Phospholipase D

Resting human neutrophils acylate 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (1-O-alkyl-2-lyso-GPC; Lyso-PAF) specifically with arachidonate (AA); upon stimulation, however, the specificity is lost and other fatty acid residues are added. The major goals of this study were to compare the various acylation reactions present in the cells and to determine the cause of the specificity loss upon stimulation. The CoA-independent transacylase was active in neutrophil homogenates and was found to be both highly specific for AA and stereospecific, requiring 1-O-alkyl-2-lyso-GPC for activity. Homogenates also contained acyl-CoA:1-radyl-2-lyso-sn-glycero-3-phosphocholine acyltransferase activity, which transferred acyl chains from oleoyl-, linoleoyl-, or linolenoyl-CoA to both 1-alkyl and 1-acyl acceptors, but preferred the 1-acyl acceptor when arachidonoyl-CoA was used. The CoA-dependent and -independent activities co-sedimented on a discontinuous Percoll gradient in a single band containing plasma membrane and possibly other membranes. CoA alone promoted nonspecific acylation in the homogenates. The AA-specific acylation was attenuated up to 80% in sonicates of ionophore-stimulated cells, whereas the CoA-dependent acyltransferase remained unchanged. Potential phospholipid AA donors for the transacylase were substantially depleted in the stimulated cells but could not account for the large decrease in acylation. An accumulation of 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine (alkenyl-2-lyso-GPE), which acts as a competing substrate, appeared to be the major cause of the reduced AA-specific acylation of Lyso-PAF observed in the stimulated preparations. Removal of the alkenyl-2-lyso-GPE restored the activity, whereas the addition of alkenyl-2-lyso-GPE (2 microM) to resting membrane preparations resulted in a marked decrease in transacylation of lyso-PAF [1].

---

Mycobacterium tuberculosis (M.tb) infection results in approximately 1.3 million human deaths each year. M.tb resides primarily inside macrophages, and maintains persistent infection. In response to infection and inflammation, platelet activating factor C-16 (PAF C-16), a phospholipid compound, is released by various cells including neutophils and monocytes. We have recently shown that PAF C-16 can directly inhibit the growth of two representative non-pathogenic mycobacteria, Mycobacterium bovis BCG and Mycobacterium smegmatis (M. smegmatis), by damaging the bacterial cell membrane. Here, we have examined the effect of PAF C-16 on M. smegmatis residing within macrophages, and identified mechanisms involved in their growth inhibitory function. Our results demonstrated that exogenous PAF C-16 inhibited the growth of M. smegmatis inside phagocytic cells of monocytic cell line, THP-1; this effect was partially blocked by PAF receptor antagonists, suggesting the involvement of PAF receptor-mediated signaling pathways. Arachidonic acid, a downstream metabolite of PAF C-16 signaling pathway, directly inhibited the growth of M. smegmatis in vitro. Moreover, the inhibition of phospholipase C and phospholipase A2 activities, involved in PAF C-16 signaling pathway, increased survival of intracellular M. smegmatis. Interestingly, we also observed that inhibition of inducible nitric oxide synthase (iNOS) enzyme and antibody-mediated neutralization of TNF-α partially mitigated the intracellular growth inhibitory effect of PAF C-16. Use of a number of PAF C-16 structural analogs, including Lyso-PAF, 2-O-methyl PAF, PAF C-18 and Hexanolamino PAF, revealed that the presence of acetyl group (CH3CO) at sn-2 position of the glycerol backbone of PAF is important for the intracellular growth inhibition activity against M. smegmatis. Taken together, these results suggest that exogenous PAF C-16 treatment inhibits intracellular M. smegmatis growth, at least partially, in a nitric oxide and TNF-α dependent manner[3].

The newly revealed substrate specificity of lysoplasmalogen-specific phospholipase D (lysophospholipase D (LysoPLD)) was exploited. Lp-PLA2 hydrolyzes 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16 PAF) to 1-O-Hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF). LysoPLD acted on LysoPAF, and the hydrolytically released choline was detected by choline oxidase [2].

Intracellular Growth Inhibition Assay for M. smegmatis Using THP-1 Cells [3]
Intracellular bacterial growth inhibition assays were performed to investigate the effect of different test compounds, including PAF C-16 and PAF structure analogs, on the growth of phagocytosed M. smegmatis inside THP-1 cells. THP-1 cells, grown at a density of 0.5–0.75 × 106/ml, were washed twice with plain RPMI medium and adjusted to ∼0.25 × 106 cells/ml in cRPMI without antibiotics. One ml of this cell suspension was added to individual Eppendorf tubes. Approximately 1.25 × 106 M. smegmatis (bacteria to THP1 ratio; 5:1) was then added to each tube. These tubes were incubated at 37°C in a CO2 incubator for 2 h with intermittent shaking to allow phagocytosis of bacteria.

Pan anti-mouse IgG coated magnetic Dynabeads, bound to mouse pan anti-human MHC class I (HLA A, B, and C) antibody W6/32, were prepared to remove non-phagocytosed M. smegmatis. For each sample, 106 Dynabeads were incubated with 1 μg of W6/32 for 90 min on ice to allow the binding of the beads with W6/32 antibody. Later, the beads were washed twice with plain RPMI by applying a Dynal® magnet and this reagent (Dynabeads-W6/32) was resuspended in 25 μl of RPMI. The ‘Dynabeads-W6/32’ were then added to the previously prepared tube, containing THP-1 cells with M. smegmatis (beads to cells ratio; 4:1). The Eppendorf tubes were then placed on ice for 45 min with intermittent shaking to allow the binding of W6/32 component of ‘Dynabeads-W6/32’ with MHC Class I molecules present on THP-1 cells. After incubation, extracellular M. smegmatis was removed by applying a Dynal® magnet and washing the cells twice with plain RPMI. This was followed by resuspending the cells in 1ml cRPMI without antibiotic. For each experiment, a solvent control and test compounds treated samples were included and the tubes were incubated at 37°C in a CO2 incubator for another 24 h. After incubation, Dynal® magnet was applied to the Eppendorf tubes. The ‘Dynabeads-W6/32’attached to THP-1 cells migrated to the side of Eppendorf tube. The supernatant was removed and stored in 15 ml falcon tubes to collect any extracellular bacteria released from dying cells. The THP-1 cells left in the Eppendorf tubes were lysed by adding 1ml of 1% w/v saponin solution in water and vortexing the mixture for 15 min. The cell lysate for each condition was transferred to the respective 15 ml falcons already containing 1 ml of bacterial supernatant that was collected earlier. The contents of each falcon tube were mixed by vortexing for 10 s and serial dilutions (10–1, 10–2, and 10–3) were prepared in sterile PBS.

Finally, the bacterial suspensions, at dilutions of 10–2 and 10–3, were used for plating. 200 μl of bacterial suspension was plated for each experimental condition in triplicates using LB-agar plates. Plates were incubated at 37°C for 72 h, after which the number of bacterial CFUs were counted. A comparison of CFUs between test compound treated plates and the solvent control was done to determine the effect of the test compound on the growth of intracellular M. smegmatis inside THP-1 cells.

---

Assessing the Effects of PAF Receptor Antagonists on PAF C-16 Induced Intracellular M. smegmatis Growth Inhibition [3]
Two PAFR antagonists, ABT-491 and WEB-2086, were used to examine their effects on PAF C-16 induced intracellular M. smegmatis growth inhibition. The assays were carried out according to the intracellular growth inhibition assay as described above, with a difference that M. smegmatis infected THP-1 cells were initially treated with PAFR antagonists (ABT-491 or WEB-2086, 2 μg/ml each) for 1 h before adding PAF C-16 (1 μg/ml) to the cell culture, the samples were incubated further for 24 h at 37°C in a CO2 incubator. Solvent control along with an additional control comprising of M. smegmatis infected THP-1 cells treated with PAFR antagonists only, were also included in the experimental design.

Assessing the Effects of Chemical Inhibitors of PLC, PLA2 and iNOS and Anti-TNF-α Antibody on PAF C-16 Induced Intracellular M. smegmatis Growth Inhibition Assays using inhibitors of PLC (U-73122), cPLA2 (benzenesulfonamide) and iNOS (aminoguanidine hemisulfate) were also performed similar to the intracellular growth inhibition assay. The only difference was in the treatment step where M. smegmatis infected THP-1 cells were first treated with U-73122 (2 μM) (Macmillan and McCarron, 2010), benzenesulfonamide (56 nM) (Farooqui et al., 2006) or aminoguanidine hemisulfate (1 mM) (Nascimento et al., 2002) for 1 h. Subsequently, PAF C-16 (1 μg/ml) was added and the THP-1 cells containing phagocytosed M. smegmatis were further incubated for 24 h at 37°C in a CO2 incubator. An additional control comprising of M. smegmatis infected THP-1 cells treated with U-73122 (2 μM), benzenesulfonamide (56 nM) or aminoguanidine hemisulfate (1 mM), was also included in the respective experimental designs.

Anti-TNF-α neutralizing antibody as used to investigate the role of TNF-α in PAF C-16 induced growth inhibition of intracellular M. smegmatis. M. smegmatis infected THP-1 cells were treated with 10 μg/ml of mouse anti-human TNF-α antibody, an isotype antibody control (10 μg/ml mouse IgG) or ethanol (PAF C-16 solvent) for 1 h prior to treatment with 1 μg/ml PAF C-16, and the cells were further incubated for another 24 h before cell lysis and plating.

---

Direct Growth Inhibition Assay [3]
This assay was carried out as described previously (Riaz et al., 2018), to investigate the direct effect of arachidonic acid on M. smegmatis growth. Briefly, the diluted stock of M. smegmatis (2.5 × 104) in suspensions of 1ml LB broth was exposed to a range of concentrations of arachidonic acid for 2 h at 37°C with mixing every 15 min. Appropriate solvent control (10 μl/ml) for the test compound was included. After incubation, 200 μl of bacterial suspensions from test and control tubes were seeded on LB agar plates in triplicate and the plates were incubated at 37°C for 72 h. Colony counting method was used to detect the direct growth inhibitory effects of the test compounds.

[1]. Enzymatic studies of lyso platelet-activating factor acylation in human neutrophils and changes upon stimulation. J Biol Chem. 1993 Apr 15;268(11):7965-75.
[2]. Novel enzymatic method for assaying Lp-PLA2 in serum. Clin Chim Acta. 2018 Jun;481:184-188.
[3]. Dissecting the Mechanism of Intracellular Mycobacterium smegmatis Growth Inhibition by Platelet Activating Factor C-16. Front Microbiol. 2020 Jun 10:11:1046.

Lysophosphatidylcholine O-16:0/0:0 is a lysophosphatidylcholine O-16:0e in which the hexadecyl group at C-1 contains 16 carbons of which none are unsaturated. It has a role as a metabolite. It is a lysophosphatidylcholine O-16:0 and a 1-alkyl-sn-glycero-3-phosphocholine.

---

Background: Measurement of lipoprotein-associated phospholipase A2 (Lp-PLA2) can be used as an adjunct to traditional cardiovascular risk factors for identifying individuals at higher risk of cardiovascular events. This can be performed by quantification of the protein concentration using an ELISA platform or by measuring Lp-PLA2 activity using platelet-activating factor (PAF) analog as substrate. Here, an enzymatic Lp-PLA2 activity assay method using 1-O-Hexadecyl-2-acetyl-rac-glycero-3-phosphocholine (rac C16 PAF) was developed.
Methods: The newly revealed substrate specificity of lysoplasmalogen-specific phospholipase D (lysophospholipase D (LysoPLD)) was exploited. Lp-PLA2 hydrolyzes 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16 PAF) to 1-O-Hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF). LysoPLD acted on LysoPAF, and the hydrolytically released choline was detected by choline oxidase.
Results: Regression analysis of Lp-PLA2 activity measured by the enzymatic Lp-PLA2 activity assay vs. two chemical Lp-PLA2 activity assays, i.e. LpPLA2 FS and PLAC® test, and ELISA, gave the following correlation coefficients: 0.990, 0.893 and 0.785, respectively (n = 30).
Conclusion: Advantages of this enzymatic Lp-PLA2 activity assay compared with chemical Lp-PLA2 methods include the following; (i) only requires two reagents enabling a simple two-point linear calibration method with one calibrator (ii) no need for inhibitors of esterase-like activity in serum. [2]

C24H52NO6P

481.6548

481.353

C, 59.85; H, 10.88; N, 2.91; O, 19.93; P, 6.43

52691-62-0

Lyso-PAF C-16-d4;201216-37-7

162126

White to off-white solid powder

3.23

1

6

24

32

450

1

CCCCCCCCCCCCCCCCOC[C@H](COP(=O)([O-])OCC[N+](C)(C)C)O

VLBPIWYTPAXCFJ-XMMPIXPASA-N

InChI=1S/C24H52NO6P/c1-5-6-7-8-9-10-11-12-13-14-15-16-17-18-20-29-22-24(26)23-31-32(27,28)30-21-19-25(2,3)4/h24,26H,5-23H2,1-4H3/t24-/m1/s1

3,5,9-Trioxa-4-phosphapentacosan-1-aminium, 4,7-dihydroxy-N,N,N-trimethyl-, inner salt, 4-oxide, (R)-

LPC O-16; 52691-62-0; 1-O-Hexadecyl-sn-glycero-3-phosphocholine; Lyso-PAF C-16; 1-hexadecyl-sn-glycero-3-phosphocholine; 1-O-Hexadecyl-2-lyso-glycero-3-phosphorylcholine; lyso-Platelet-activating factor; 1-O-Palmityl-sn-glycero-3-phosphocholine; FSF9VMH5MK;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL (~103.81 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)