GO:0004464 leukotriene-C4 synthase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004464 (leukotriene-C4 synthase activity) catalyzes the conjugation of leukotriene A4 (LTA4) with glutathione to form leukotriene C4 (LTC4), the parent cysteinyl leukotriene.
• The reaction is an epoxide-ring-opening glutathione transfer, converting the unstable LTA4 epoxide into LTC4.
• LTC4 synthase (LTC4S) is a nuclear/perinuclear membrane protein that is the pivotal and committed step in cellular cysteinyl leukotriene biosynthesis.
• Enzyme activity is regulated post-translationally; phosphorylation of LTC4S at serine 36 impairs catalytic activity.
• LTC4 synthase activity is present in multiple cell types including platelets, and its species distribution has been characterized.
• Deficiency of LTC4 synthase causes spontaneous emphysema in female mice, linking this activity to lung structural homeostasis.
Description
Leukotriene-C4 synthase activity (GO:0004464) is a molecular function that produces leukotriene C4 (LTC4), the first committed cysteinyl leukotriene, by conjugating leukotriene A4 (LTA4) to reduced glutathione. This activity sits at the branch point of eicosanoid biosynthesis where the 5-lipoxygenase pathway is diverted toward cysteinyl leukotrienes, a family of lipid mediators with potent bronchoconstrictive and pro-inflammatory actions. Because LTC4 is the precursor of LTD4 and LTE4, the catalytic step defined by GO:0004464 controls the entire cysteinyl leukotriene output of a cell. Researchers study GO:0004464 because it is the terminal, dedicated enzymatic step for cysteinyl leukotriene synthesis and therefore a focal point for understanding allergic, asthmatic, and inflammatory disease mechanisms. The enzyme is a membrane-embedded glutathione transferase that uses LTA4, an unstable epoxide, as its substrate, and its activity can be measured biochemically in cell lysates and intact cells. Beyond inflammation, recent work has connected LTC4 synthesis to lung structural maintenance, since LTC4 synthase deficiency causes spontaneous emphysema in female mice, and to neurobiology, where modulation of leukotriene-C4 synthesis accompanies microglial repopulation and recovery of cognitive function after radiation. This article summarizes the QuickGO definition and authoritative literature on GO:0004464, covering its catalytic mechanism, the genes and proteins that carry or regulate the activity, its role in disease, and the experimental and CRISPR-based methods used to interrogate it.
leukotriene-C4 synthase activity At A Glance
| GO ID | GO:0004464 |
|---|---|
| GO term | leukotriene-C4 synthase activity |
| Ontology | molecular_function |
| Synonym | LTC4 synthase activity; leukotriene C4 synthetase activity; leukotriene A4:glutathione S-leukotrienyltransferase activity; leukotriene-C4 glutathione-lyase (leukotriene-A4-forming) |
| Definition | Catalysis of the reaction: leukotriene C(4) = glutathione + leukotriene A(4) |
| Major function | Conjugation of LTA4 with glutathione to form LTC4, the committed step in cysteinyl leukotriene biosynthesis |
| Substrate | Leukotriene A4 (LTA4) and reduced glutathione |
| Product | Leukotriene C4 (LTC4) |
| Representative enzyme | Leukotriene C4 synthase (LTC4S), a membrane-bound glutathione transferase |
| Related activity | Microsomal glutathione S-transferase with LTC4 synthase activity (e.g., MGST2) |
What Is GO:0004464?
In plain terms, GO:0004464 describes the catalytic activity that joins glutathione to leukotriene A4 to make leukotriene C4. Formally, the term is defined as the catalysis of the reaction: leukotriene C(4) = glutathione + leukotriene A(4). This is an epoxide-ring-opening glutathione transfer in which the thiol of glutathione attacks the epoxide of LTA4, yielding the glutathione conjugate LTC4. The activity is also known as LTC4 synthase activity, leukotriene C4 synthetase activity, and leukotriene A4:glutathione S-leukotrienyltransferase activity.
Why Is leukotriene-C4 synthase activity Important in Cell Biology?
GO:0004464 is important because it defines the committed enzymatic step that generates all cysteinyl leukotrienes, a class of lipid mediators centrally implicated in asthma, allergic inflammation, and other inflammatory conditions. Without this activity, LTA4 cannot be converted to LTC4, and the downstream cysteinyl leukotrienes LTD4 and LTE4 cannot be produced. The activity is also emerging as relevant to tissue homeostasis and neurobiology, as LTC4 synthase deficiency causes spontaneous emphysema in female mice and modulation of leukotriene-C4 synthesis accompanies microglial repopulation and cognitive recovery after radiation. Understanding GO:0004464 therefore informs both inflammatory disease biology and broader physiological roles of cysteinyl leukotrienes.
• Defines the committed step in cysteinyl leukotriene biosynthesis, producing LTC4 from LTA4 and glutathione.
• Controls the supply of LTC4, the precursor of LTD4 and LTE4, which mediate bronchoconstriction and inflammation.
• Provides a biochemical target for understanding allergic and asthmatic disease mechanisms.
• Is regulated by phosphorylation at serine 36, linking signaling to catalytic output.
• Is detectable in platelets and shows species-specific distribution, informing comparative studies.
• Loss of LTC4 synthase activity causes spontaneous emphysema in female mice, implicating it in lung structural homeostasis.
• Modulation of leukotriene-C4 synthesis is associated with microglial repopulation and recovery of cognitive function after radiation.
• The enzyme belongs to the MAPEG family, sharing sequence identity with 5-lipoxygenase-activating protein and related transferases.
• Mouse LTC4 synthase has been cloned and characterized, enabling genetic models.
• Measuring this activity supports drug discovery and biomarker development in inflammation research.
Molecular Mechanism of leukotriene-C4 synthase activity
Substrate binding and epoxide activation
In simple terms: The enzyme grabs an unstable lipid epoxide and holds it ready for a chemical attack.
Leukotriene C4 synthase binds leukotriene A4 (LTA4), an unstable epoxide intermediate generated by 5-lipoxygenase, and positions it for nucleophilic attack by glutathione. The enzyme is a membrane-bound glutathione transferase that acts on this lipid epoxide substrate rather than on typical xenobiotic substrates.
Glutathione conjugation and ring opening
In simple terms: Glutathione attacks the epoxide, opening the ring and attaching itself to the lipid.
The catalytic event is an epoxide-ring-opening glutathione transfer: the thiol of reduced glutathione attacks the LTA4 epoxide, forming a thioether bond and yielding leukotriene C4. This reaction is the defining chemistry of GO:0004464 and converts the unstable LTA4 into the stable, exportable cysteinyl leukotriene LTC4.
Product formation and downstream metabolism
In simple terms: The product LTC4 is the starting material for other cysteinyl leukotrienes.
LTC4 produced by this activity is subsequently converted to LTD4 and LTE4, so GO:0004464 controls the entry point into the cysteinyl leukotriene family. Because LTC4 is the parent compound, the activity of LTC4 synthase determines the cellular capacity for cysteinyl leukotriene production.
Enzyme identity and family context
In simple terms: Several related proteins can carry this activity, but LTC4 synthase is the dedicated enzyme.
The canonical enzyme carrying GO:0004464 is leukotriene C4 synthase (LTC4S), a member of the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) family. A related human microsomal glutathione S-transferase, MGST2, also displays LTC4 synthase activity and shares significant sequence identity with 5-lipoxygenase-activating protein and LTC4 synthase. Mouse LTC4 synthase has been cloned and characterized, confirming conservation of the activity.
Post-translational regulation of catalytic activity
In simple terms: Adding a phosphate tag to the enzyme can turn its activity down.
Phosphorylation of LTC4 synthase at serine 36 impairs its catalytic activity, providing a direct post-translational mechanism for regulating GO:0004464. This modification links upstream kinase signaling to the output of cysteinyl leukotriene biosynthesis.
Key Genes Involved in GO:0004464 leukotriene-C4 synthase activity
The following genes and proteins are directly implicated in leukotriene-C4 synthase activity (GO:0004464) or in its regulation and downstream context, based on the cited literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| LTC4S | Canonical leukotriene C4 synthase enzyme catalyzing LTA4 conjugation with glutathione | Core enzyme for GO:0004464; target for KO, point mutation, and overexpression studies |
| MGST2 | Microsomal glutathione S-transferase with LTC4 synthase activity | Alternative enzyme carrying the activity; comparative studies |
| ALOX5 | 5-lipoxygenase, generates LTA4 upstream of LTC4 synthase | Upstream pathway context for LTC4 production |
| ALOX5AP | 5-lipoxygenase-activating protein, shares sequence identity with LTC4S | Family context and pathway regulation |
| GGT1 | Gamma-glutamyl transpeptidase, converts LTC4 to LTD4 | Downstream metabolism of LTC4 |
| GGT5 | Gamma-glutamyl transpeptidase family member, downstream of LTC4 | Downstream cysteinyl leukotriene processing |
| DPEP1 | Dipeptidase converting LTD4 to LTE4 | Downstream metabolism of cysteinyl leukotrienes |
| CysLT1R | Cysteinyl leukotriene receptor 1, mediates LTC4/LTD4/LTE4 signaling | Receptor-level readout of LTC4 synthase activity |
| CysLT2R | Cysteinyl leukotriene receptor 2 | Alternative receptor for cysteinyl leukotrienes |
| PRKACA | Protein kinase A catalytic subunit, can phosphorylate LTC4S at Ser36 | Regulation of LTC4 synthase activity by phosphorylation |
| PRKACB | Protein kinase A catalytic subunit beta | Potential kinase for LTC4S Ser36 phosphorylation |
| MAPEG family members | Membrane-associated eicosanoid and glutathione metabolism proteins | Structural and evolutionary context of LTC4S |
| FLAP | 5-lipoxygenase-activating protein, related to LTC4S | Sequence identity and pathway coordination |
| LTA4H | Leukotriene A4 hydrolase, competes for LTA4 | Determines LTA4 fate toward LTB4 vs LTC4 |
| GCLC | Glutamate-cysteine ligase catalytic subunit, glutathione synthesis | Supplies glutathione substrate for LTC4 synthesis |
| GCLM | Glutamate-cysteine ligase modifier subunit | Regulates glutathione availability |
| GSS | Glutathione synthetase | Glutathione biosynthesis supporting LTC4 production |
| ABCC1 | Multidrug resistance protein 1, exports LTC4 | Transport of LTC4 out of cells |
How Is leukotriene-C4 synthase activity Regulated?
Leukotriene-C4 synthase activity is regulated at least in part by post-translational phosphorylation: phosphorylation of LTC4S at serine 36 impairs catalytic activity, providing a direct brake on LTC4 production. Because the enzyme is the committed step in cysteinyl leukotriene biosynthesis, its regulation determines the cellular output of LTC4 and downstream mediators. The activity also depends on the availability of its substrates, LTA4 and reduced glutathione, which are supplied by upstream 5-lipoxygenase and glutathione biosynthetic pathways, respectively. In addition, the enzyme is a membrane protein of the MAPEG family, and its membrane environment and family context influence its function.
leukotriene-C4 synthase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| LTC4S | Spontaneous emphysema in female mice upon deficiency | LTC4S knockout mouse; lung histology and function |
| LTC4S | Radiation-induced cognitive dysfunction and neuroinflammation | Microglial repopulation models with LTC4S modulation |
| LTC4S | Allergic and asthmatic inflammation | LTC4S knockout or point-mutant cell lines; LTC4 production assays |
| MGST2 | Alternative LTC4 synthase activity in inflammation | MGST2 overexpression and knockout cell models |
| LTC4S | Platelet cysteinyl leukotriene production | Platelet lysate enzyme activity assays |
Pulmonary emphysema and lung structural homeostasis
Leukotriene C4 synthase deficiency causes spontaneous emphysema in female mice, demonstrating that loss of GO:0004464 activity can disrupt lung structural integrity in a sex-dependent manner. This finding links the enzymatic activity to maintenance of alveolar architecture and suggests that cysteinyl leukotriene synthesis has homeostatic roles beyond classical inflammation.
Radiation-induced cognitive dysfunction and neuroinflammation
Microglial repopulation reverses radiation-induced cognitive dysfunction by restoring medial prefrontal cortex activity and modulating leukotriene-C4 synthesis, implicating GO:0004464 in neuroinflammatory and cognitive recovery processes. This connects the activity to brain function and suggests that modulating LTC4 synthesis may influence cognitive outcomes after radiation injury.
Allergic and asthmatic inflammation
As the committed step in cysteinyl leukotriene biosynthesis, LTC4 synthase activity is central to the production of LTC4, LTD4, and LTE4, which are potent mediators of bronchoconstriction and allergic inflammation. Consequently, the activity is a long-standing focus in asthma and allergic disease research.
From leukotriene-C4 synthase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of LTC4S abolish LTC4 production? | LTC4S knockout cell line or mouse |
| Does Ser36 phosphorylation regulate catalytic activity? | LTC4S S36A or S36D point-mutation knock-in cell lines |
| Can LTC4S be tagged for localization studies? | Tagged knock-in of LTC4S (e.g., FLAG or GFP) |
| Does overexpression increase cysteinyl leukotriene output? | LTC4S overexpression cell model |
| Is MGST2 sufficient to carry LTC4 synthase activity? | MGST2 overexpression and knockout models |
| Does LTC4S deficiency alter lung structure? | LTC4S knockout mouse with lung phenotyping |
How to Study the leukotriene-C4 synthase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| LTC4 synthase activity assay | Conversion of LTA4 and glutathione to LTC4 | Enzyme characterization and inhibitor testing |
| Heterologous expression | Recombinant enzyme activity in defined cells | Structure-function and species comparison |
| Phospho-specific immunoblot | Phosphorylation status of LTC4S at Ser36 | Regulation of catalytic activity |
| Knockout mouse phenotyping | Physiological consequences of lost activity | Emphysema and lung homeostasis studies |
| Microglial repopulation models | Cognitive and neuroinflammatory outcomes | Radiation-induced cognitive dysfunction |
| Lipid mediator profiling | Levels of LTC4, LTD4, LTE4 | Cysteinyl leukotriene pathway analysis |
| Platelet enzyme assays | LTC4 synthase activity in platelets | Cell-type distribution studies |
| Sequence and family analysis | Identity with MAPEG family members | Evolutionary and structural context |
Enzymatic activity assays
LTC4 synthase activity can be measured biochemically by incubating cell lysates or intact cells with LTA4 and glutathione and quantifying LTC4 formation, as demonstrated in platelets and species-distribution studies. Such assays directly report GO:0004464 catalytic output and are foundational for enzyme characterization.
Molecular cloning and expression
Cloning and heterologous expression of LTC4 synthase, as performed for the mouse enzyme, allow controlled study of the activity in defined cellular backgrounds. Expression systems also enable structure-function analysis of the enzyme and its relatives.
Phosphorylation and signaling analysis
Because phosphorylation at serine 36 impairs catalytic activity, methods that detect LTC4S phosphorylation and kinase signaling are used to study regulation of GO:0004464. Combining phospho-specific detection with activity assays links post-translational modification to function.
Genetic and phenotypic models
Knockout and transgenic models, such as LTC4S-deficient mice, reveal physiological consequences of losing the activity, including spontaneous emphysema in female mice. Microglial repopulation models further connect LTC4 synthesis to cognitive and neuroinflammatory phenotypes.
How CRISPR Can Be Used to Study GO:0004464 leukotriene-C4 synthase activity
Knockout
CRISPR knockout of LTC4S or MGST2 can eliminate leukotriene-C4 synthase activity, enabling loss-of-function studies of LTC4 production and downstream cysteinyl leukotriene biology. Such models complement LTC4S-deficient mouse phenotypes such as spontaneous emphysema.
Point Mutation
Point-mutation knock-in of LTC4S at serine 36 (for example, S36A or S36D) allows precise testing of how phosphorylation regulates catalytic activity, based on the finding that Ser36 phosphorylation impairs activity. These models separate catalytic function from regulatory modification.
Knock-in
Tagged knock-in of LTC4S with epitope or fluorescent tags supports localization and interaction studies of the enzyme in its native genomic context. Knock-in approaches also allow physiological expression levels to be preserved while introducing reporters.
Overexpression
CRISPR-based overexpression or cDNA overexpression of LTC4S or MGST2 increases cellular capacity for LTC4 synthesis, useful for pathway flux and inhibitor studies. Overexpression models help determine whether the enzyme is rate-limiting for cysteinyl leukotriene output.
How EDITGENE Supports leukotriene-C4 synthase activity Research
Researchers studying leukotriene-C4 synthase activity-related genes often need to determine whether a candidate gene is causally involved in LTC4 production, how post-translational modifications such as Ser36 phosphorylation alter catalysis, and whether loss or gain of the activity changes disease-relevant phenotypes. EDITGENE provides the CRISPR cell models and screening services needed to answer these questions with rigor.
Contact EDITGENE today to design your custom CRISPR model for leukotriene-C4 synthase activity research.
Frequently Asked Questions About leukotriene-C4 synthase activity
What is leukotriene-C4 synthase activity?
Leukotriene-C4 synthase activity (GO:0004464) is the catalysis of the reaction leukotriene C4 = glutathione + leukotriene A4, producing the cysteinyl leukotriene LTC4.
What does GO:0004464 mean?
GO:0004464 is the Gene Ontology molecular function term for leukotriene-C4 synthase activity, defined as catalysis of the reaction leukotriene C(4) = glutathione + leukotriene A(4).
What genes are involved in leukotriene-C4 synthase activity?
The canonical gene is LTC4S, encoding leukotriene C4 synthase; MGST2 encodes a related microsomal glutathione S-transferase that also displays this activity.
What reaction does leukotriene-C4 synthase catalyze?
It conjugates leukotriene A4 with glutathione to form leukotriene C4 via an epoxide-ring-opening glutathione transfer.
How is leukotriene-C4 synthase activity regulated?
Phosphorylation of LTC4S at serine 36 impairs catalytic activity, providing a post-translational regulatory mechanism.
Which cells have leukotriene-C4 synthase activity?
The activity has been demonstrated in platelets, and its species distribution has been characterized.
What diseases are linked to leukotriene-C4 synthase activity?
It is linked to allergic and asthmatic inflammation, spontaneous emphysema in female mice upon deficiency, and radiation-induced cognitive dysfunction.
How can I measure leukotriene-C4 synthase activity?
Activity is measured by incubating LTA4 and glutathione with cell lysates or intact cells and quantifying LTC4 formation.
What is the difference between LTC4S and MGST2?
LTC4S is the dedicated leukotriene C4 synthase, while MGST2 is a related microsomal glutathione S-transferase that also exhibits LTC4 synthase activity and shares sequence identity with LTC4S and FLAP.
How do CRISPR models help study GO:0004464?
CRISPR knockout, point mutation, knock-in, and overexpression models allow causal testing of LTC4S and MGST2 function, including the regulatory role of Ser36 phosphorylation.
Conclusion
Leukotriene-C4 synthase activity (GO:0004464) is the committed enzymatic step that converts leukotriene A4 and glutathione into leukotriene C4, the parent cysteinyl leukotriene. Its catalytic mechanism, regulation by Ser36 phosphorylation, and roles in inflammation, lung homeostasis, and neurobiology make it a compelling target for basic and translational research. CRISPR-based knockout, point-mutation, knock-in, and overexpression models, combined with activity assays and screening, provide the tools needed to dissect this activity in health and disease.
References
- 1. Ishii Y et al.. 2026. Leukotriene C4 Synthase Deficiency Causes Spontaneous Emphysema in Female Mice.. Am J Respir Cell Mol Biol 74(1):27-39 PMID: 40587881
- 2. Ahmad S et al.. 2016. Phosphorylation of Leukotriene C4 Synthase at Serine 36 Impairs Catalytic Activity.. J Biol Chem 291(35):18410-8 PMID: 27365393
- 3. Hu Y et al.. 2025. Microglial repopulation reverses radiation-induced cognitive dysfunction by restoring medial prefrontal cortex activity and modulating leukotriene-C4 synthesis.. Acta Neuropathol Commun 13(1):105 PMID: 40390112
- 4. Lam BK. 2003. Leukotriene C(4) synthase.. Prostaglandins Leukot Essent Fatty Acids 69(2-3):111-6 PMID: 12895593
- 5. Tornhamre S et al.. 1998. Demonstration of leukotriene-C4 synthase in platelets and species distribution of the enzyme activity.. Eur J Biochem 251(1-2):227-35 PMID: 9492288
- 6. Lam BK et al.. 2002. Leukotriene C4 synthase: a pivotal enzyme in cellular biosynthesis of the cysteinyl leukotrienes.. Prostaglandins Other Lipid Mediat 68-69:511-20 PMID: 12432940
- 7. Lam BK et al.. 1996. Molecular cloning, expression and characterization of mouse leukotriene C4 synthase.. Eur J Biochem 238(3):606-12 PMID: 8706658
- 8. Jakobsson PJ et al.. 1996. Identification and characterization of a novel human microsomal glutathione S-transferase with leukotriene C4 synthase activity and significant sequence identity to 5-lipoxygenase-activating protein and leukotriene C4 synthase.. J Biol Chem 271(36):22203-10 PMID: 8703034
