 can be performed using tools such as Phyre2 with the Sulfotransferase domain from the Curacin biosynthetic pathway as a template (Protein Data Bank ID: 4GBM) . This approach allows researchers to predict the structural consequences of specific mutations identified in patients.

How does CHST3 influence cell differentiation and tissue regeneration in musculoskeletal disorders?

Recent research has identified CHST3 as a key player in musculoskeletal disorders beyond skeletal dysplasias. Transcriptomic analyses have identified CHST3 as one of three potential diagnostic genes for both osteoporosis and sarcopenia . Gene set enrichment analysis (GSEA) revealed that CHST3 is involved in pathways related to:

• Cell cycle regulation

• Fatty acid metabolism

• DNA replication

• Carbohydrate synthesis

• Immune response regulation

In the context of intervertebral disc degeneration, CHST3 overexpression in cartilage endplate-derived stem cells (CESCs) appears to modulate molecular mechanisms related to tissue repair . Experimental approaches to study this include:

• Gene expression manipulation (overexpression or knockdown) in CESCs

• Co-culture systems with bone marrow cells

• Differentiation assays (Alizarin red and Alcian blue staining)

• Transmission electron microscopy (TEM) for ultrastructural analysis

• Western blot analysis of specific biomarkers including RUNX, OC (in osteoblasts) and aggrecan, collagen II (in chondroblasts)

What factors should be considered when designing activity assays for recombinant CHST3?

When designing activity assays for recombinant CHST3, researchers should consider:

• Substrate selection:

  • Polymer chondroitin is the preferred substrate

  • Alternative substrates include various CS variants, keratan sulfate, and sialyl lactosamine oligosaccharides

  • The choice of substrate should align with the specific research question

• Reaction conditions:

  • PAPS concentration and purity are critical for accurate results

  • Buffer composition affects enzyme stability and activity

  • Temperature and pH optimization is essential

  • Incubation time (typically 1 hour) should be validated for linearity

• Detection methods:

  • Radiometric assays using [³⁵S]PAPS offer high sensitivity

  • HPLC analysis of reaction products provides detailed structural information

  • Chondroitinase digestion followed by disaccharide analysis confirms specific sulfation patterns

• Controls:

  • Include wild-type recombinant CHST3 as a positive control

  • Use known inactive mutants (e.g., R304Q) as negative controls

  • Include substrate-free and enzyme-free reactions

How can molecular interactions between CHST3 and its substrates or regulatory partners be studied?

Investigating molecular interactions involving CHST3 requires multiple complementary approaches:

• Co-immunoprecipitation (Co-IP) can be used to detect protein-protein interactions, as demonstrated in studies examining the interaction between CHST3 and CSPG4 .

• Structural analysis through:

  • X-ray crystallography of recombinant CHST3 with substrates

  • Molecular modeling using templates such as the Sulfotransferase domain from the Curacin biosynthetic pathway (PDB: 4GBM)

  • Site-directed mutagenesis to validate predicted interaction sites

• Binding assays:

  • Surface plasmon resonance (SPR)

  • Isothermal titration calorimetry (ITC)

  • Fluorescence polarization for smaller ligands

• Functional genomics approaches:

  • Transcriptomic analysis of cells with modified CHST3 expression

  • GO and KEGG pathway analysis to identify functional networks

  • Integration of differential gene expression data with protein interaction networks

What analytical methods are most appropriate for characterizing the products of CHST3 activity?

The sulfated chondroitin products of CHST3 activity can be characterized using several analytical techniques:

• Anion-exchange HPLC analysis of disaccharide composition:

  • This method effectively separates and quantifies different sulfated disaccharides

  • Can detect specific patterns like ΔHexA-GalNAc(6S), ΔHexA(2S)-GalNAc(6S), and ΔHexA-GalNAc(4S,6S)

• Enzymatic digestion approaches:

  • Chondroitinase ABC digestion generates unsaturated disaccharides

  • These products can be analyzed to determine sulfation position and degree

• Mass spectrometry:

  • Provides detailed structural information about sulfation patterns

  • Can identify novel or unexpected modifications

• Biological activity assays:

  • Functional assessment of sulfated products in cell culture systems

  • Evaluation of binding to relevant receptors or growth factors

How should researchers interpret differences in sulfation patterns observed in CHST3 functional studies?

Interpretation of sulfation pattern differences requires careful consideration of multiple factors:

• Quantitative analysis:

  • Compare relative abundance of different disaccharide units

  • Example from patient studies shows significant reduction in ΔHexA-GalNAc(6S) and ΔHexA(2S)-GalNAc(6S) with increased ΔHexA-GalNAc(4S,6S)

• Tissue-specific patterns:

  • Normal sulfation profiles vary between tissue types

  • Interpret changes in the context of tissue-specific norms

• Compensatory mechanisms:

  • Altered activity of other sulfotransferases (e.g., C4ST) may occur

  • Changes in substrate availability can impact observed patterns

• Biological consequences:

  • Correlate sulfation changes with functional outcomes

  • Consider effects on binding to growth factors, cytokines, and cell surface receptors

A typical analytical approach would include comparative disaccharide composition analysis of CS chains by anion-exchange HPLC, looking for significant changes in specific sulfated disaccharides such as ΔHexA-GalNAc(6S) and ΔHexA(2S)-GalNAc(6S) .

What are the implications of CHST3 activity for tissue engineering and regenerative medicine applications?

CHST3's role in chondroitin sulfation makes it highly relevant for tissue engineering and regenerative medicine approaches focused on cartilage, bone, and intervertebral disc repair:

• Stem cell differentiation:

  • CHST3 expression influences the differentiation potential of cartilage endplate-derived stem cells (CESCs)

  • Modulation of CHST3 can direct CESCs toward osteogenic or chondrogenic lineages

• Extracellular matrix composition:

  • Proper sulfation of chondroitin is critical for matrix assembly and function

  • Engineered tissues require appropriate sulfation patterns for mechanical properties

• Therapeutic approaches:

  • Gene therapy to restore CHST3 function in skeletal disorders

  • Small molecule modulators of CHST3 activity for osteoporosis and sarcopenia

  • Cell-based therapies using CHST3-modified stem cells for intervertebral disc repair

• Biomarker development:

  • CHST3 expression and activity as potential biomarkers for musculoskeletal disorders

  • Sulfation patterns as indicators of disease progression or treatment response

How does recombinant partial CHST3 compare to full-length protein in research applications?

When using partial recombinant CHST3 compared to full-length protein, researchers should consider:

The construction of a soluble form of CHST3 by substituting the first NH₂-terminal 48 amino acids with an insulin signal sequence provides a practical example of engineering partial recombinant CHST3 while maintaining functionality .

What emerging technologies could enhance CHST3 research?

Several cutting-edge technologies hold promise for advancing CHST3 research:

• CRISPR-Cas9 genome editing:

  • Generation of precise CHST3 mutations to model disease variants

  • Creation of reporter cell lines for high-throughput screening

  • Knock-in of tagged CHST3 for localization and interaction studies

• Single-cell technologies:

  • Analysis of CHST3 expression in heterogeneous tissues

  • Correlation with cell state and differentiation potential

  • Integration with spatial transcriptomics for tissue context

• Advanced structural biology techniques:

  • Cryo-EM for structure determination of CHST3 complexes

  • Hydrogen-deuterium exchange mass spectrometry for dynamic structural information

  • In-cell NMR for studying CHST3 in its native environment

• Computational approaches:

  • Molecular dynamics simulations of CHST3-substrate interactions

  • Machine learning for predicting functional consequences of variants

  • Systems biology integration of CHST3 into broader regulatory networks

What are the current knowledge gaps in understanding CHST3 function and regulation?

Despite significant progress, several important questions remain about CHST3:

• Tissue-specific regulation:

  • Why does CHST3 deficiency predominantly affect skeletal tissues despite broad expression?

  • What accounts for the different phenotypes between CHST3-deficient mice and humans?

  • How is CHST3 expression regulated during development and in disease states?

• Functional interactions:

  • What is the full interactome of CHST3 in different cell types?

  • How do these interactions modulate enzyme activity and specificity?

  • What signaling pathways regulate CHST3 function?

• Disease mechanisms:

  • How does abnormal chondroitin sulfation lead to progressive skeletal abnormalities?

  • What is the molecular basis for hearing loss observed in some patients with CHST3 mutations?

  • How does CHST3 contribute to common age-related disorders like osteoporosis and sarcopenia?

• Therapeutic potential:

  • Can recombinant CHST3 or gene therapy effectively treat CHST3-related disorders?

  • What small molecules can modulate CHST3 activity for therapeutic benefit?

  • How can CHST3-related pathways be targeted for musculoskeletal tissue regeneration?

How can integration of multi-omics data enhance our understanding of CHST3 biology?

Multi-omics integration offers powerful opportunities to comprehensively understand CHST3 biology:

• Transcriptomics combined with proteomics:

  • Identification of co-regulated genes and proteins

  • Correlation between CHST3 expression and broader cellular states

  • CHST3 has been identified in transcriptomic analyses as a key gene in both osteoporosis and sarcopenia

• Glycomics and glycoproteomics:

  • Comprehensive profiling of chondroitin sulfation patterns

  • Correlation of sulfation changes with functional outcomes

  • Identification of novel CHST3 substrates

• Metabolomics:

  • Analysis of PAPS availability and metabolism

  • Effects of metabolic state on CHST3 activity

  • Identification of metabolic biomarkers associated with CHST3 function

• Integration frameworks:

  • Machine learning approaches to predict CHST3 activity from multi-omics data

  • Network analysis to position CHST3 within broader regulatory systems

  • Patient stratification based on integrated biomarker profiles