Recombinant Bovine Abhydrolase domain-containing protein 16A (ABHD16A)

What is ABHD16A and what are its primary enzymatic functions in bovine systems?

ABHD16A is a member of the α/β hydrolase domain-containing (ABHD) protein family expressed in a variety of animal cells. This 63 kDa protein (containing approximately 558 amino acid residues in humans) functions primarily as a lipid-metabolizing enzyme with two key activities:

• Phosphatidylserine (PS) hydrolase activity: ABHD16A serves as the main brain phosphatidylserine hydrolase, converting phosphatidylserine to lysophosphatidylserine (LPS) .

• Acylglycerol lipase activity: The enzyme demonstrates capability in catalyzing the hydrolysis of monoacylglycerols .

Methodologically, these enzymatic activities can be assessed through targeted lipidomic approaches using Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS), which allows for quantification of substrate (PS) and product (LPS) levels in cellular systems expressing bovine ABHD16A .

How is expression of ABHD16A regulated across different bovine tissues?

While the search results don't provide bovine-specific expression data, comparative analysis across species indicates that ABHD16A shows differential expression patterns across tissues. The human ortholog demonstrates high expression in:

• Brain tissue

• Muscle tissue

• Testes

• Heart

For researchers studying bovine ABHD16A, quantitative tissue expression profiling can be conducted using:

• Quantitative PCR (qPCR) to measure transcript levels

• Western blotting with specific antibodies for protein detection

• Immunohistochemistry for spatial localization within tissues

When designing expression studies, researchers should consider using multiple reference genes for normalization, and validate antibody specificity against recombinant bovine ABHD16A to ensure accurate tissue distribution assessment.

What are the key structural domains and active sites that characterize bovine ABHD16A?

Bovine ABHD16A, like its orthologs in other species, is characterized by:

• An α/β hydrolase fold domain - the catalytic core structure common to the ABHD family

• A phosphatidylserine lipase ABHD16 N-terminal domain

• Catalytic triad of amino acids (likely Ser, His, Asp) forming the active site, though the exact positions may vary slightly from human ABHD16A

Domain structure analysis methods:

• Bioinformatic sequence analysis using tools like InterPro, as applied to zebrafish ABHD16A, reveals the presence of these conserved domains

• Site-directed mutagenesis of predicted active site residues, followed by activity assays, can confirm the functional importance of specific amino acids

• Homology modeling based on crystal structures of related α/β hydrolases can predict three-dimensional arrangement of catalytic residues

How does bovine ABHD16A differ from human ABHD16A in terms of sequence homology and substrate specificity?

While the search results don't provide direct sequence comparison between bovine and human ABHD16A, cross-species analysis suggests:

• Substantial sequence conservation in the catalytic domains across mammalian species

• Potential species-specific variations in N-terminal regions that may influence membrane association or substrate binding

For researchers investigating these differences:

• Perform sequence alignment using tools like CLUSTAL or MUSCLE to identify conserved and divergent regions

• Express both bovine and human recombinant proteins and conduct comparative kinetic analyses with various PS and acylglycerol substrates

• Assess thermal stability differences between the orthologs using thermal shift assays to identify structural distinctions

What are the optimal expression systems and purification strategies for producing recombinant bovine ABHD16A?

For efficient production of functional recombinant bovine ABHD16A:

Expression Systems:

• Bacterial systems (E. coli): May require optimization due to the membrane-associated nature of ABHD16A . Consider using specialized strains like Rosetta™ or BL21(DE3) with chaperones to aid proper folding.

• Insect cell systems (Sf9, Hi5): Often superior for mammalian membrane-associated proteins, using baculovirus expression vectors.

• Mammalian cell lines (HEK293, CHO): Provide native-like post-translational modifications but at lower yields.

Purification Strategy:

• Affinity chromatography using N or C-terminal tags (His6, GST, FLAG)

• Size exclusion chromatography to separate monomeric protein from aggregates

• Ion exchange chromatography as a polishing step

Key Optimization Considerations:

• Include detergents (CHAPS, DDM) during extraction to maintain solubility of this membrane-associated protein

• Consider adding glycerol (10-15%) to stabilize during purification

• Validate activity after each purification step to ensure functionality is maintained

How can researchers accurately measure ABHD16A enzymatic activity in vitro?

To measure bovine ABHD16A phosphatidylserine hydrolase and lipase activities:

Lysophosphatidylserine (LPS) Production Assay:

• Incubate purified enzyme with various PS substrates (varying in fatty acid chain length)

• Extract lipids using modified Bligh-Dyer method

• Quantify PS and LPS species by UPLC-MS

Fluorogenic Substrate Assay:

• Utilize substrates like 4-methylumbelliferyl-based lipid analogs

• Monitor fluorescence increase as substrate is hydrolyzed

• Calculate kinetic parameters (Km, Vmax)

Data Analysis Approach:

• For each PS species, calculate the ratio of product (LPS) to substrate (PS)

• Compare activity with specific LPS species of different chain lengths (C18-C22 vs shorter chains)

• Create a substrate specificity profile to identify preferred fatty acid compositions

Note: Table values are hypothetical examples based on observed patterns from ABHD16A-deficient human cells

What genetic manipulation approaches are most effective for studying ABHD16A function?

Researchers investigating bovine ABHD16A function through genetic manipulation should consider:

CRISPR-Cas9 Knockout/Knockdown:

• Design guide RNAs targeting early exons of bovine ABHD16A

• Verify knockout efficiency by Western blot and activity assays

• Analyze phenotypic changes, particularly in lipid metabolism

Rescue Experiments:

• Re-express wild-type or mutant bovine ABHD16A in knockout cells

• Compare PS/LPS profiles between knockout and rescued cells

• Identify structure-function relationships by analyzing point mutations in the catalytic domain

Inducible Expression Systems:

• Use Tet-On/Off systems to control expression timing

• Monitor temporal changes in lipid profiles upon induction/repression

• Assess acute versus chronic effects of ABHD16A modulation

These approaches have been successfully applied to human ABHD16A to demonstrate its role in PS metabolism, as evidenced by altered PS and LPS levels in patient-derived fibroblasts with ABHD16A loss-of-function variants .

How does ABHD16A deficiency affect phosphatidylserine metabolism and what methods detect these changes?

ABHD16A deficiency leads to specific alterations in phosphatidylserine metabolism, which can be detected and quantified using sophisticated lipidomic approaches:

Metabolic Consequences:

• Decreased levels of long-chain lysophosphatidylserine (LPS) species (particularly C18-C22)

• Increased levels of multiple phosphatidylserine (PS) species

• Most significant changes observed in PS species with long-chain fatty acids

Detection Methodology:

• Sample Preparation:

  • Extract total lipids from cells using chloroform/methanol extraction

  • Separate phospholipid classes using solid-phase extraction

• Analysis Techniques:

  • UPLC-MS/MS with multiple reaction monitoring for targeted quantification of PS and LPS species

  • Internal standards for each lipid class to ensure accurate quantification

• Data Processing:

  • Normalize to total protein content or cell number

  • Compare specific PS/LPS species ratios between wild-type and ABHD16A-deficient samples

Representative Data from ABHD16A-Deficient Cells:

Note: Values adapted from trends reported in ABHD16A-deficient fibroblasts; AU = arbitrary units

How do mutations in ABHD16A contribute to neurological disorders, and what model systems best study these mechanisms?

ABHD16A loss-of-function mutations have been directly implicated in hereditary spastic paraplegia 86 (SPG86), a rare neurological disorder:

Disease Mechanisms:

• Altered PS/LPS Homeostasis: Loss of ABHD16A causes accumulation of PS species and reduction in LPS species, particularly those with long fatty acid chains

• Neuronal Impact: These lipid imbalances likely affect membrane composition, signaling pathways, and synapse function in neurons

• ABHD16A-ABHD12 Axis: Disruption of the metabolic balance between these two enzymes contributes to neurodegenerative phenotypes

Optimal Research Models:

• Patient-Derived Cells: Fibroblasts from patients with ABHD16A mutations show characteristic lipid profile changes

• CRISPR-Modified Bovine Cell Lines: Creating bovine cell models with ABHD16A knockout to study species-specific effects

• Mouse Models: ABHD16A knockout mice exhibit reduced brain LPS, particularly in the cerebellum and olfactory bulb

• iPSC-Derived Neurons: Differentiate patient-derived iPSCs into neurons to study cell-type specific effects

Experimental Approaches:

• Electrophysiology to assess neuronal function

• Advanced imaging to examine axonal transport

• Lipidomics to quantify PS/LPS levels in different brain regions

• Behavioral tests to assess motor function in animal models

These approaches help connect biochemical alterations (PS/LPS imbalance) to clinical manifestations (complex hereditary spastic paraplegia).

What is the relationship between ABHD16A and ABHD12 in phosphatidylserine metabolism, and how might this be therapeutically relevant?

ABHD16A and ABHD12 function in a metabolic axis that regulates phosphatidylserine metabolism:

Metabolic Relationship:

• Sequential Processing: ABHD16A hydrolyzes PS to produce LPS, which is then further processed by ABHD12

• Complementary Activities: ABHD16A primarily generates LPS, while ABHD12 degrades LPS

• Balancing Act: The relative activities of these enzymes maintain appropriate PS/LPS ratios in tissues, particularly in the brain

Therapeutic Implications:

• ABHD12 Inhibition for ABHD16A Deficiency: Selective ABHD12 inhibitors could normalize low LPS levels in ABHD16A-deficient patients

• Known Inhibitors:

  • DO264: An in vivo selective inhibitor of ABHD12 that elevates LPS in a dose-dependent manner in human macrophages and mouse brain

  • Ursolic acid: Identified as having selective inhibition of ABHD12 with minimal non-selectivity

Research Approaches to Study This Axis:

• Co-expression Studies: Express varying levels of both enzymes to determine optimal ratios

• Activity Assays: Compare PS/LPS profiles in systems with modified ABHD16A/ABHD12 ratios

• Inhibitor Screening: Test combinations of selective modulators of both enzymes

Therapeutic Development Strategy:

• Determine optimal LPS species and concentrations for neuronal health

• Develop selective ABHD12 inhibitors based on structure-activity relationships

• Test in cellular and animal models of ABHD16A deficiency

• Monitor both lipid profiles and functional outcomes

What are the methodological challenges in studying ABHD16A structure and developing selective modulators?

Researchers face several technical challenges when investigating ABHD16A structure and developing modulators:

Structural Characterization Challenges:

• Membrane Association: ABHD16A is predicted to be membrane-associated , complicating crystallization

• Protein Stability: The enzyme may require specific lipid environments to maintain native conformation

• Active Site Flexibility: The catalytic pocket likely undergoes conformational changes during substrate binding

Methodological Solutions:

• Cryo-EM Approaches: Suitable for membrane proteins that resist crystallization

• Nanodiscs Technology: Incorporate purified ABHD16A into synthetic lipid bilayers

• Hydrogen-Deuterium Exchange Mass Spectrometry: Map dynamic regions and ligand binding sites

• Homology Modeling: Leverage structures of related α/β hydrolases to predict ABHD16A structure

Modulator Development Challenges:

• Selectivity Issues: Ensuring compounds don't affect related ABHD family members

• Species Differences: Bovine vs. human ABHD16A may respond differently to modulators

• Membrane Penetration: Compounds must reach the enzyme in its native membrane environment

Screening Approaches:

• Activity-Based Protein Profiling (ABPP): Using active site-directed probes to assess selectivity

• Fragment-Based Screening: Building modulators from small fragments that bind distinct sites

• In silico Docking: Virtual screening against homology models to identify potential binding compounds

What lipidomic approaches best characterize the ABHD16A-dependent lipidome in bovine tissues?

To comprehensively characterize the ABHD16A-dependent lipidome in bovine tissues:

Sample Preparation Considerations:

• Tissue Homogenization: Optimize buffers to prevent ex vivo lipid degradation

• Fractionation: Separate membrane fractions to enrich for PS/LPS

• Extraction Methods: Use modified Bligh-Dyer or MTBE extraction optimized for phospholipids

Analytical Platforms:

• Untargeted Lipidomics:

  • High-resolution LC-MS/MS (Q-TOF or Orbitrap) for broad lipid profiling

  • Data-independent acquisition for improved coverage

• Targeted Lipidomics:

  • Triple quadrupole MS with multiple reaction monitoring for PS/LPS quantification

  • Internal standards for each PS/LPS species for accurate quantitation

Data Analysis Workflow:

• Feature detection and alignment across samples

• Identification based on accurate mass, retention time, and MS/MS fragmentation

• Statistical analysis comparing wild-type vs. ABHD16A-deficient samples

• Pathway enrichment analysis to identify broader metabolic impacts

Validation Approaches:

• Orthogonal measurement of key lipids by enzymatic assays

• Stable isotope tracing to confirm altered PS/LPS metabolism

• Spatial distribution using MALDI imaging mass spectrometry

Based on findings from ABHD16A-deficient human fibroblasts, researchers should focus particularly on PS species with chain lengths of 34:1, 36:2, 36:1, and 42:1, which showed the most significant changes .

How can researchers develop specific antibodies against bovine ABHD16A for immunological applications?

Developing specific antibodies against bovine ABHD16A requires careful antigen design and validation:

Antigen Design Strategies:

• Recombinant Full-Length Protein:

  • Express and purify full-length bovine ABHD16A

  • Challenge: Maintaining native conformation of this membrane-associated protein

• Unique Peptide Epitopes:

  • Identify sequences unique to bovine ABHD16A vs. other ABHD family members

  • Select regions with high predicted antigenicity and surface exposure

  • Synthesize KLH-conjugated peptides for immunization

Production Methods:

• Polyclonal Antibodies:

  • Immunize rabbits with purified antigen

  • Advantages: Multiple epitope recognition, higher sensitivity

• Monoclonal Antibodies:

  • Hybridoma technology following mouse immunization

  • Advantages: Consistent production, single epitope specificity

• Recombinant Antibodies:

  • Phage display selection against the target

  • Advantages: No animals required, customizable affinity

Rigorous Validation Protocol:

• ELISA against recombinant protein and peptide antigens

• Western blot against:

  • Recombinant ABHD16A (positive control)

  • Tissue lysates from multiple species

  • ABHD16A-knockout samples (negative control)

• Immunoprecipitation followed by mass spectrometry confirmation

• Immunohistochemistry with peptide competition controls

Application-Specific Optimization:

• For Western blotting: Determine optimal dilution and blocking conditions

• For immunoprecipitation: Test various lysis conditions to maintain native conformation

• For immunofluorescence: Optimize fixation methods (chemical vs. freezing)

What computational approaches can predict functional impacts of ABHD16A variants identified in genetic studies?

For researchers studying bovine ABHD16A variants or comparing to human disease-associated variants:

Sequence-Based Prediction Methods:

• Conservation Analysis:

  • Multiple sequence alignment across species

  • Calculate conservation scores (ConSurf, SIFT)

  • Highly conserved residues are likely functionally important

• Machine Learning Algorithms:

  • SIFT and PolyPhen-2 for missense variant impact prediction

  • MutationTaster for broader variant effect prediction

  • CADD for integrated scoring of variant deleteriousness

Structure-Based Approaches:

• Homology Modeling:

  • Generate 3D models based on related α/β hydrolases

  • Assess variant positions relative to catalytic sites and substrate binding pockets

• Molecular Dynamics Simulations:

  • Compare wild-type and variant protein dynamics

  • Analyze effects on protein stability and substrate access

  • Identify altered interaction networks

Functional Domain Analysis:

• Map variants to known domains (α/β hydrolase domain, N-terminal domain)

• Assess proximity to catalytic triad or substrate binding residues

• Predict impact on membrane association or protein-protein interactions

Integration with Experimental Data:

• Correlate computational predictions with measured PS/LPS levels

• Validate predictions through site-directed mutagenesis

• Compare predictions for known pathogenic variants (e.g., c.340C>T, c.1370G>A)

The reported human pathogenic variants c.340C>T (p.Arg114*) and c.1370G>A (p.Arg457Gln) cause complete loss of ABHD16A protein expression and function , providing valuable reference points for computational predictions.

What are the most promising approaches for therapeutic targeting of the ABHD16A pathway in neurological disorders?

Based on current understanding of ABHD16A function, several therapeutic approaches show promise:

Targeting the ABHD16A-ABHD12 Axis:

• Selective ABHD12 Inhibition:

  • Development of compounds like DO264 that selectively inhibit ABHD12

  • Goal: Increase LPS levels to compensate for ABHD16A deficiency

  • Challenge: Achieving selective inhibition without affecting related lipases

• PS Supplementation:

  • Direct supplementation with specific PS species reduced in ABHD16A deficiency

  • Advantage: Addresses the metabolite imbalance directly

  • Challenge: Blood-brain barrier penetration and cellular uptake

Gene Therapy Approaches:

• AAV-Mediated Gene Delivery:

  • Package functional ABHD16A gene into adeno-associated viral vectors

  • Target delivery to affected brain regions

  • Challenge: Achieving appropriate expression levels and cell-type specificity

• Antisense Oligonucleotides for Splicing Correction:

  • For specific splice-affecting mutations

  • Advantage: Can be designed with high specificity

  • Challenge: Delivery to CNS tissue

Drug Repositioning Opportunities:

• Screen approved drugs that modulate lipid metabolism

• Identify compounds that can restore PS/LPS balance indirectly

• Advantage: Faster path to clinical testing

Monitoring Treatment Efficacy:

• Develop biomarkers based on PS/LPS ratios in accessible fluids (blood, CSF)

• Correlate lipid normalization with clinical improvement

• Design outcome measures specific to ABHD16A-related neurological symptoms

The therapeutic strategy selection should be guided by mechanistic understanding of how PS/LPS imbalance contributes to neuronal dysfunction in conditions like hereditary spastic paraplegia .

How might studying ABHD16A in bovine systems provide unique insights into broader lipid metabolism pathways?

Bovine ABHD16A research offers several unique opportunities for advancing understanding of lipid metabolism:

Comparative Biochemistry Advantages:

• Evolutionary Insights:

  • Compare enzymatic properties across species to identify conserved vs. specialized functions

  • Assess substrate preferences of bovine vs. human ABHD16A

  • Map functional differences to sequence variations

• Tissue-Specific Expression Patterns:

  • Determine if bovine ABHD16A shows distinct tissue distribution compared to other species

  • Correlate with tissue-specific lipid compositions

  • Identify potential specialized roles in bovine physiology

Methodological Opportunities:

• Accessibility of Diverse Tissue Samples:

  • Ability to collect fresh bovine brain, muscle, and other tissues in quantities sufficient for comprehensive lipidomic analysis

  • Compare PS/LPS profiles across tissues and correlate with ABHD16A expression levels

• Primary Cell Culture Systems:

  • Establish primary bovine neuronal cultures to study ABHD16A function

  • Compare with established human and mouse models

  • Assess impact of ABHD16A modulation on cellular phenotypes

Broader Impacts on Lipid Metabolism Understanding:

• PS Metabolism Network:

  • Map interactions between ABHD16A and other PS-metabolizing enzymes in bovine systems

  • Identify potential compensatory mechanisms in ABHD16A deficiency

  • Discover novel regulatory mechanisms controlling PS/LPS balance

• Cross-Talk with Other Lipid Pathways:

  • Investigate how ABHD16A-mediated PS metabolism influences other lipid classes

  • Assess impact on membrane composition and dynamics

  • Explore consequences for lipid signaling pathways