Recombinant Rat Group XVI phospholipase A1/A2 (Pla2g16)
Description
Genetic and Molecular Identity
Phospholipase A2 Group XVI (Pla2g16) belongs to the diverse phospholipase A2 enzyme family that catalyzes the hydrolysis of phospholipids. In rats, this enzyme is encoded by the Pla2g16 gene (Gene ID: 24913) with mRNA reference sequence NM_017060.2 and protein reference sequence NP_058756.2 . The rat Pla2g16 protein shares significant homology with human PLA2G16, though species-specific differences exist in regulation and tissue distribution. The enzyme is classified within group XVI of phospholipases, distinguishing it from more extensively studied phospholipase groups through its unique structural and catalytic properties. Taxonomically, it represents a distinct evolutionary branch within phospholipase enzymes while maintaining core functional properties related to lipid metabolism and cellular signaling .
Synonyms and Related Terminology
The enzyme is known by several alternative designations in scientific literature, reflecting its discovery context and functional associations. In rat species specifically, common synonyms include H-rev107, Hrasls3, Hrev107, and HRSL3 . These multiple designations stem from the enzyme's identification in different research contexts, including studies on HRAS-related suppressor genes and reversion of transformed phenotypes. The human ortholog shows an even wider range of synonyms including adipose-specific phospholipase A2 (AdPLA), H-REV107-1, HRAS-like suppressor 3, and Ca-independent phospholipase A1/2 . This nomenclature diversity highlights the enzyme's multifunctional nature and its identification through various experimental approaches across different research fields.
Enzymatic Activities and Substrate Preferences
Pla2g16 demonstrates a distinctive enzymatic profile characterized by dual phospholipase A1 and A2 activities, with the A1 activity predominating for most substrates. This enzymatic behavior distinguishes it from classical phospholipases that typically exhibit higher specificity for either A1 or A2 positions. The enzyme catalyzes the calcium-independent hydrolysis of acyl groups in various phosphatidylcholines (PC) and phosphatidylethanolamine (PE) . Its calcium independence represents another significant distinguishing feature from many other phospholipases that require calcium ions for catalytic activity.
The enzyme exhibits notable substrate selectivity, preferentially acting on phospholipids in adipose tissue to release fatty acids. This tissue-specific activity suggests adapted functionality related to lipid storage and mobilization in adipocytes. Additionally, Pla2g16 shows weak lysophospholipase activity, further expanding its potential impact on lipid metabolism beyond primary phospholipase functions . The enzyme's N- and O-acylation activities are described as hardly detectable, indicating specialized evolutionary adaptation toward phospholipid hydrolysis rather than lipid modification.
Catalytic Mechanisms
The primary catalytic reaction facilitated by Pla2g16 can be represented as:
Phosphatidylcholine + H₂O → 1-acylglycerophosphocholine + carboxylate
This hydrolysis occurs through nucleophilic attack on the phospholipid ester bond, facilitated by the enzyme's active site architecture. Unlike many phospholipases that require calcium for stabilizing the tetrahedral intermediate during catalysis, Pla2g16 employs alternative mechanisms for substrate binding and transition state stabilization. The enzyme's active site likely contains specific amino acid residues that position the substrate and activate the water molecule for nucleophilic attack, enabling efficient catalysis without metal ion cofactors.
Tissue Distribution and Cellular Localization
Pla2g16 exhibits a distinctive tissue distribution pattern with predominant expression in adipose tissue, particularly within the adipocyte fraction rather than the stromal vascular fraction. This contrasts sharply with phospholipase A2 group IIA (Pla2g2a), which is primarily expressed in the stromal vascular component containing immune cells . The adipocyte-specific expression pattern suggests specialized functions related to lipid metabolism and adipocyte biology. Within cells, the enzyme likely associates with membrane structures given its substrate specificity for phospholipids, though specific subcellular localization data from the provided sources is limited.
Regulation of Expression
A notable characteristic of Pla2g16 is its stable expression pattern under metabolic challenges. Unlike Pla2g2a, which shows dramatic upregulation (approximately 20-fold increase) in response to high-carbohydrate, high-fat (HCHF) feeding, Pla2g16 expression remains unchanged by dietary manipulation in rats . This differential regulation suggests distinct physiological roles and regulatory mechanisms between these phospholipase family members. The stability of Pla2g16 expression during metabolic stress indicates potential constitutive functions in adipocyte biology rather than responsive roles in inflammation or metabolic adaptation. Similar findings were reported in diet-induced obese mice, where Pla2g16 gene expression remained constant despite significant adiposity development .
Role in Adipocyte Function and Metabolism
Pla2g16 plays significant autocrine and paracrine roles in regulating adipocyte function and lipid metabolism. Research indicates it participates in regulating lipolysis through a prostaglandin E₂ (PGE₂)–prostaglandin EP3 receptor–cyclic adenosine monophosphate (cAMP) pathway . This signaling cascade represents an important mechanism for modulating fat storage and mobilization in adipose tissue. The enzyme's activity leads to the release of fatty acids from phospholipids specifically in adipose tissue, potentially contributing to local lipid availability for metabolic processes or signaling functions.
Involvement in Biochemical Pathways
Pla2g16 participates in multiple biochemical pathways critical to cellular lipid metabolism and signaling. According to pathway analyses, the enzyme is involved in acyl chain remodeling of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) . These remodeling processes are essential for maintaining membrane phospholipid composition and generating lipid signaling molecules. Additionally, Pla2g16 relates to linoleic acid metabolism and regulation of lipolysis in adipocytes, further emphasizing its multifunctional role in lipid homeostasis . The enzyme functions alongside other pathway components including PNPLA8, PLB1, PLBD1, and PLA2G4B in these metabolic processes .
| Pathway Name | Related Proteins |
|---|---|
| Acyl chain remodelling of PC | PNPLA8, PLB1, PLBD1, PLA2G4B |
| Acyl chain remodelling of PE | PNPLA8, PLA2G4B, PLBD1 |
| Acyl chain remodelling of PI | PLBD1 |
| Linoleic acid metabolism | Multiple enzymes |
| Regulation of lipolysis in adipocytes | Multiple regulators |
Applications in Research
Recombinant rat Pla2g16 protein serves multiple research purposes in biochemical and cellular studies. Primary applications include:
-
Enzyme activity assays to characterize catalytic properties and substrate preferences
-
Structural studies examining protein-protein interactions and conformational changes
-
Development and validation of inhibitors for potential therapeutic applications
-
Cell-based assays investigating physiological functions in adipocytes and other tissues
These recombinant preparations enable controlled experimental conditions that would be difficult to achieve with native protein isolated from tissues, providing researchers with standardized tools for investigating Pla2g16 biology.
Distinction from Phospholipase A2 Group IIA (Pla2g2a)
Important distinctions exist between Pla2g16 and other phospholipase family members, particularly Pla2g2a. While both enzymes participate in phospholipid metabolism, they differ markedly in tissue distribution, regulation, and physiological functions. Pla2g16 is predominantly expressed in adipocytes and maintains stable expression levels under metabolic stress conditions. In contrast, Pla2g2a is primarily expressed in the stromal vascular fraction of adipose tissue containing immune cells and shows dramatic upregulation (approximately 20-fold) in response to high-carbohydrate, high-fat feeding in rats .
These differences extend to their respective roles in inflammation and metabolism. Pla2g2a is strongly associated with inflammatory responses and generation of prostaglandin E₂ after immune cell activation, while Pla2g16 appears more specifically involved in constitutive adipocyte functions related to lipid metabolism . This functional specialization is further evidenced by their differential responses to inhibitors - the compound KH064 effectively inhibits Pla2g2a without affecting Pla2g16 activity, allowing selective pharmacological targeting .
Enzyme Activity Comparison
From an enzymatic perspective, Pla2g16 exhibits unique characteristics compared to other phospholipases. It displays dual phospholipase A1 and A2 activities with predominant A1 activity, while many classical phospholipases show more specific positional preferences . Additionally, Pla2g16 functions in a calcium-independent manner, distinguishing it from calcium-dependent phospholipases such as group IIA phospholipase A2 (Pla2g2a) . These enzymatic distinctions reflect evolutionary specialization for specific physiological niches, with Pla2g16 adapted for functions in adipose tissue metabolism rather than inflammatory responses typically associated with other phospholipase family members.
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized fulfillment.
Note: Standard shipping includes blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us for preferential development.
Target Background
Recombinant Rat Group XVI phospholipase A1/A2 (Pla2g16) exhibits phospholipase A1/A2 and acyltransferase activities. It catalyzes the calcium-independent release of fatty acids from the sn-1 or sn-2 position of glycerophospholipids, demonstrating both phospholipase A1 (PLA1) and A2 (PLA2) activity. PLA1 activity generally surpasses PLA2 activity for most substrates. Additionally, it displays O-acyltransferase activity, transferring a fatty acyl group from glycerophospholipid to the hydroxyl group of lysophospholipid. The enzyme also exhibits N-acyltransferase activity, catalyzing the calcium-independent transfer of a fatty acyl group at the sn-1 position of phosphatidylcholine (PC) and other glycerophospholipids to the primary amine of phosphatidylethanolamine (PE), producing N-acylphosphatidylethanolamine (NAPE), a precursor for N-acylethanolamines (NAEs). It exhibits significantly higher N-acyltransferase activity compared to its phospholipase A1/2 activity. Pla2g16 is crucial for complete organelle rupture and degradation during eye lens terminal differentiation. In this process, lens fiber cells degrade all membrane-bound organelles, contributing to lens transparency and light passage. This organelle membrane degradation is likely mediated by the enzyme's phospholipase activity.
Q&A
What is PLA2G16 and what are its primary biological functions?
PLA2G16 (Group XVI Phospholipase A1/A2) is a thiol hydrolase belonging to the HRASLS (HRAS-like suppressor) family that primarily functions as a phospholipase. It is an intercellular, single-pass transmembrane protein with a molecular weight of approximately 18 kDa that predominantly hydrolyzes the sn-2 fatty acyl chain of phosphatidylcholine . PLA2G16 plays critical roles in several biological processes, including regulation of lipolysis in adipose tissue and serving as a host factor that enables cellular entry of picornaviruses . Recent research has also implicated PLA2G16 in cancer metabolism, particularly in pancreatic adenocarcinoma where it promotes aerobic glycolysis and tumor growth . Unlike traditional phospholipases that target only one position on the glycerol backbone, PLA2G16 exhibits dual PLA1/PLA2 activity, contributing to the rapid turnover and remodeling of cellular glycerophospholipids .
What is the structural organization of PLA2G16?
PLA2G16 has a distinctive papain-fold motif consisting of three α-helices and five antiparallel β-sheets organized in a circular permutation . The enzyme contains a highly conserved catalytic triad made up of Cys113, His23, and His35, as determined by X-ray crystallography (PDB code: 4DOT) and confirmed through site-directed mutagenesis studies . This catalytic triad is essential for its enzymatic function. The enzyme's structural features enable it to interact with membrane phospholipids and carry out its hydrolytic function. Understanding this structure has been crucial for the development of specific inhibitors like α-ketoamides that can form hemithioacetal adducts with the active site Cys113 .
How is recombinant PLA2G16 typically expressed and purified for research applications?
Recombinant PLA2G16 is commonly produced using bacterial expression systems, particularly E. coli. For example, recombinant human PLA2G16 can be expressed by targeting the gene encoding amino acids Asp12-Asp132 with an N-terminal 6His tag to facilitate purification . The expression construct typically includes only the catalytic domain, omitting the transmembrane region to improve solubility and expression efficiency.
For rat PLA2G16, a similar approach can be employed with appropriate adjustments for species-specific sequence variations. The purification process typically involves:
-
Cell lysis under native or denaturing conditions depending on protein solubility
-
Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices
-
Size exclusion chromatography to enhance purity
-
Assessment of protein quality using SDS-PAGE and Western blotting
-
Enzyme activity assays to confirm functional integrity
The purified recombinant protein can then be used for structural studies, enzymatic assays, inhibitor screening, and other research applications .
What methods are available for measuring PLA2G16 enzymatic activity?
Several complementary approaches can be employed to measure PLA2G16 activity:
-
Fluorescent lipase probes: MB064 is a fluorescent probe that effectively labels both recombinant and endogenously expressed PLA2G16, making it valuable for activity-based protein profiling (ABPP) assays . This allows for visualization of enzyme activity in complex biological samples.
-
Biochemical assays: Direct measurement of PLA2G16 hydrolytic activity can be performed using artificial substrates or natural phospholipids. For kinetic analysis, researchers have established assays yielding Ki values for inhibitors, such as the 20 nM Ki determined for LEI110 .
-
Lipidomic analysis: Cellular products of PLA2G16 activity, such as arachidonic acid, can be quantified using targeted lipidomics. For example, transfection of human PLA2G16 in U2OS cells leads to a time-dependent increase in arachidonic acid that can be quantified and used to assess enzyme activity or inhibitor efficacy .
-
Lipid droplet visualization: In cellular models such as HepG2 cells, PLA2G16 activity can be indirectly assessed by measuring lipid droplet formation after oleic acid treatment, as PLA2G16 modulates lipolysis in these cells .
How can activity-based protein profiling (ABPP) be utilized to study PLA2G16?
Activity-based protein profiling is a powerful technique for studying PLA2G16 function and inhibition:
-
Probe selection: MB064, a fluorescent lipase probe, has been demonstrated to effectively label PLA2G16 in both recombinant systems and endogenous contexts .
-
Competitive ABPP: This approach involves pre-incubating samples with potential inhibitors before adding the activity-based probe. Reduction in probe labeling indicates inhibitor binding to the active site. This method was instrumental in identifying α-ketoamides as the first selective PLA2G16 inhibitors .
-
Gel-based ABPP: After labeling with fluorescent probes like MB064, proteins can be separated by SDS-PAGE and visualized using a fluorescence scanner. This approach allows for the assessment of inhibitor selectivity by examining effects on multiple enzymes simultaneously .
-
Chemical proteomics: By combining ABPP with mass spectrometry, researchers can comprehensively identify proteins targeted by inhibitors. This approach has shown that LEI110 acts as a selective pan-inhibitor of the HRASLS family of thiol hydrolases (PLA2G16, HRASLS2, RARRES3, and iNAT) .
-
Tissue-specific profiling: ABPP has been successfully applied to visualize PLA2G16 activity in both brown and white adipose tissue, demonstrating the technique's utility for studying the enzyme in its physiological context .
What are the known inhibitors of PLA2G16 and how were they discovered?
The first selective PLA2G16 inhibitors were identified through competitive activity-based protein profiling (ABPP) using the fluorescent lipase probe MB064. Key inhibitors include:
-
Compound 1 (N-phenethylbutanamide): An α-ketoamide identified as an initial hit that almost completely abolished PLA2G16 labeling at 10 μM with a pIC50 of 6.0 ± 0.1 .
-
LEI110 (2-oxo-5-phenyl-N-(4-((5-(trifluoromethyl)pyridin-2-yl)oxy)-phenethyl)pentanamide): A significantly improved inhibitor developed through optimization of Compound 1, with a 10-fold increased potency (pIC50 = 7.0 ± 0.1) and a Ki value of 20 nM (95% CI: 17–24 nM) .
The discovery process involved:
-
Initial screening using competitive ABPP
-
Hit optimization (64 analogues were synthesized)
-
Biochemical validation of inhibitory potency
-
Selectivity profiling against other hydrolases
-
Molecular dynamic simulations to understand binding interactions
The table below summarizes the potency of these inhibitors against the HRASLS protein family members:
| Compound | PLA2G16 pIC50 ± SEM | HRASLS2 pIC50 ± SEM | RARRES3 pIC50 ± SEM | iNAT pIC50 ± SEM |
|---|---|---|---|---|
| 1 | 6.0 ± 0.1 | 6.2 ± 0.1 | 6.2 ± 0.1 | 6.4 ± 0.1 |
| LEI110 | 7.0 ± 0.1 | 6.8 ± 0.1 | 6.8 ± 0.1 | 7.6 ± 0.1 |
What is the mechanism of action for PLA2G16 inhibitors?
The α-ketoamide inhibitors of PLA2G16, such as Compound 1 and LEI110, are believed to act through a reversible covalent mechanism:
-
The electrophilic ketone of the inhibitor reacts with the active site Cys113, forming a hemithioacetal adduct .
-
This mechanism is similar to other reported α-ketoamide inhibitors of thiol hydrolases.
-
Molecular dynamic simulations using the PLA2G16 crystal structure (PDB: 4DOT) have provided insights into the potential ligand-protein interactions that explain binding specificity .
-
The inhibitors demonstrate selectivity for the HRASLS family of thiol hydrolases while sparing other serine hydrolases, as confirmed by ABPP experiments in mouse brain proteomes using broad-spectrum serine hydrolase probes like fluorophosphonate (FP)-TAMRA .
-
The inhibitory effect translates to cellular contexts, where LEI110 (10 μM) effectively blocks PLA2G16-mediated arachidonic acid production in transfected U2OS cells and reduces oleic acid-induced lipid droplet formation in HepG2 cells .
How does PLA2G16 contribute to cancer progression, particularly in pancreatic cancer?
PLA2G16 plays a significant role in cancer progression, particularly in pancreatic adenocarcinoma (PAAD):
-
Upregulation in cancer: PLA2G16 expression is significantly upregulated in PAAD tissue compared to normal pancreatic tissue, as confirmed by transcriptional profiling comparing TCGA-PAAD and GTEx-pancreas datasets .
-
Prognostic significance: Higher PLA2G16 expression is associated with unfavorable survival outcomes in PAAD patients .
-
Functional effects: Inhibition of PLA2G16 suppresses pancreatic cancer cell growth both in vitro and in vivo, indicating its pro-tumorigenic role .
-
Metabolic reprogramming: PLA2G16 promotes aerobic glycolysis in cancer cells, contributing to the Warburg effect that supports rapid cancer cell proliferation .
-
Regulation by oncogenic pathways: PLA2G16 is a transcriptional target of KLF5 (Krüppel-like factor 5), with mutant p53 enhancing the binding of KLF5 to the PLA2G16 promoter. This creates a mutant p53/KLF5-PLA2G16 regulatory axis that drives tumor growth and glycolysis in PAAD .
-
Genetic and epigenetic alterations: DNA copy number amplification and promoter hypomethylation contribute to PLA2G16 upregulation in PAAD. Specifically, PLA2G16 expression shows a strong positive correlation with its copy number (Pearson's r = 0.51, P < 0.001) and a strong negative correlation with the methylation level of cg09518969 (Pearson's r = −0.64, P < 0.001), a CpG site within its gene locus .
What is the role of PLA2G16 in viral infections?
PLA2G16 has been identified as a host factor enabling the cellular entry of picornaviruses . This finding highlights the enzyme's significance beyond its canonical roles in lipid metabolism:
-
PLA2G16 appears to facilitate specific steps in the viral entry process, potentially by modifying membrane phospholipids to create a favorable environment for viral penetration.
-
This function of PLA2G16 suggests that inhibitors like LEI110 might have potential applications not only in metabolic disorders and cancer but also as antiviral agents targeting host factors rather than viral proteins.
-
The dual role of PLA2G16 in both lipid metabolism and viral entry underscores the complex interplay between cellular metabolism and susceptibility to infection.
-
Further research is needed to fully characterize the specific mechanisms by which PLA2G16 facilitates viral entry and to determine whether this function extends to other virus families beyond picornaviruses.
How can gene expression regulation of PLA2G16 be studied in research models?
Studying the regulation of PLA2G16 gene expression involves several complementary approaches:
-
Promoter analysis: The PLA2G16 promoter region can be analyzed using bioinformatic tools like JASPAR to identify potential transcription factor binding sites. For example, potential KLF5 binding sites in the PLA2G16 promoter have been identified by setting the relative profile score threshold to 90% .
-
Luciferase reporter assays: PLA2G16 promoter segments can be cloned into reporter vectors like pGL3 basic vector to assess transcriptional activity. For instance, cells can be transfected with constructs containing different lengths of PLA2G16 promoter fragments along with a normalization vector like pRL-CMV to control for transfection efficiency .
-
Chromatin Immunoprecipitation (ChIP): ChIP-qPCR assays can confirm direct binding of transcription factors to the PLA2G16 promoter, as demonstrated for KLF5 binding .
-
Co-immunoprecipitation: This technique can identify protein-protein interactions between transcription factors and potential co-activators, such as the interaction between mutant p53 and KLF5 .
-
Epigenetic analysis: Methylation status of the PLA2G16 promoter can be assessed, as methylation level (particularly at cg09518969) strongly correlates with PLA2G16 expression (Pearson's r = −0.64, P < 0.001) .
-
Copy number variation analysis: Examining genomic amplifications can provide insights into expression changes, as PLA2G16 expression positively correlates with its copy number (Pearson's r = 0.51, P < 0.001) in PAAD cases .
What experimental models are most suitable for studying PLA2G16 function?
Several experimental models have proven valuable for studying PLA2G16 function:
-
Cell lines:
-
U2OS cells transfected with human PLA2G16 provide a clean system for studying enzyme activity through targeted lipidomics of arachidonic acid production
-
HepG2 cells serve as an in vitro model for studying fatty liver diseases and PLA2G16's role in lipolysis
-
MIA-PaCa-2 and PANC-1 pancreatic cancer cell lines are suitable for studying PLA2G16's role in cancer metabolism
-
-
Tissue models:
-
Animal models:
-
Recombinant protein systems:
Each model offers distinct advantages depending on the specific research question, ranging from mechanistic biochemical studies to physiological and pathological investigations.
What are the current challenges and future directions in PLA2G16 research?
Despite significant advances, several challenges and opportunities remain in PLA2G16 research:
-
Substrate specificity: Many questions regarding the biochemical properties of PLA2G16, including the identification of its genuine in vivo substrates, remain elusive . Further research is needed to comprehensively map the lipid substrates across different tissues and physiological states.
-
Inhibitor specificity: While LEI110 represents a significant advance as a selective pan-inhibitor of the HRASLS family, developing compounds with absolute specificity for PLA2G16 over related family members remains challenging . Structure-guided design could potentially yield more selective inhibitors.
-
Therapeutic applications: The roles of PLA2G16 in cancer metabolism, viral entry, and lipid homeostasis suggest multiple potential therapeutic applications. Developing these applications requires further investigation of tissue-specific functions and potential side effects of inhibition.
-
Structural dynamics: While the crystal structure of PLA2G16 is available (PDB: 4DOT), understanding the dynamic aspects of substrate binding, catalysis, and inhibitor interactions would benefit from advanced techniques like molecular dynamics simulations and cryo-electron microscopy.
-
Translational research: Bridging the gap between basic research findings and clinical applications will require more studies in patient-derived samples and appropriate animal models that recapitulate human pathophysiology.
-
Systems biology approaches: Integrating PLA2G16 into broader lipid metabolism networks and understanding its crosstalk with other metabolic pathways presents opportunities for systems-level insights into its functions.
Future research directions should aim to address these challenges while exploring the therapeutic potential of PLA2G16 modulation in cancer, metabolic disorders, and viral infections.
