Recombinant Macaca fascicularis Abhydrolase domain-containing protein 16A (ABHD16A)

What is ABHD16A and what are its primary biochemical characteristics?

ABHD16A belongs to the α/β hydrolase domain-containing protein family, a diverse group of enzymes participating in lipid metabolism and intracellular signaling. It functions primarily as a serine hydrolase containing a conserved α/β hydrolase domain with specific catalytic activity toward phospholipids . Human ABHD16A is a 63 kDa protein comprising 558 amino acid residues, with high sequence conservation expected in the Macaca fascicularis ortholog . The protein contains characteristic motifs including an acyltransferase motif HXXXXD (where H is histidine, D is aspartic acid, and X is any residue) and lipase-like motifs GXSXXG (where G is glycine, S is serine, and X is any residue) .

For researchers working with recombinant Macaca fascicularis ABHD16A, it's important to note that mammalian ABHD16A proteins typically have multiple splicing variants. In humans, two main protein-coding transcripts exist: NM_021160 (558 amino acids) and NM_001177515 (525 amino acids) . Similar transcript diversity likely exists in Macaca fascicularis, necessitating careful construct design when expressing recombinant protein.

What are the enzymatic activities of ABHD16A and how might they be conserved in Macaca fascicularis?

ABHD16A demonstrates multiple enzymatic activities that are likely conserved across primate species:

• Phosphatidylserine hydrolase activity: ABHD16A functions as the main brain phosphatidylserine (PS) hydrolase . This activity is critical for maintaining proper PS and lysophosphatidylserine (lyso-PS) balance.

• Acylglycerol lipase activity: 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) .

These enzymatic activities appear highly conserved across mammalian species, suggesting that Macaca fascicularis ABHD16A would exhibit similar substrate preferences and catalytic efficiencies . When designing assays for recombinant Macaca fascicularis ABHD16A, researchers should consider using these established substrates as positive controls.

Where is ABHD16A predominantly expressed and what does this suggest about its function?

Expression pattern analysis indicates that ABHD16A is widely distributed across tissues, with particularly high expression in neurological tissues, immune cells, muscles, testes, and heart . The broad tissue distribution suggests multiple physiological roles.

The predominant transcript (equivalent to human NM_021160) shows high expression in brain, muscles, testes, and heart, while the alternative isoform (equivalent to human NM_001177515) shows highest expression in testes with minimal brain expression . When studying Macaca fascicularis ABHD16A, tissue-specific expression profiling would help determine whether similar expression patterns exist in this primate species.

How is ABHD16A implicated in neurological disorders and what relevance might this have for Macaca fascicularis models?

Recent evidence has established ABHD16A as a critical factor in neurological function, with pathogenic variants causing a novel form of complex hereditary spastic paraplegia (HSP) . This neurological condition is characterized by:

• Spasticity in lower limbs

• Psychomotor developmental delay and/or regression

• Intellectual impairment

Loss-of-function mutations in ABHD16A disrupt phospholipid metabolism, particularly altering the balance between phosphatidylserine species and lysophosphatidylserine species . In patient-derived fibroblasts, ABHD16A dysfunction reduced levels of certain long-chain lysophosphatidylserine species while increasing levels of multiple phosphatidylserine species .

For researchers working with Macaca fascicularis models, this presents an opportunity to develop non-human primate models of hereditary spastic paraplegia that may more closely recapitulate human pathophysiology than rodent models. The high conservation of ABHD16A across species suggests that similar lipid metabolic disruptions would occur in Macaca fascicularis with ABHD16A mutations.

What is the relationship between ABHD16A and immune function?

ABHD16A's gene location within the major histocompatibility complex (MHC) III gene cluster suggests a role in immune regulation . Further evidence supporting this function includes:

• ABHD16A and ABHD12 dynamically regulate immunomodulatory lysophosphatidylserines (lyso-PSs), affecting lipopolysaccharide-induced proinflammatory cytokine release from macrophages .

• The interplay between these enzymes appears critical for proper immune homeostasis, with ABHD16A hydrolyzing PS to lyso-PS, while ABHD12 degrades lyso-PS .

• ABHD16A inhibition has been shown to suppress neuroinflammation in models where ABHD12 is deficient .

When designing studies with recombinant Macaca fascicularis ABHD16A, researchers should consider immune cell-based assays to evaluate conservation of these immunomodulatory functions.

How does ABHD16A interact with ABHD12 in lipid metabolism pathways?

The functional relationship between ABHD16A and ABHD12 represents a key regulatory mechanism in phospholipid metabolism:

• ABHD16A serves as the principal PS hydrolase, generating lyso-PS species .

• ABHD12 functions as a lyso-PS lipase, degrading lyso-PS .

• The balance between these enzymes controls lyso-PS levels, which function as signaling molecules in inflammation .

This enzymatic partnership has significant pathophysiological implications, as ABHD12 mutations cause PHARC syndrome (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract) . In ABHD12-deficient models, elevated lyso-PS levels contribute to neuroinflammation and cerebellar atrophy .

For researchers working with recombinant Macaca fascicularis ABHD16A, co-expression studies with ABHD12 could provide valuable insights into the conservation of this regulatory pathway in non-human primates.

What expression systems are most effective for producing recombinant Macaca fascicularis ABHD16A?

Based on research with human ABHD16A, several expression systems can be considered for Macaca fascicularis ABHD16A production:

• Mammalian expression systems: HEK293 or CHO cells provide appropriate post-translational modifications and are likely to yield functionally active enzyme. These systems are preferred when studying enzymatic activity or protein-protein interactions.

• Insect cell systems: Baculovirus-infected Sf9 or High Five cells offer a good compromise between yield and post-translational modifications for structural studies.

• Bacterial expression systems: E. coli systems may be suitable for producing protein fragments for antibody generation but typically struggle with full-length, properly folded ABHD16A due to its membrane association properties.

When expressing Macaca fascicularis ABHD16A, researchers should verify that the construct contains the complete α/β hydrolase domain and catalytic triad (typically Ser, His, Asp) essential for enzymatic function . Expression constructs should be designed with appropriate tags (His, FLAG, etc.) positioned to avoid interference with the catalytic domain.

What are effective methods for measuring ABHD16A enzymatic activity?

Based on established protocols for human ABHD16A, the following assays can be adapted for Macaca fascicularis ABHD16A:

• Phosphatidylserine hydrolase assay: This assay measures the conversion of PS to lyso-PS using mass spectrometry-based lipidomics. Typically, the enzyme is incubated with PS substrates (varying acyl chain lengths), and the production of lyso-PS species is quantified .

• Acylglycerol lipase activity assay: ABHD16A's preference for medium and long-chain fatty acids can be assessed using substrates such as 15d-PGJ2-G or monoacylglycerols with varying fatty acid chain lengths. Product formation can be measured by liquid chromatography-mass spectrometry .

• Activity-based protein profiling: Selective ABHD16A inhibitors identified through comparative activity-based protein profiling can be used to confirm the identity and activity of the recombinant enzyme .

For all enzymatic assays, appropriate controls should include heat-inactivated enzyme and known ABHD16A inhibitors to confirm specificity.

How can researchers evaluate the effects of ABHD16A dysfunction in cellular models?

To study the consequences of ABHD16A dysfunction in cellular models related to Macaca fascicularis:

• Targeted lipidomics: Measure PS and lyso-PS species profiles using liquid chromatography-mass spectrometry to detect changes in lipid metabolism. Loss of ABHD16A function typically reduces certain long-chain lyso-PS species while increasing multiple PS species .

• Immune response assays: Measure cytokine production in response to lipopolysaccharide stimulation in cells with ABHD16A knockdown or overexpression to assess effects on inflammatory responses .

• CRISPR/Cas9 gene editing: Generate ABHD16A knockout or knockin (disease-associated variants) in relevant cell lines to study cellular phenotypes.

• Primary cell isolation: For more physiologically relevant models, consider isolating primary cells from Macaca fascicularis tissues, though this approach requires appropriate ethical approvals and specialized facilities.

What are the critical domains and active sites of ABHD16A for structure-function studies?

Key structural elements that should be preserved in recombinant Macaca fascicularis ABHD16A include:

• α/β hydrolase domain: The core catalytic domain essential for enzymatic function .

• Catalytic triad: Typically comprises serine, histidine, and aspartic acid residues forming the nucleophile center .

• Acyltransferase motif (HXXXXD): A conserved motif where H is histidine, D is aspartic acid, and X represents any amino acid residue .

• Lipase-like motifs (GXSXXG): Conserved glycine-rich sequences where G is glycine, S is serine, and X is any amino acid residue .

When designing mutagenesis studies, these conserved regions should be primary targets for investigating structure-function relationships. The high conservation of these domains across species suggests that findings from human ABHD16A studies likely apply to Macaca fascicularis ABHD16A as well.

How can researchers identify potential binding partners and interacting proteins of ABHD16A?

Previous studies with human ABHD16A have identified several interaction partners using techniques that could be applied to Macaca fascicularis ABHD16A:

• Yeast two-hybrid screening: This approach has successfully identified potential interactors including HLA-B, POU5F1, NAT1, TUBB, LECT1, NONO, PGAM1, CCDC107, SLC12A7, and NAD3 .

• Co-immunoprecipitation followed by mass spectrometry: This technique can validate interactions in mammalian cell systems and potentially identify cell-type specific interactors.

• Proximity labeling approaches: BioID or APEX2 fusion proteins can identify proximal proteins in living cells, providing insights into the ABHD16A interactome under physiological conditions.

The diverse nature of the identified interaction partners suggests ABHD16A may participate in multiple physiological processes beyond lipid metabolism . Researchers working with Macaca fascicularis ABHD16A should consider validating these interactions to determine conservation across species.

How can Macaca fascicularis ABHD16A be used to model hereditary spastic paraplegia?

Recent identification of ABHD16A loss-of-function variants in hereditary spastic paraplegia patients provides new opportunities for disease modeling :

• Patient-derived mutations: Introducing equivalent mutations in Macaca fascicularis ABHD16A can help determine if similar enzymatic defects occur in the non-human primate ortholog.

• Lipidomic profiling: Comparing the lipid profiles (particularly PS and lyso-PS species) between wild-type and mutant ABHD16A can help validate disease mechanisms .

• Neuronal cell models: Evaluating the effects of ABHD16A dysfunction in neuronal cells can provide insights into the cellular pathophysiology underlying hereditary spastic paraplegia.

When designing disease models, researchers should consider the two homozygous variants identified in human patients that segregated with disease in the studied families .

What considerations are important when developing inhibitors or modulators of ABHD16A?

For researchers interested in developing compounds targeting Macaca fascicularis ABHD16A:

• Known inhibitors: Several selective inhibitors of ABHD16A have been identified through comparative activity-based protein profiling analyses . These compounds provide starting points for structure-activity relationship studies.

• Inhibitor selectivity: Due to the similarity between ABHD family members, inhibitor selectivity should be carefully evaluated across multiple ABHD proteins, particularly ABHD12 given its functional relationship with ABHD16A .

• Assay development: Establishing robust enzymatic assays with recombinant Macaca fascicularis ABHD16A is essential for screening and validating potential inhibitors.

• Physiological effects: The dual roles of ABHD16A in neurological function and immune regulation necessitate careful evaluation of inhibitor effects in both contexts.

Development of ABHD16A inhibitors could have therapeutic potential for conditions where excess lyso-PS contributes to pathology, though careful consideration of the ABHD16A-ABHD12 axis is essential to avoid unintended consequences .