Recombinant Human Monoacylglycerol lipase ABHD2 (ABHD2)

What is the primary enzymatic function of ABHD2?

ABHD2 functions as a progesterone (P4)-dependent 2-arachidonoylglycerol (2-AG) hydrolase. Upon progesterone stimulation, ABHD2 hydrolyzes 2-AG, which is an endogenous inhibitor of the CatSper channel in spermatozoa. This hydrolysis leads to CatSper channel opening, triggering sperm hyperactivation and enabling fertilization capacity . This mechanism represents an unusual regulatory paradigm where 2-AG signaling is continuously "on" (blocking CatSper) until progesterone terminates it by stimulating ABHD2-mediated 2-AG hydrolysis . This challenges the conventional "on demand" synthesis model for endocannabinoid signaling in certain tissues.

What tissues demonstrate the highest ABHD2 expression?

ABHD2 shows varied expression across tissues, with particularly high expression in testis where it plays a critical role in sperm fertility . Additionally, ABHD2 is expressed in atherosclerotic lesions, with significantly higher expression observed in patients with unstable angina compared to those with stable angina . Within these lesions, ABHD2 shows abundant expression in macrophages but relatively low expression in smooth muscle cells (SMCs), with expression significantly increasing during monocyte-to-macrophage differentiation .

How does ABHD2 differ from other ABHD family members?

While all ABHD proteins contain conserved structural motifs predicting roles in lipid metabolism, ABHD2 has distinctive functions related to reproductive biology and inflammatory processes. Unlike some family members primarily involved in general lipid homeostasis, ABHD2 specifically regulates 2-AG signaling in reproductive tissues and has been implicated in atherosclerosis progression through its expression in macrophages . The targeted inhibition studies demonstrate that ABHD2 can be selectively inhibited without significantly affecting other ABHD family members, highlighting structural and functional differences within this enzyme family .

What approaches can be used to identify selective ABHD2 inhibitors?

Activity-based protein profiling (ABPP) using library-versus-library screening has proven effective for identifying selective ABHD2 inhibitors . This approach employs activity-based probes (ABPs) such as β-lactone-based MB064 and MB108 that target serine hydrolases and assess their functional state in complex proteomes . For initial screening, a gel-based assay using MB064 can be employed with recombinant proteins to avoid limitations associated with variable tissue distribution or enzyme labeling intensities . Once candidate inhibitors are identified, their selectivity should be further validated in native proteomes using competitive ABPP with broad-spectrum fluorophosphonate-based serine hydrolase probes (FP-TAMRA and FP-biotin) alongside ABPs MB064 and MB108 .

How can researchers assess ABHD2 inhibition in sperm function studies?

To evaluate the effect of ABHD2 inhibition on sperm function, researchers should employ acrosome reaction (AR) assays in vitro. The methodology involves:

• Collection and capacitation of mouse cauda epididymal spermatozoa in capacitation medium

• Treatment with potential ABHD2 inhibitors at varying concentrations (e.g., 1-20 μM)

• Stimulation with progesterone (typically 3 μM)

• Assessment of acrosome reaction rates

Control experiments should include baseline AR rates (~20%) and progesterone-stimulated AR rates (~35%). The broad-spectrum serine hydrolase inhibitor MAFP serves as an effective positive control . A dose-dependent reduction in progesterone-induced AR confirms ABHD2 inhibition. Researchers should also monitor intracellular calcium levels as a secondary measurement, as ABHD2 inhibition affects calcium signaling through CatSper channels .

What expression systems are recommended for producing recombinant ABHD2?

For recombinant ABHD2 production, mammalian expression systems (particularly HEK293T cells) are recommended to ensure proper folding and post-translational modifications essential for enzymatic activity. Expression constructs should incorporate a FLAG or His tag for purification purposes while including the full-length human ABHD2 sequence. Transient transfection followed by expression for 48-72 hours yields suitable protein levels for most applications . When expressing multiple ABHD family members for comparative studies, standardized expression conditions are crucial for meaningful cross-comparisons in inhibitor screening or activity assays.

How does ABHD2 contribute to viral infection processes?

ABHD2 plays a critical role in hepatitis B virus (HBV) propagation. Studies have demonstrated that antisense oligonucleotides (ASODNs) targeting ABHD2 can significantly downregulate ABHD2 mRNA and protein expression in HepG2.2.15 cells . This downregulation results in reduced HBV DNA, hepatitis B surface antigen, and hepatitis B antigen protein expression levels without affecting cell viability . This suggests ABHD2 is essential for HBV replication and expression, making it a potential novel target for anti-HBV drug development. The mechanism likely involves ABHD2's lipid-metabolizing functions, which may facilitate viral replication processes, though the exact mechanisms require further elucidation.

What is the significance of ABHD2 in cardiovascular disease models?

ABHD2 demonstrates significant relevance in cardiovascular disease through two key mechanisms:

• Smooth muscle cell (SMC) migration regulation: Studies in ABHD2-knockout mice revealed increased SMC migration and neointimal thickening, suggesting ABHD2 normally suppresses these processes .

• Atherosclerotic plaque stability: ABHD2 expression is significantly elevated in vulnerable plaques from patients with unstable angina compared to those with stable angina . Within these lesions, ABHD2 is primarily expressed in macrophages rather than SMCs, with expression increasing proportionally with monocyte-to-macrophage differentiation .

These findings suggest ABHD2 functions differently depending on cell type: suppressing migration in SMCs while potentially promoting inflammatory processes in macrophages within atherosclerotic plaques. This dual role makes ABHD2 a complex but promising target for cardiovascular disease interventions.

What pulmonary phenotypes are associated with ABHD2 deficiency?

ABHD2-deficient mice develop age-related pulmonary emphysema characterized by gradual progression, mimicking the pace of emphysema development in humans . This phenotype stems from decreased phosphatidylcholine levels in bronchoalveolar lavage, increased macrophage infiltration, elevated inflammatory cytokines, protease/anti-protease imbalance, and enhanced apoptosis . ABHD2 expression in alveolar type II cells appears crucial for maintaining proper phospholipid metabolism in the lung. These findings suggest that derangements in alveolar phospholipid metabolism can induce emphysema, highlighting ABHD2's critical role in maintaining lung structural integrity. This model provides valuable insights for studying the gradual progression of emphysema in humans.

How can selectivity profiles of ABHD2 inhibitors be comprehensively characterized?

Comprehensive characterization of ABHD2 inhibitor selectivity requires a multi-tiered approach:

• Primary screening using gel-based ABPP with recombinant ABHD family proteins

• Secondary validation using competitive chemoproteomics in native proteomes (particularly testis)

• Functional validation in cellular assays (e.g., progesterone-induced acrosome reaction)

For the most reliable selectivity profiling, researchers should employ both:

• ABPs with different reactive groups (β-lactone-based MB064/MB108 and fluorophosphonate-based FP-TAMRA/FP-biotin)

• Analysis across multiple tissue types where ABHD2 and related enzymes are expressed

This approach has revealed that compound 183, for example, demonstrates remarkable selectivity for ABHD2, with minimal cross-reactivity observed in testis proteome . The ideal methodology combines these chemical proteomics approaches with targeted functional assays specific to ABHD2's biological roles.

What mechanisms explain the tissue-specific effects of ABHD2 in different disease models?

The diverse pathophysiological roles of ABHD2 across tissues likely stem from:

• Substrate specificity variations: While ABHD2 hydrolyzes 2-AG in reproductive tissues, it may target different lipid substrates in vascular or pulmonary tissues.

• Differential expression patterns: ABHD2 shows cell-type-specific expression within tissues (e.g., high in macrophages, low in SMCs in atherosclerotic lesions).

• Interaction with tissue-specific signaling networks: In sperm, ABHD2 regulates CatSper channels through 2-AG, while in lungs, it maintains phosphatidylcholine levels.

These tissue-specific mechanisms must be considered when designing therapeutic strategies targeting ABHD2. Understanding these differences requires comprehensive lipidomic analyses across tissues in ABHD2-deficient models, coupled with tissue-specific conditional knockout studies to isolate ABHD2 functions in individual cell populations.

How might the ABHD2-regulated endocannabinoid system interface with steroid hormone signaling in other tissues?

The discovery that ABHD2 functions as a progesterone-dependent 2-AG hydrolase in sperm suggests potential unrecognized mechanisms linking steroid signaling and endocannabinoid metabolism in other tissues. This represents a novel cross-talk whereby steroid hormones like progesterone can directly modulate endocannabinoid signaling through enzymatic regulation . This mechanism may extend beyond reproductive tissues, potentially operating in immune cells, vascular tissues, or the central nervous system where both systems are active. Further research should investigate whether other steroid hormones (estrogens, androgens, corticosteroids) might similarly regulate ABHD2 activity in different cellular contexts, potentially explaining some of the rapid non-genomic effects of steroid hormones observed in various tissues.

What approaches could identify the complete substrate profile of ABHD2?

Comprehensive substrate profiling for ABHD2 requires untargeted lipidomic analyses comparing wild-type and ABHD2-deficient or ABHD2-inhibited systems. An integrated approach should include:

• Untargeted LC-MS/MS-based lipidomics on tissues from ABHD2-knockout mice versus wild-type controls

• Activity-based metabolite profiling using inhibitor 183 in cell and tissue lysates

• In vitro enzymatic assays with recombinant ABHD2 and candidate lipid substrates

• Stable isotope-labeled precursor studies to track lipid metabolism

This multi-faceted approach would help identify both direct substrates and downstream metabolic changes resulting from ABHD2 activity across different tissues and cell types. Given ABHD2's known activity on 2-AG in reproductive tissues, particular attention should be given to various monoacylglycerols and related lipid species.

What strategies could improve the specificity and efficacy of ABHD2-targeted interventions?

Enhancing ABHD2-targeted interventions requires several complementary approaches:

• Structure-guided inhibitor optimization: Using compound 183 as a starting point, structure-activity relationship studies can improve potency while maintaining selectivity .

• Tissue-specific delivery systems: Given ABHD2's diverse roles across tissues, targeted delivery to specific tissues (e.g., reproductive tract for contraceptive applications) would minimize off-target effects.

• Allosteric modulation: Developing modulators that affect ABHD2 activity only in the presence of specific stimuli (e.g., progesterone) would provide contextual control over enzyme inhibition.

• Gene therapy approaches: For chronic conditions like pulmonary emphysema, AAV-mediated delivery of ABHD2 to specific cell types (alveolar type II cells) could restore function while minimizing systemic effects.

Each approach requires careful validation in relevant disease models before clinical translation.