HPGDS Human

What is the molecular characterization of human HPGDS?

Human HPGDS is a cytosolic enzyme consisting of 199 amino acids (Pro2-Leu199) with a molecular weight of approximately 25-28 kDa as detected in Western blots . It is the only mammalian member of the class Sigma glutathione S-transferase family, showing broad specificity towards standard transferase substrates . The protein requires glutathione as a cofactor for its enzymatic activity, distinguishing it from the lipocalin-type PGDS which is glutathione-independent. HPGDS is encoded by the human gene with the accession number O60760 .

How does HPGDS function within the arachidonic acid metabolic pathway?

HPGDS catalyzes the conversion of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2) in the arachidonic acid cascade. In mast cells, this biosynthetic pathway is predominantly initiated by COX-1 . When activated, mast cells efficiently convert PGH2 to PGD2, which then mediates various biological effects through three different receptors, including bronchoconstriction, vasodilation, and immune cell regulation . Experimental evidence shows that exogenous PGH2 added to mast cells is predominantly converted to PGD2 under normal conditions, indicating the high efficiency of this enzymatic conversion .

In which cell types and tissues is HPGDS primarily expressed?

HPGDS expression is predominantly found in:

• Mast cells, including laboratory models LAD2, cord blood-derived mast cells (CBMC), peripheral blood-derived mast cells (PBMC), and human lung mast cells (HLMC)

• Antigen-presenting cells

• Leukemic cell lines such as HEL 92.1.7 erythroleukemic cells and KG-1 acute myelogenous leukemia cells

• Human lung tissue

• Human placental tissue

Immunofluorescence studies demonstrate that HPGDS localizes primarily to the cytoplasm of positive cells, consistent with its function as a cytosolic enzyme .

What cell models are most appropriate for studying HPGDS function?

Researchers investigating HPGDS function should consider the following validated human cell models:

• Human mast cell models:

  • LAD2 (Laboratory of Allergic Diseases 2) cells

  • Cord blood-derived mast cells (CBMC)

  • Peripheral blood-derived mast cells (PBMC)

  • Human lung mast cells (HLMC)

These models can be effectively activated using either anti-IgE (mimicking allergic stimulation) or ionophore A23187 . All four cell types have been shown to almost exclusively release PGD2 when activated, with biosynthesis entirely initiated by COX-1, making them ideal for studying HPGDS function .

For studies focused on hematological malignancies, HEL 92.1.7 human erythroleukemic cell line and KG-1 human acute myelogenous leukemia cell line have demonstrated HPGDS expression and can serve as alternative models .

What analytical techniques should be employed to measure HPGDS activity and prostanoid production?

UPLC-MS/MS (Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry) represents the gold standard for measuring prostanoid production in HPGDS research . This technique allows:

• Simultaneous quantification of multiple prostanoids (PGD2, PGE2, TXB2 as a stable metabolite of TXA2)

• High sensitivity and specificity for detecting even small changes in prostanoid levels

• Accurate assessment of metabolic shifts when HPGDS is inhibited or when cells are exposed to different stimuli

For protein expression analysis, Western blot using specific antibodies against human HPGDS is recommended, with expected band detection at approximately 25-28 kDa . Immunofluorescence can be employed for cellular localization studies, with appropriate controls to confirm specificity .

How can researchers effectively validate HPGDS antibodies for experimental use?

Validation of HPGDS antibodies should follow a multi-step approach:

• Western blot validation:

  • Test antibody against positive control samples (e.g., NCI-H460 human large cell lung carcinoma, human lung tissue, human placenta tissue, or KG-1 cells)

  • Confirm detection at the expected molecular weight (25-28 kDa)

  • Include negative controls lacking HPGDS expression

• Immunofluorescence/immunocytochemistry validation:

  • Test in known HPGDS-expressing cells (e.g., HEL 92.1.7 cells)

  • Confirm cytoplasmic localization pattern

  • Include appropriate negative controls and blocking peptides

• Specificity testing:

  • Perform pre-absorption with recombinant HPGDS to confirm specificity

  • Test cross-reactivity with related proteins, particularly L-PGDS

Commercial antibodies such as Mouse Anti-Human HPGDS Monoclonal Antibody (Clone #735301) and Sheep Anti-Human HPGDS Polyclonal Antibody have been validated for detecting human HPGDS in research applications .

What is the significance of HPGDS-derived PGD2 in allergic and inflammatory diseases?

HPGDS-derived PGD2 plays a central role in allergic and inflammatory diseases through multiple mechanisms:

• Receptor-mediated effects:

  • PGD2 acts through three different receptors to mediate bronchoconstriction, vasodilation, and immune cell regulation

  • These diverse biological actions make HPGDS a key target for therapy of asthma and other inflammatory diseases

• Mast cell activation pathway:

  • Mast cells almost exclusively release PGD2 when activated during allergic responses

  • This release is primarily mediated by COX-1 activity followed by HPGDS-catalyzed conversion of PGH2 to PGD2

• Inflammatory amplification:

  • PGD2 can recruit and activate additional inflammatory cells

  • It may contribute to chronic inflammation through sustained signaling cascades

The therapeutic potential of targeting HPGDS lies in the ability to inhibit the production of PGD2, thereby potentially affecting all downstream receptor-mediated effects simultaneously, which could overcome the need for multiple receptor antagonists .

How does inhibition of HPGDS affect the broader prostanoid network?

Inhibition of HPGDS has significant consequences for the broader prostanoid network through metabolic shunting:

These findings have important implications for drug development, as inhibiting HPGDS could have unintended consequences through increased production of other prostanoids with distinct biological activities.

What evidence supports dual roles for PGD2 in inflammation?

Research suggests that PGD2 may play both pro-inflammatory and anti-inflammatory roles depending on context:

• Pro-inflammatory effects:

  • Mediates bronchoconstriction in asthma

  • Contributes to vasodilation in allergic responses

  • Recruits and activates inflammatory cells

• Anti-inflammatory properties:

  • Studies referenced in suggest PGD2 can attenuate anaphylactic reactions in mice

  • May play a role in resolving acute inflammation

  • Could influence regulatory T cell induction

• Receptor-specific effects:

  • Different PGD2 receptors may mediate opposing effects

  • Activation of the D prostanoid 1 receptor has been linked to suppression of asthma via modulation of lung dendritic cell function

This dual nature highlights the complexity of targeting the HPGDS-PGD2 pathway and suggests that context-specific approaches may be needed for different disease states.

What computational approaches are utilized in designing selective HPGDS inhibitors?

Development of selective HPGDS inhibitors employs several computational approaches:

• Molecular modeling and docking:

  • Virtual screening of compound libraries against the HPGDS active site

  • Evaluation of binding affinity and interaction patterns

  • Identification of key protein-ligand interactions

• Molecular dynamics simulations:

  • Analysis of protein flexibility and conformational changes upon ligand binding

  • Evaluation of stability of protein-inhibitor complexes over time

  • Identification of transient binding pockets not evident in static structures

• Structure-based design:

  • Utilization of HPGDS crystal structure to design compounds that exploit unique features of the enzyme

  • Focus on interactions with the glutathione-binding site, as HPGDS is glutathione-dependent

  • Optimization of scaffolds to improve selectivity over related enzymes

As evidenced in search result , researchers have employed these approaches to evaluate sets of molecules with different molecular scaffolds as potential HPGDS inhibitors, demonstrating the value of computational methods in targeting this enzyme.

What methodological approaches can detect metabolic shunting following HPGDS inhibition?

Detecting and quantifying metabolic shunting after HPGDS inhibition requires several methodological approaches:

• Comprehensive prostanoid profiling:

  • UPLC-MS/MS analysis of multiple prostanoids simultaneously

  • Measurement of PGD2, PGE2, TXB2 (stable metabolite of TXA2), and other relevant eicosanoids

  • Temporal profiling to capture dynamic changes in the prostanoid network

• Enzymatic pathway manipulation:

  • Selective inhibition of HPGDS followed by prostanoid quantification

  • Complementary inhibition of alternative enzymes (e.g., thromboxane synthase)

  • Addition of exogenous PGH2 to directly assess metabolic fate

• Isotope labeling studies:

  • Use of isotope-labeled arachidonic acid to track metabolic conversion

  • Determination of flux through different branches of the prostanoid pathway

  • Quantification of labeled intermediates and products

• Enzyme activity assays:

  • Direct measurement of HPGDS and alternative enzyme activities

  • Assessment of compensatory changes in enzyme expression or activity

  • Correlation with prostanoid levels

Research has demonstrated that when HPGDS is inhibited, redirected use of PGH2 leads to increased production of TXA2 and potential non-enzymatic formation of PGE2, highlighting the importance of comprehensive prostanoid analysis when evaluating HPGDS inhibitors .

How should researchers approach the evaluation of HPGDS inhibitor selectivity?

Evaluating HPGDS inhibitor selectivity requires a multi-faceted approach:

• Enzymatic selectivity assessment:

  • Testing against purified recombinant HPGDS

  • Comparative analysis against related enzymes (other GSTs, L-PGDS)

  • Determination of IC50 values and selectivity ratios

• Cellular validation:

  • Quantification of PGD2 reduction in relevant cell models

  • Measurement of other prostanoids to detect potential shunting

  • Functional assays to confirm biological consequences of HPGDS inhibition

• Mechanistic evaluation:

  • Assessment of competitive vs. non-competitive inhibition

  • Investigation of potential interactions with the glutathione cofactor

  • Structural studies to confirm binding mode

• Off-target screening:

  • Profiling against a panel of pharmacologically relevant targets

  • Assessment of effects on related enzymes in the arachidonic acid cascade

  • Evaluation of potential interactions with PGD2 receptors

A robust evaluation should include the assessment of metabolic shunting, as inhibition of HPGDS has been shown to redirect PGH2 metabolism toward increased TXA2 production, which could have significant biological consequences .

What emerging approaches might address the challenge of prostanoid shunting?

Several innovative approaches could address the challenge of prostanoid shunting following HPGDS inhibition:

• Dual-action inhibitors:

  • Development of compounds that simultaneously inhibit HPGDS and thromboxane synthase

  • Creation of balanced inhibitory profiles to minimize adverse redirected metabolism

  • Design of molecules with appropriate selectivity for multiple targets in the prostanoid pathway

• Partial inhibition strategies:

  • Identification of allosteric modulators that reduce but do not eliminate HPGDS activity

  • Development of compounds that modify enzyme kinetics without complete inhibition

  • Creation of context-dependent inhibitors active primarily under inflammatory conditions

• Targeted delivery approaches:

  • Design of cell type-specific delivery systems for HPGDS inhibitors

  • Development of prodrugs activated in specific tissues or microenvironments

  • Creation of nanocarriers that preferentially accumulate in target tissues

• Combination therapies:

  • Pairing HPGDS inhibitors with agents that prevent or mitigate consequences of metabolic shunting

  • Developing regimens that block both PGD2 production and TXA2 activity

  • Exploring synergistic combinations with anti-inflammatory agents

These approaches could help overcome the limitation identified in research where inhibiting HPGDS led to redirected use of PGH2 and increased biosynthesis of TXA2 .

How might single-cell technologies advance HPGDS research?

Single-cell technologies offer promising opportunities to advance HPGDS research:

• Single-cell transcriptomics:

  • Identification of HPGDS expression patterns in rare cell populations

  • Characterization of heterogeneity among HPGDS-expressing cells

  • Correlation of HPGDS expression with other inflammatory mediators

• Single-cell proteomics:

  • Quantification of HPGDS protein levels at single-cell resolution

  • Analysis of post-translational modifications affecting enzyme activity

  • Correlation of HPGDS protein levels with functional outcomes

• Single-cell metabolomics:

  • Measurement of PGD2 and other prostanoids at single-cell level

  • Analysis of cell-to-cell variability in prostanoid production

  • Identification of metabolic signatures associated with HPGDS activity

• Spatial transcriptomics:

  • Mapping HPGDS expression within tissue microenvironments

  • Understanding spatial relationships between HPGDS-expressing and receptor-expressing cells

  • Analysis of regional differences in HPGDS expression in disease models

These technologies could help resolve conflicting findings regarding the role of PGD2 in different disease contexts and identify specific cell populations that might be targeted therapeutically.

What potential roles might HPGDS play in diseases beyond allergic inflammation?

Emerging research suggests HPGDS may play important roles in multiple disease contexts beyond allergic inflammation:

• Autoimmune diseases:

  • PGD2 might amplify lupus disease through basophil accumulation in lymphoid organs

  • HPGDS-derived PGD2 could modulate dendritic cell function and regulatory T cell induction

• Cancer biology:

  • HPGDS expression in leukemic cell lines suggests potential roles in hematological malignancies

  • Altered prostanoid metabolism may influence tumor microenvironment and immune surveillance

  • PGD2 signaling might affect cancer cell proliferation or apoptosis

• Cardiovascular disorders:

  • PGD2's vasodilatory effects suggest potential roles in vascular regulation

  • Research indicates differential roles of HPGDS vs. L-PGDS in thrombogenesis and hypertension

• Neurological conditions:

  • Immune cells expressing HPGDS may contribute to neuroinflammation

  • PGD2 signaling could influence microglial activation states

  • Potential involvement in pain processing and neurogenic inflammation

Understanding these broader roles could expand the therapeutic potential of HPGDS inhibitors and identify new research directions for targeting this enzyme in diverse disease states.