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

What is the basic structure of human ABHD16A?

Human ABHD16A is a 63 kDa protein containing 558 amino acid residues in its primary isoform. Structural analysis reveals four transmembrane regions located at residues 59-85, 91-113, 204-229, and 350-365. The protein contains three highly conserved domains similar to Abhydrolase 1 (residues 280-408), BioH (residues 276-428), and PldB (residues 302-398) . The protein structure follows the typical α/β-hydrolase fold pattern with 8 β-strands and 6 α-helices, with the hydrolytic enzyme active center formed by histidine residues surrounded by helices and loops linking the β-strands .

What are the known enzymatic activities of ABHD16A?

ABHD16A demonstrates multiple enzymatic activities, functioning primarily as:

• A phosphatidylserine (PS) lipase, particularly in brain tissue

• An acylglycerol lipase

• A lysophospholipase (via its PldB domain)

The protein contains key catalytic motifs including an acyltransferase motif HXXXXD (H, histidine; D, aspartic acid and X, any residues) and lipase-like motifs GXSXXG (G, glycine; S, serine and X, any residues) . The catalytic triad typically consists of a serine residue located in a compact loop, with the highly conserved histidine residue present in a variable loop behind β8 .

What are effective methods for expressing recombinant human ABHD16A in laboratory settings?

For successful expression of recombinant human ABHD16A:

• Expression System Selection:

  • Prokaryotic systems: E. coli BL21(DE3) strains are suitable for expressing soluble domains

  • Eukaryotic systems: HEK293 or insect cells (Sf9, Hi5) are recommended for full-length protein with proper post-translational modifications

• Expression Vectors:

  • For bacterial expression: pET series vectors with His-tag or GST-tag

  • For mammalian expression: pcDNA3.1 or pCMV with appropriate signal sequences

• Optimization Considerations:

  • Codon optimization for the expression system

  • Temperature reduction during induction (16-18°C) to enhance proper folding

  • Including chaperones for transmembrane protein expression

  • Using detergents for solubilization (CHAPS, DDM, or Triton X-100)

• Purification Strategy:

  • Two-step purification combining affinity chromatography followed by size exclusion

  • Critical buffer components include glycerol (10-15%) and reducing agents to maintain stability

What are reliable assays for measuring ABHD16A enzymatic activity?

Several assay systems have been developed to measure ABHD16A activity:

• Fluorogenic Substrate Assays:

  • Using 4-methylumbelliferyl substrates with varying acyl chain lengths

  • Monitoring product release via fluorescence (Ex: 355nm/Em: 460nm)

• Radiometric Assays:

  • Using radiolabeled phosphatidylserine as substrate

  • Quantifying lysophosphatidylserine production by TLC separation

• LC-MS Based Assays:

  • Most accurate for physiological substrate processing

  • Sample preparation: Lipid extraction followed by LC-MS/MS analysis

  • Quantification of both substrates and products simultaneously

• Coupled Enzyme Assays:

  • Measuring free fatty acid release using acyl-CoA oxidase coupling

  • Colorimetric or fluorometric endpoint measurements

Optimal assay conditions include pH 7.4-8.0, 37°C, with appropriate detergent concentrations to maintain enzyme solubility without inhibiting activity .

How can researchers effectively generate ABHD16A knockout models?

Generation of reliable ABHD16A knockout models requires specific approaches:

• Cell Line Models:

  • CRISPR/Cas9 system targeting conserved exons (particularly exons encoding catalytic domains)

  • Recommended guide RNA design: Target regions encoding the Ser-His-Asp catalytic triad

  • Verification methods: Western blot, enzymatic activity assays, and genomic sequencing

• Animal Models:

  • Conditional knockout recommended due to potential embryonic lethality

  • Tissue-specific Cre-loxP systems particularly useful for neurological studies

  • Alternative: CRISPR/Cas9 with tissue-specific promoters

• Validation Requirements:

  • Protein level verification by Western blot analysis

  • Enzymatic activity measurements to confirm functional loss

  • Phenotypic characterization focusing on lipid metabolism and neurological parameters

  • Analysis of compensatory mechanisms (other ABHD family members upregulation)

What is the role of ABHD16A in hereditary spastic paraplegia?

ABHD16A deficiency causes a complicated form of hereditary spastic paraplegia (HSP86). The disease mechanism involves:

• Clinical Manifestations:

  • Global developmental delay/intellectual disability

  • Progressive spasticity affecting upper and lower limbs

  • Corpus callosum and white matter anomalies

• Molecular Pathogenesis:

  • Disruption of phosphatidylserine metabolism

  • Reduced synthesis of lysophosphatidylserine, an important signaling lipid in the central nervous system

  • Altered lipid homeostasis affecting axonal maintenance

• Genotype-Phenotype Correlations:

  • Bi-allelic deleterious variants in ABHD16A are causal

  • The mean age of onset for lower limb spasticity is approximately 3 years

  • Progressive worsening of symptoms over time

• Pathophysiological Significance:

  • ABHD16A joins other HSP-associated genes in highlighting the importance of lipid metabolism in neuronal physiology

  • Altered lipid metabolism contributes to axonal degeneration

  • ABHD16A serves as the major phosphatidylserine lipase in the brain, making its function critical for neuronal health

How is ABHD16A connected to immune system regulation?

ABHD16A has significant immunomodulatory functions:

• Genomic Context:

  • Located in the main histocompatibility complex (MHC) III gene cluster

  • Previously known as human leucocyte antigen B (HLA-B) associated transcript 5 (BAT5)

  • This genomic location suggests inherent immunological functions

• Immunological Associations:

  • Involved in inflammatory response regulation

  • Associated with Kawasaki disease and coronary artery aneurysm

  • Potential role in neurodegenerative diseases with inflammatory components

• Mechanistic Considerations:

  • Production of bioactive lipid mediators that regulate immune cell function

  • Involvement in phosphatidylserine metabolism, which affects apoptotic cell clearance

  • Potential role in membrane lipid composition affecting immune receptors

What other diseases have been associated with ABHD16A dysfunction?

Beyond hereditary spastic paraplegia, ABHD16A has been implicated in:

• Neurological Disorders:

  • Neurodegenerative diseases (specific mechanisms still under investigation)

  • Potential involvement in axonal maintenance disorders

• Inflammatory Conditions:

  • Kawasaki disease

  • Coronary artery aneurysm

  • Potential role in other inflammatory disorders due to its immunomodulatory functions

• Metabolic Disorders:

  • Potential involvement in lipid metabolism disorders

  • May affect cellular signaling through bioactive lipid regulation

Research is ongoing to fully characterize the spectrum of disorders associated with ABHD16A dysfunction, as the field is still developing .

How do post-translational modifications affect ABHD16A function?

Post-translational modifications significantly impact ABHD16A function:

• Phosphorylation Sites:

  • Multiple serine and threonine residues can be phosphorylated

  • Phosphorylation may regulate enzymatic activity and protein-protein interactions

  • Key kinases involved include PKC and MAPK pathways

• Glycosylation:

  • N-linked glycosylation sites present in the luminal domains

  • Affects protein folding, stability, and trafficking

  • May influence interaction with other membrane proteins

• Palmitoylation:

  • Affects membrane localization and microdomain targeting

  • May regulate association with lipid rafts

  • Potentially critical for accessing specific substrate pools

• Methodological Approaches:

  • Mass spectrometry-based proteomics for identification of modification sites

  • Site-directed mutagenesis to assess functional importance

  • Inhibitor studies targeting specific modification enzymes to determine regulatory mechanisms

What are the challenges in developing selective inhibitors for ABHD16A?

Developing selective ABHD16A inhibitors faces several challenges:

• Structural Homology Issues:

  • High similarity with other ABHD family members (particularly ABHD6 and ABHD12)

  • Challenge of achieving selectivity while maintaining potency

  • Need for high-resolution structural data to guide design

• Current Inhibitor Development Approaches:

  • Irreversible inhibitors targeting the catalytic serine residue

  • Piperidyl-1,2,3-triazole urea scaffold showing promise

  • Structure-activity relationship studies highlighting importance of specific moieties

• Selectivity Assessment:

  • Comprehensive profiling against all ABHD family members required

  • Activity-based protein profiling (ABPP) using terminal alkyne-containing probes

  • Click chemistry approaches for target visualization

• Design Strategies:

  • Exploiting unique active site features of ABHD16A

  • Targeting non-catalytic regulatory sites

  • Development of allosteric modulators with improved selectivity profiles

How can researchers effectively study ABHD16A protein-protein interactions?

Several approaches can be employed to investigate ABHD16A interactions:

• Affinity-Based Methods:

  • Tandem affinity purification combined with mass spectrometry

  • BioID or APEX proximity labeling to identify neighboring proteins

  • Co-immunoprecipitation with specific antibodies against endogenous ABHD16A

• Imaging-Based Approaches:

  • Fluorescence resonance energy transfer (FRET)

  • Bimolecular fluorescence complementation (BiFC)

  • Super-resolution microscopy to visualize co-localization

• Functional Validation Strategies:

  • Mutational analysis of interaction interfaces

  • Competition assays with peptides derived from interacting regions

  • CRISPR-mediated knockdown/knockout of interaction partners

• Computational Methods:

  • Molecular docking simulations

  • Protein-protein interaction network analysis

  • Evolutionary conservation mapping to identify potential interaction interfaces

What are promising therapeutic applications targeting ABHD16A?

Several therapeutic strategies targeting ABHD16A show promise:

• Neurological Disorders:

  • Development of ABHD16A modulators for hereditary spastic paraplegia

  • Potential applications in other neurodegenerative disorders

  • Approaches focusing on restoring lysophosphatidylserine levels

• Inflammatory Conditions:

  • ABHD16A inhibitors as anti-inflammatory agents

  • Dual ABHD16A/ABHD12 modulators for enhanced effect

  • Targeted delivery systems for tissue-specific activity

• Drug Development Approaches:

  • Structure-guided design of reversible inhibitors

  • Development of degraders (PROTACs) targeting ABHD16A

  • Gene therapy approaches for hereditary conditions

• Therapeutic Considerations:

  • Need for careful assessment of on-target effects on lipid metabolism

  • Potential compensatory mechanisms through other ABHD family members

  • Tissue-specific targeting to minimize systemic effects

How can multi-omics approaches enhance our understanding of ABHD16A function?

Integrated multi-omics approaches offer powerful insights into ABHD16A biology:

• Lipidomics:

  • Comprehensive profiling of lipid changes in ABHD16A-deficient models

  • Identification of physiological substrates and products

  • Temporal dynamics of lipid alterations following perturbation

• Proteomics:

  • Interaction networks in different cellular compartments

  • Changes in protein expression and modification in response to ABHD16A modulation

  • Activity-based protein profiling to identify functional changes

• Transcriptomics:

  • Gene expression changes in ABHD16A-deficient models

  • Identification of compensatory mechanisms

  • Pathway analysis to understand broader biological impact

• Integration Strategies:

  • Machine learning approaches to identify patterns across data types

  • Network analysis to map functional relationships

  • Systems biology modeling of ABHD16A in lipid homeostasis

What experimental approaches are recommended for characterizing ABHD16A variants found in clinical settings?

Comprehensive characterization of clinical ABHD16A variants requires:

• Functional Assessment Workflow:

  • Expression analysis in relevant cell types

  • Protein stability and localization studies

  • Enzymatic activity measurements against multiple substrates

  • Interaction profiling with known partners

• Structural Impact Analysis:

  • In silico modeling of variant effects on protein structure

  • Molecular dynamics simulations to assess conformational changes

  • Experimental validation using biophysical methods (CD, DSF, NMR)

• Cellular Phenotype Characterization:

  • Creation of isogenic cell lines using CRISPR-Cas9 knock-in

  • Lipid profiling to identify metabolic consequences

  • Assessment of cellular stress responses and viability

• Translational Research Approaches:

  • Patient-derived iPSCs differentiated into relevant cell types

  • Organoid models to study tissue-specific effects

  • Correlation of variant characteristics with clinical phenotypes