Recombinant Rat Abhydrolase domain-containing protein 16A (Abhd16a)

What is the molecular structure of rat ABHD16A?

Rat ABHD16A, like its human ortholog, is a member of the α/β hydrolase domain-containing protein family. The protein contains approximately 558 amino acids and has a molecular weight of approximately 63 kDa. The structure features a typical α/β-hydrolase fold consisting of 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 .

The protein contains multiple conserved domains similar to:

• Abhydrolase 1 domain (amino acid residues ~280-408)

• BioH domain (amino acid residues ~276-428)

• PldB domain (amino acid residues ~302-398)

Analysis of ABHD16A across species reveals four transmembrane regions, typically at residues 59-85, 91-113, 204-229, and 350-365 in the human ortholog, with similar regions expected in the rat protein due to high sequence conservation .

What are the primary enzymatic functions of ABHD16A?

ABHD16A functions primarily as a lipase with multiple enzymatic activities:

• Phosphatidylserine hydrolase: ABHD16A is the main brain phosphatidylserine (PS) hydrolase, catalyzing the conversion of phosphatidylserine to lysophosphatidylserine (LPS) .

• Acylglycerol lipase: The enzyme shows preference for medium-chain and long-chain fatty acids, especially long-chain unsaturated monoglycerides and 15-deoxy-Δ12,14-prostaglandin J2–2-glycerol ester (15d-PGJ2-G) .

• Prostaglandin-glycerol (PG-G) hydrolase: In neutrophils, ABHD16A catalyzes the hydrolysis of prostaglandin-glycerol, suggesting a role in inflammatory regulation .

Experimental evidence from patient-derived fibroblasts with ABHD16A loss-of-function mutations shows accumulation of phosphatidylserine substrate species and reduction of lysophosphatidylserine product species, confirming its phosphatidylserine hydrolase function .

How conserved is ABHD16A across species?

ABHD16A is highly conserved across mammalian species, suggesting its fundamental biological importance. Analysis of amino acid sequences from 13 mammalian species revealed:

• 412 conserved sites

• 146 variable sites

• 58 parsimony-informative sites

• 88 singleton sites

Phylogenetic analysis shows that ABHD16A protein sequences can be divided into three categories. Despite variations in the length of the polypeptide chain due to alternative splicing, the functional domains remain highly conserved across species, including:

• The alpha/beta hydrolytic enzyme domain

• Acyltransferase motif HXXXXD (H, histidine; D, aspartic acid; X, any residue)

• Lipase-like motifs GXSXXG (G, glycine; S, serine; X, any residue)

• Nucleophile centers (Ser, Cys, or Asp)

This high conservation facilitates translational research between rat models and human applications.

Where is the ABHD16A gene located in the rat genome?

The rat ABHD16A gene, similar to the mouse ortholog, is located near the tumor necrosis factor (TNF) and Heat shock protein 70 (HSP70) genes in the major histocompatibility complex class III (MHC III) region . This location is analogous to the human ABHD16A gene (formerly known as BAT5), which is positioned within the human MHC III region between TNF and complement gene cluster C2 genes .

This genomic location within the MHC III region suggests a potential role for ABHD16A in immune regulation and inflammation processes, which should be considered when designing experiments with recombinant rat ABHD16A .

What expression systems are optimal for producing functional recombinant rat ABHD16A?

When expressing recombinant rat ABHD16A, researchers should consider:

• Mammalian expression systems: HEK293 or CHO cells often provide proper post-translational modifications for enzymatically active ABHD16A.

• Transmembrane considerations: Since ABHD16A contains four transmembrane regions (residues approximately 59-85, 91-113, 204-229, and 350-365 in human ortholog), expression systems must support proper membrane integration .

• Purification tags: N-terminal tags are generally preferred over C-terminal tags to avoid interference with the catalytic domain located toward the C-terminus.

• Alternative splicing: Consider which isoform to express, as multiple isoforms exist due to variable splicing during post-transcriptional processing of ABHD16A mRNA. For example, in mice, isoforms a and b have 558 and 339 amino acids, respectively .

• Enzymatic activity verification: After expression, verify lipase activity using specific substrates like phosphatidylserine or long-chain monoglycerides to ensure proper folding and function.

How can researchers measure ABHD16A enzymatic activity in experimental settings?

Several methodological approaches can be used to measure rat ABHD16A enzyme activity:

• Phosphatidylserine hydrolase assay: Measure the conversion of phosphatidylserine to lysophosphatidylserine using liquid chromatography-mass spectrometry (LC-MS). This directly reflects ABHD16A's primary activity in the brain .

• Lipidomics analysis: Compare levels of phosphatidylserine (PS) and lysophosphatidylserine (LPS) species in systems with normal versus altered ABHD16A expression. In ABHD16A-deficient systems, expect accumulation of PS substrate species and reduction of LPS product species, particularly long-chain LPS (18:1, 20:4, 20:0, and 20:1) .

• Inhibitor studies: Use selective ABHD16A inhibitors identified through comparative activity-based protein profiling analyses to confirm specificity of observed enzymatic activities .

• PG-G hydrolysis assay: For studying ABHD16A's role in inflammation, measure hydrolysis of prostaglandin-glycerol in neutrophil-based systems .

When analyzing experimental data, maintain appropriate controls and consider that ABHD16A shows higher specific activity with phosphatidylserine than with other substrates including lysophospholipids, diacylated phospholipids, and neutral lipids .

What disease models can benefit from studies with recombinant rat ABHD16A?

Based on current evidence, recombinant rat ABHD16A could be valuable in studying several disease models:

• Neurodegenerative disorders: Homozygous null variants in ABHD16A have been linked to a novel form of complex hereditary spastic paraplegia characterized by developmental delay, intellectual impairment, spasticity, and skeletal deformities .

• Inflammatory conditions: The interplay between ABHD16A and ABHD12 dynamically regulates immunomodulatory lysophosphatidylserines and affects the release of lipopolysaccharide-induced proinflammatory cytokines from macrophages .

• PHARC-related research: While mutations in ABHD12 (not ABHD16A) cause PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract), ABHD16A's functional relationship with ABHD12 suggests relevance to this condition .

• Metabolic disorders: Correlation between ABHD16A gene polymorphisms and back fat thickness in Sus scrofa suggests a potential role in adipose tissue metabolism .

• Viral infection models: ABHD16A has been shown to inhibit Japanese encephalitis virus in vitro .

For disease model development, consider that complete loss of ABHD16A function leads to severe phenotypes, while partial inhibition or overexpression might produce more subtle metabolic effects.

How does ABHD16A loss of function affect phospholipid profiles?

ABHD16A loss of function significantly alters phospholipid metabolism, particularly affecting phosphatidylserine and lysophosphatidylserine levels. Studies in patient-derived fibroblasts with ABHD16A loss-of-function mutations revealed:

• Increased phosphatidylserine levels: Multiple PS species accumulated in ABHD16A-deficient cells compared to control cells .

• Decreased lysophosphatidylserine levels: Levels of long-chain LPS species (18:1, 20:4, 20:0, and 20:1) were significantly reduced (p < 0.0001) in patient fibroblasts compared to control cells .

This lipid profile alteration confirms that ABHD16A functions primarily as a phosphatidylserine hydrolase, converting PS to LPS. The specific changes in long-chain LPS species suggest that ABHD16A preferentially processes certain PS substrates, which may have implications for membrane composition and signaling in various tissues, particularly in the brain .

When studying ABHD16A function in rat models, similar phospholipid profile changes should be anticipated and can serve as biomarkers for successful genetic manipulation or pharmacological inhibition.

What protein interactions should be considered when working with recombinant rat ABHD16A?

ABHD16A participates in multiple protein-protein interactions that may affect its function in experimental systems:

• ABHD12 functional relationship: ABHD16A and ABHD12 work in tandem to regulate lysophosphatidylserine levels. While ABHD16A produces LPS by hydrolyzing PS, ABHD12 further metabolizes LPS. This interplay dynamically regulates immunomodulatory lysophosphatidylserines and affects inflammatory responses .

• Known interaction partners: Several ABHD16A interaction partners have been identified through yeast two-hybrid screening, including:

  • IFITM1 (interferon-induced transmembrane proteins)

  • hnRNP1 (heterogeneous nuclear ribonucleoprotein A1)

  • RNF5 (RING zinc-finger domain protein)

  • SPP (signal peptide peptidase)

  • COX II (Mitochondrial cytochrome oxidase II)

  • ATP5G3 (subunit of mitochondrial ATP synthase H+ transporter)

  • NAD3 (Mitochondrial NADH dehydrogenase)

• MicroRNA regulation: MiR-155 has been shown to reduce ABHD16A mRNA expression in activated B cells, suggesting regulatory mechanisms that should be considered when studying ABHD16A function .

These interactions suggest ABHD16A may participate in multiple physiological processes beyond its enzymatic function, including immune regulation, mitochondrial function, and cellular signaling pathways.

What are the key challenges in purifying active recombinant rat ABHD16A?

Purifying enzymatically active recombinant rat ABHD16A presents several challenges:

• Membrane association: ABHD16A contains multiple transmembrane domains, making it difficult to solubilize while maintaining its native conformation and activity .

• Lipid environment requirements: As a lipid-metabolizing enzyme, ABHD16A may require specific lipid environments for optimal activity.

• Structural complexity: The presence of multiple conserved domains (Abhydrolase 1, BioH, and PldB) increases the complexity of proper folding during recombinant expression .

• Alternative splicing considerations: Different isoforms resulting from alternative splicing may have different properties and activities, requiring careful selection of the appropriate isoform for specific research purposes .

Strategies to address these challenges include:

• Using mild detergents for solubilization

• Expressing the protein in lipid nanodiscs

• Employing mammalian expression systems for proper post-translational modifications

• Considering truncated constructs that retain the catalytic domain while eliminating challenging transmembrane regions

How can researchers genetically modify ABHD16A in rat models?

When developing genetic modifications of ABHD16A in rat models, several approaches should be considered:

• CRISPR/Cas9 genome editing: For creating knockout or knock-in models. Target sequences should avoid the highly conserved catalytic domains to prevent off-target effects in related hydrolases.

• Conditional knockouts: Consider tissue-specific or inducible systems, particularly since complete ABHD16A knockout may cause severe neurological phenotypes similar to those observed in human patients with homozygous null variants .

• Point mutations: To study specific aspects of ABHD16A function, consider introducing mutations that affect:

  • The catalytic triad (likely involving Ser, His, and Asp residues)

  • The acyltransferase motif HXXXXD

  • The lipase-like motifs GXSXXG

• Overexpression models: Viral vector-mediated overexpression can help study gain-of-function effects, potentially relevant for therapeutic development.

When designing genetic modifications, consider that ABHD16A's location in the MHC III region may complicate genetic manipulation due to potential effects on nearby immune-related genes .

What analytical methods are best suited for studying rat ABHD16A-dependent lipid metabolism?

To effectively study rat ABHD16A-dependent lipid metabolism, researchers should consider these analytical approaches:

• Targeted lipidomics: Liquid chromatography-mass spectrometry (LC-MS) with multiple reaction monitoring (MRM) to quantify specific phosphatidylserine and lysophosphatidylserine species known to be affected by ABHD16A activity .

• Untargeted lipidomics: Broader LC-MS approaches to identify novel lipid species that may be affected by ABHD16A activity.

• Activity-based protein profiling: To assess ABHD16A activity in complex biological samples and to screen for selective inhibitors .

• Radiolabeled substrate assays: Using radiolabeled phosphatidylserine to track hydrolysis products with high sensitivity.

• Immunoblotting and immunohistochemistry: To verify ABHD16A expression levels and tissue distribution in experimental models.

When analyzing data, compare results with the pattern observed in patient-derived cells, where ABHD16A deficiency led to increased levels of phosphatidylserine species and decreased levels of specific lysophosphatidylserine species, particularly long-chain LPS (18:1, 20:4, 20:0, and 20:1) .

How can inhibitors of ABHD16A be developed and characterized?

Development of selective ABHD16A inhibitors follows several key steps:

• High-throughput screening: Screen compound libraries against recombinant rat ABHD16A using activity-based assays that measure phosphatidylserine hydrolysis.

• Structure-activity relationship studies: Refine hit compounds through medicinal chemistry approaches guided by the enzyme's structural features, particularly the conserved catalytic domains.

• Selectivity profiling: Test compounds against related hydrolases, particularly ABHD12, to ensure specificity. Comparative activity-based protein profiling can be used to identify selective inhibitors .

• Cellular validation: Verify that inhibitors recapitulate the lipid profile changes observed in ABHD16A loss-of-function models, specifically increased phosphatidylserine levels and decreased lysophosphatidylserine levels .

• In vivo testing: Evaluate pharmacokinetics and efficacy in rat models, with potential applications in inflammatory conditions based on ABHD16A's role in immune regulation .

Several selective inhibitors of ABHD16A have been identified through comparative activity-based protein profiling analyses, providing starting points for further development .

What considerations are important when studying ABHD16A in neuroinflammation models?

When investigating ABHD16A in neuroinflammation contexts, researchers should consider:

• ABHD16A-ABHD12 balance: The interplay between these enzymes regulates lysophosphatidylserine levels, which affect inflammatory responses. ABHD12 deficiency is associated with PHARC and neuroinflammation, while ABHD16A inhibition might counteract these effects .

• Cytokine production monitoring: Measure the release of lipopolysaccharide-induced proinflammatory cytokines from macrophages, which can be affected by ABHD16A-mediated lysophosphatidylserine regulation .

• Blood-brain barrier considerations: When developing ABHD16A-targeting compounds, consider blood-brain barrier penetration for neurological applications.

• Neurological phenotyping: Monitor for spasticity, developmental delays, and intellectual impairments in animal models, reflecting the symptoms observed in human patients with ABHD16A mutations .

• Imaging techniques: Consider advanced imaging to detect subtle changes in brain structure, as ABHD16A mutations in humans are associated with complex hereditary spastic paraplegia .

The relationship between ABHD16A dysfunction and complex hereditary spastic paraplegia suggests that this protein plays a crucial role in normal neurological development and function, making it a potential target for neurodegenerative disease research .

How does rat ABHD16A compare to human ABHD16A for translational research?

When conducting translational research with rat ABHD16A models, consider these comparative aspects:

• Sequence homology: Rat and human ABHD16A share high sequence homology, particularly in functional domains, facilitating translation of findings between species .

• Enzymatic activity conservation: Both rat and human ABHD16A function primarily as phosphatidylserine hydrolases, with similar substrate preferences for medium-chain and long-chain fatty acids .

• Genomic location similarities: Both rat and human ABHD16A genes are located in the MHC III region, suggesting conserved regulatory contexts and potential immunological functions .

• Expression pattern differences: While broadly similar, there may be tissue-specific expression differences between rat and human ABHD16A that should be accounted for when translating findings.

• Phenotypic relevance: Loss of function of ABHD16A in humans causes complex hereditary spastic paraplegia with developmental delay, intellectual impairment, and skeletal deformities . Rat models with ABHD16A mutations should be evaluated for analogous phenotypes to confirm translational relevance.

For maximal translational value, validation of key findings in both rat models and human cell lines or clinical samples is recommended.