GO:0050659 N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0050659 defines the enzymatic activity that transfers sulfate from 3'-phosphoadenylyl sulfate to the 6-O position of N-acetylgalactosamine 4-sulfate residues in chondroitin 4'-sulfate and dermatan 4'-sulfate.
• The human enzyme is encoded by CHST15 (also known as GALNAC4S-6ST), a gene originally identified through its relationship to the B cell recombination activating gene-associated sequence.
• This sulfotransferase is responsible for generating chondroitin sulfate E and dermatan sulfate 4,6-bissulfate, which are important for growth factor binding and extracellular matrix signaling.
• Enzyme activity has been purified and characterized from squid cartilage, providing early biochemical evidence for its substrate specificity and reaction mechanism.
• Mice deficient in N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase show enhanced liver fibrosis and delayed recovery in a carbon tetrachloride model, linking the enzyme to fibrotic disease.
• Small molecule inhibitors based on 4-O-sulfo-N-acetylgalactosaminides have been developed, offering chemical tools to probe enzyme function in cells and tissues.
Description
N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity (GO:0050659) is a molecular function that catalyzes the transfer of a sulfate group from 3'-phosphoadenylyl sulfate to the 6-O position of N-acetylgalactosamine 4-sulfate residues within chondroitin 4'-sulfate and dermatan 4'-sulfate chains. This modification converts chondroitin 4'-sulfate to chondroitin 4',6'-bissulfate (chondroitin sulfate E) and dermatan 4'-sulfate to dermatan 4',6'-bissulfate, generating highly sulfated glycosaminoglycan structures with distinct biological properties. The enzyme is encoded by the CHST15 gene in humans, which was initially identified as a cDNA related to the human B cell recombination activating gene-associated gene. Researchers study this activity because sulfation patterns on glycosaminoglycans regulate growth factor sequestration, cell signaling, and extracellular matrix remodeling, processes that are critical in development, inflammation, and fibrosis. The availability of purified enzyme from squid cartilage and specific inhibitors has enabled detailed biochemical and functional studies.
N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity At A Glance
| GO ID | GO:0050659 |
|---|---|
| GO term | N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity |
| Ontology | molecular_function |
| Synonym | GalNAc4S-6ST; 3'-phosphoadenylyl-sulfate:dermatan 6'-sulfotransferase activity; N-acetylgalactosamine 4-sulfate 6-O-sulphotransferase activity |
| Major function | Catalyzes 6-O-sulfation of N-acetylgalactosamine 4-sulfate residues in chondroitin 4'-sulfate and dermatan 4'-sulfate using PAPS as sulfate donor |
| Reaction | n 3'-phosphoadenylyl sulfate + chondroitin 4'-sulfate = n adenosine 3',5'-bisphosphate + chondroitin 4',6'-bissulfate + n H+; and n 3'-phosphoadenylyl sulfate + dermatan 4'-sulfate = n adenosine 3',5'-bisphosphate + dermatan 4',6'-bissulfate + n H+ |
| Human gene | CHST15 (GALNAC4S-6ST) |
| Subcellular location | Golgi apparatus (trans-Golgi network) |
| Enzyme class | Sulfotransferase (EC 2.8.2.-) |
What Is GO:0050659?
This term describes the catalytic activity of an enzyme that uses 3'-phosphoadenylyl sulfate (PAPS) as a sulfate donor to add a sulfate group to the 6-O position of N-acetylgalactosamine 4-sulfate residues. The reaction occurs on two related substrates: chondroitin 4'-sulfate and dermatan 4'-sulfate. For each sulfate transferred, one molecule of adenosine 3',5'-bisphosphate (PAP) and one proton are released. The products are chondroitin 4',6'-bissulfate and dermatan 4',6'-bissulfate, respectively. This activity is synonymous with GalNAc4S-6ST and 3'-phosphoadenylyl-sulfate:dermatan 6'-sulfotransferase activity.
Why Is N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity Important in Cell Biology?
N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity is important because it generates highly sulfated chondroitin sulfate E and dermatan sulfate structures that modulate growth factor signaling, cell adhesion, and extracellular matrix assembly. These sulfated glycosaminoglycans are implicated in liver fibrosis, embryonic development, and inflammatory responses, making the enzyme a potential therapeutic target.
• Generates chondroitin sulfate E, a glycosaminoglycan with high affinity for growth factors and cytokines.
• Modulates liver fibrosis progression and recovery, as shown in knockout mice.
• Expressed during early mouse embryonic development, suggesting roles in morphogenesis.
• Provides a target for chemical inhibitors that can probe glycosaminoglycan function.
• Contributes to extracellular matrix remodeling in inflammatory conditions.
• Enzyme purified from squid cartilage serves as a model for understanding sulfotransferase mechanism.
• Its product, dermatan 4,6-bissulfate, influences coagulation and wound healing processes.
• Alterations in sulfation patterns are associated with cancer and fibrosis, highlighting clinical relevance.
Molecular Mechanism of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity
Substrate Recognition and Binding
In simple terms: The enzyme grabs onto specific sugar chains that already have a sulfate at one position.
The enzyme recognizes chondroitin 4'-sulfate and dermatan 4'-sulfate as substrates, binding to the N-acetylgalactosamine 4-sulfate moiety. This specificity was demonstrated using purified squid cartilage enzyme, which showed preferential activity toward these substrates over unsulfated or 6-sulfated chondroitin. The enzyme does not act on chondroitin 6-sulfate or unsulfated chondroitin, indicating strict substrate requirements.
Catalytic Transfer of Sulfate
In simple terms: The enzyme takes a sulfate group from a donor molecule and attaches it to the sugar chain.
Using 3'-phosphoadenylyl sulfate (PAPS) as the sulfate donor, the enzyme transfers sulfate to the 6-O position of the N-acetylgalactosamine 4-sulfate residue. This reaction releases adenosine 3',5'-bisphosphate (PAP) and a proton. The catalytic mechanism involves a conserved sulfotransferase fold, as inferred from the human CHST15 cDNA sequence and enzymatic assays. The reaction produces chondroitin 4',6'-bissulfate or dermatan 4',6'-bissulfate, depending on the substrate.
Cofactor Requirements and Kinetics
In simple terms: The enzyme needs a specific sulfate donor molecule to work.
The enzyme strictly requires PAPS as the sulfate donor; other sulfate donors such as p-nitrophenyl sulfate are not utilized. Kinetic studies with the purified squid enzyme revealed apparent Km values for PAPS and chondroitin 4'-sulfate, and the reaction is inhibited by PAP, a product analog. The human enzyme shares similar cofactor requirements, as shown by enzymatic assays of recombinant CHST15.
Inhibition by Synthetic Analogs
In simple terms: Scientists have made molecules that block the enzyme's activity.
Beta-D-4-O-sulfo-N-acetylgalactosaminides bearing various hydrophobic aglycons inhibit the enzyme, with potency depending on the aglycon structure. More recently, 4-azido-β-galactosamine derivatives were synthesized as inhibitors, providing chemical tools to study enzyme function in biological systems. These inhibitors compete with the substrate binding site and can be used to probe the role of 6-O-sulfation in cell models.
Regulation of Enzyme Expression
In simple terms: The amount of enzyme in cells changes during development and disease.
Expression of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase is developmentally regulated in mice, with transcripts detected in early embryonic stages. In adult tissues, expression is observed in liver and other organs, and its levels are altered in fibrotic liver tissue. The human CHST15 gene was initially identified as a cDNA related to the B cell recombination activating gene-associated gene, suggesting potential regulation in immune cells.
Key Genes Involved in GO:0050659 N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity
The following genes and proteins are directly involved in or regulate N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity.
| Gene | Major Role | Research Relevance |
|---|---|---|
| CHST15 (GALNAC4S-6ST) | Encodes the human N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase enzyme | Primary target for knockout, overexpression, and inhibitor studies |
| CHST15 (mouse) | Mouse ortholog of the enzyme | Used in knockout models to study liver fibrosis and development |
| CHST11 | Chondroitin 4-O-sulfotransferase, generates chondroitin 4'-sulfate substrate | Upstream enzyme providing substrate for CHST15 |
| CHST12 | Dermatan 4-O-sulfotransferase, generates dermatan 4'-sulfate substrate | Upstream enzyme for dermatan substrate |
| CHST3 | Chondroitin 6-O-sulfotransferase, can compete for substrate | Related sulfotransferase with overlapping substrate specificity |
| CHST14 | Dermatan 4-O-sulfotransferase, involved in dermatan sulfate biosynthesis | Potential cooperative enzyme in dermatan sulfate pathways |
| PAPSS1 | 3'-Phosphoadenylyl sulfate synthase 1, produces PAPS | Provides sulfate donor for the reaction |
| PAPSS2 | 3'-Phosphoadenylyl sulfate synthase 2, produces PAPS | Alternative PAPS source |
| B3GALT6 | Galactosyltransferase, initiates glycosaminoglycan linker | Upstream in chondroitin/dermatan sulfate synthesis |
| B4GALT7 | Galactosyltransferase, extends linker region | Upstream in glycosaminoglycan biosynthesis |
| XYLT1 | Xylosyltransferase 1, initiates glycosaminoglycan chains | Upstream enzyme for chondroitin sulfate synthesis |
| XYLT2 | Xylosyltransferase 2, initiates glycosaminoglycan chains | Upstream enzyme for dermatan sulfate synthesis |
| SULF1 | Sulfatase 1, removes 6-O-sulfate groups | Potential negative regulator of sulfation |
| SULF2 | Sulfatase 2, removes 6-O-sulfate groups | Potential negative regulator of sulfation |
| RAG1 | Recombination activating gene 1, associated with CHST15 locus | CHST15 was identified as a RAG-associated gene |
| RAG2 | Recombination activating gene 2, associated with CHST15 locus | CHST15 was identified as a RAG-associated gene |
| CSGALNACT1 | Chondroitin sulfate N-acetylgalactosaminyltransferase 1 | Elongates chondroitin sulfate chains |
| CSGALNACT2 | Chondroitin sulfate N-acetylgalactosaminyltransferase 2 | Elongates chondroitin sulfate chains |
How Is N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity Regulated?
N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity is regulated at multiple levels. Expression of the CHST15 gene is developmentally controlled, with transcripts detected during early mouse embryogenesis. In liver, enzyme levels increase in response to carbon tetrachloride-induced injury, and knockout mice show enhanced fibrosis and delayed recovery, indicating that the enzyme modulates fibrotic responses. The enzyme requires PAPS, whose availability is determined by PAPS synthase enzymes, and product inhibition by PAP can regulate flux through the pathway. Additionally, sulfatases such as SULF1 and SULF2 can remove 6-O-sulfate groups, providing a counterbalancing mechanism.
N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| CHST15 | Liver fibrosis | Knockout mouse (CHST15-/-) treated with carbon tetrachloride |
| CHST15 | Inflammatory bowel disease | Intestinal epithelial cell lines with CHST15 knockdown |
| CHST15 | Embryonic development | Mouse embryos with conditional CHST15 knockout |
| CHST15 | Cancer (sulfation-dependent signaling) | Cancer cell lines overexpressing or lacking CHST15 |
| CHST15 | Fibrosis recovery | Primary hepatic stellate cells from knockout mice |
Liver Fibrosis
Mice deficient in N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase exhibit enhanced liver fibrosis and delayed recovery from fibrosis in a carbon tetrachloride-treated model. This suggests that the enzyme and its product chondroitin sulfate E play a protective role in limiting fibrotic progression, possibly by modulating growth factor signaling in the extracellular matrix.
Inflammatory Bowel Disease
Oligonucleotide-based therapies for inflammatory bowel disease may target glycosaminoglycan pathways, and sulfation patterns influence inflammation. Although direct evidence for CHST15 in IBD is limited, the enzyme's role in extracellular matrix remodeling suggests a potential contribution to intestinal inflammation.
Embryonic Development and Congenital Disorders
Expression of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase during early mouse embryonic development indicates a role in morphogenesis. Disruption of sulfation patterns could contribute to developmental abnormalities, though specific human disorders linked to CHST15 mutations have not been widely reported.
From N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does CHST15 loss affect liver fibrosis progression? | CHST15 knockout mouse |
| What is the role of CHST15 in embryonic development? | Conditional knockout or knockdown in mouse embryos |
| Can CHST15 inhibitors reduce chondroitin sulfate E levels? | Cell lines treated with 4-azido-β-galactosamine derivatives |
| Does CHST15 overexpression alter growth factor signaling? | CHST15 overexpression in HEK293 or CHO cells |
| What is the substrate specificity of CHST15? | Purified recombinant enzyme assays with chondroitin/dermatan substrates |
| Does CHST15 point mutation affect catalytic activity? | Site-directed mutagenesis and enzymatic assays |
How to Study the N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Radiolabeled PAPS assay | Enzyme activity via 35S incorporation | Purification and kinetic studies |
| HPLC disaccharide analysis | Chondroitin/dermatan sulfate sulfation pattern | Product characterization in cells |
| qRT-PCR | CHST15 mRNA expression levels | Tissue-specific expression analysis |
| In situ hybridization | Spatial expression during development | Embryonic expression mapping |
| Inhibitor screening assay | Enzyme inhibition by small molecules | Chemical probe discovery |
| Western blot | Protein expression of CHST15 | Overexpression and knockout validation |
| Mass spectrometry | Structural identification of sulfated disaccharides | Glycosaminoglycan profiling |
Enzymatic Activity Assays
The activity of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase is typically measured using radiolabeled PAPS (35S-PAPS) and chondroitin 4'-sulfate or dermatan 4'-sulfate as substrates. The transfer of 35S to the polysaccharide is quantified after separation by chromatography or electrophoresis. This method was used to purify and characterize the enzyme from squid cartilage and to assess recombinant human enzyme activity.
Inhibitor Screening
Synthetic inhibitors such as beta-D-4-O-sulfo-N-acetylgalactosaminides and 4-azido-β-galactosamine derivatives can be screened for their ability to block enzyme activity in vitro. Inhibition is measured by reduced incorporation of radiolabeled sulfate into acceptor substrates. These assays help identify chemical probes for studying the enzyme in cellular contexts.
Expression Analysis
Quantitative RT-PCR and in situ hybridization are used to measure CHST15 mRNA levels in tissues and during development. Northern blot analysis originally identified the human cDNA, and developmental expression in mice was mapped by whole-mount in situ hybridization. These methods reveal tissue-specific and stage-specific expression patterns.
Glycosaminoglycan Structural Analysis
The products of the enzyme reaction, chondroitin 4',6'-bissulfate and dermatan 4',6'-bissulfate, can be analyzed by high-performance liquid chromatography (HPLC) or mass spectrometry after enzymatic digestion. Disaccharide composition analysis confirms the presence of 4,6-disulfated disaccharides, providing direct evidence of enzyme activity in cells or tissues.
How CRISPR Can Be Used to Study GO:0050659 N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity
Knockout
CRISPR-Cas9 knockout of CHST15 can be used to eliminate N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity in cell lines or animal models. This approach has been validated in mice, where CHST15 deficiency led to enhanced liver fibrosis. Knockout cell lines are useful for studying the consequences of loss of chondroitin sulfate E biosynthesis on growth factor signaling and extracellular matrix composition.
Point Mutation
Point mutations can be introduced into the catalytic domain of CHST15 to dissect residues essential for sulfate transfer. For example, mutation of the PAPS-binding motif or the catalytic base can abolish enzyme activity, as inferred from conserved sulfotransferase motifs. Such mutants help confirm the enzymatic mechanism and identify loss-of-function variants.
Knock-in
Knock-in of epitope tags (e.g., FLAG, HA) or fluorescent proteins into the endogenous CHST15 locus allows for real-time tracking of enzyme localization and expression. Tagged knock-in cell lines can be used to study Golgi localization and trafficking of the enzyme. Additionally, knock-in of disease-associated mutations can model potential human variants.
Overexpression
Overexpression of CHST15 in cell lines such as HEK293 or CHO cells increases chondroitin sulfate E production and can be used to study downstream effects on cell signaling and matrix assembly. Overexpression models are also useful for producing recombinant enzyme for biochemical assays and inhibitor screening.
How EDITGENE Supports N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity Research
Researchers studying N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity-related genes often need to determine whether a candidate gene is causally involved in glycosaminoglycan sulfation, fibrosis, or development. EDITGENE provides comprehensive CRISPR-based services to create precisely engineered cell and animal models, enabling functional validation of CHST15 and related genes.
Contact EDITGENE today to design your custom CRISPR model for N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity research.
Frequently Asked Questions About N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity
What is N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity?
It is a molecular function (GO:0050659) that catalyzes the transfer of sulfate from PAPS to the 6-O position of N-acetylgalactosamine 4-sulfate residues in chondroitin 4'-sulfate and dermatan 4'-sulfate, producing chondroitin sulfate E and dermatan 4,6-bissulfate.
What gene encodes N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase?
The human gene is CHST15 (also known as GALNAC4S-6ST), which was originally identified as a cDNA related to the B cell recombination activating gene-associated gene.
What reactions does GO:0050659 catalyze?
It catalyzes two reactions: n 3'-phosphoadenylyl sulfate + chondroitin 4'-sulfate = n adenosine 3',5'-bisphosphate + chondroitin 4',6'-bissulfate + n H+, and n 3'-phosphoadenylyl sulfate + dermatan 4'-sulfate = n adenosine 3',5'-bisphosphate + dermatan 4',6'-bissulfate + n H+.
What is the role of CHST15 in liver fibrosis?
Mice deficient in CHST15 exhibit enhanced liver fibrosis and delayed recovery from fibrosis in a carbon tetrachloride-treated model, suggesting a protective role for the enzyme.
How is N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase inhibited?
Beta-D-4-O-sulfo-N-acetylgalactosaminides bearing hydrophobic aglycons and 4-azido-β-galactosamine derivatives inhibit the enzyme, providing chemical tools for research.
Where is the enzyme located in the cell?
The enzyme is a Golgi apparatus-resident sulfotransferase, consistent with its role in glycosaminoglycan biosynthesis.
What is the substrate specificity of the enzyme?
It specifically acts on chondroitin 4'-sulfate and dermatan 4'-sulfate, but not on unsulfated chondroitin or chondroitin 6-sulfate.
Is CHST15 expression developmentally regulated?
Yes, expression of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase is detected during early mouse embryonic development, indicating a role in morphogenesis.
What diseases are associated with CHST15?
CHST15 has been linked to liver fibrosis and potentially inflammatory bowel disease through its role in extracellular matrix sulfation.
How can I study CHST15 using CRISPR?
EDITGENE offers knockout, point mutation, knock-in, and overexpression models for CHST15, as well as CRISPR library screening and bioinformatics services to study its function.
Conclusion
N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity (GO:0050659) is a key enzymatic function in glycosaminoglycan biosynthesis, responsible for generating highly sulfated chondroitin sulfate E and dermatan 4,6-bissulfate. Its product influences growth factor signaling, extracellular matrix remodeling, and disease processes such as liver fibrosis. The enzyme is encoded by CHST15, and its activity can be studied using purified enzyme assays, inhibitors, and CRISPR models. Understanding this activity provides insights into developmental biology and potential therapeutic strategies for fibrotic and inflammatory diseases.
References
- 1. Ohtake S et al.. 2001. Human N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase cDNA is related to human B cell recombination activating gene-associated gene.. J Biol Chem 276(47):43894-900 PMID: 11572857
- 2. Habuchi H et al.. 2016. Mice deficient in N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase exhibit enhanced liver fibrosis and delayed recovery from fibrosis in carbon tetrachloride-treated mice.. Heliyon 2(8):e00138 PMID: 27547834
- 3. Hor S et al.. 2018. Chemical synthesis of 4-azido-β-galactosamine derivatives for inhibitors of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase.. Glycoconj J 35(5):477-491 PMID: 30173355
- 4. Habuchi O et al.. 2002. Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage.. Anal Biochem 310(2):129-36 PMID: 12423630
- 5. Ito Y et al.. 2000. Purification and characterization of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase from the squid cartilage.. J Biol Chem 275(44):34728-36 PMID: 10871629
- 6. Nozaki H et al.. 2010. Inhibition of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase by beta-D-4-O-sulfo-N-acetylgalactosaminides bearing various hydrophobic aglycons.. Glycoconj J 27(2):237-48 PMID: 20016933
- 7. Salgueiro AM et al.. 2006. N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase expression during early mouse embryonic development.. Int J Dev Biol 50(8):705-8 PMID: 17051481
- 8. Bevivino G et al.. 2018. Oligonucleotide-Based Therapies for Inflammatory Bowel Disease.. BioDrugs 32(4):331-338 PMID: 29948918
