PLA2G4A protein, human

The caspase-1 activity was determined with the fluorometric substrate Z-YVAD 7-Amido-4-trifluoromethylcoumarin (Z-YVAD-AFC, caspase-1 substrate VI, Calbiochem) as described previously20 (link)28 (link). In brief, 25–35 larvae were lysed in hypotonic cell lysis buffer (25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, 5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, 5 mM dithiothreitol, 1:20 protease inhibitor cocktail (Sigma-Aldrich), pH 7.5) on ice for 10 min. For each reaction, 10 μg protein were incubated for 90 min at 23 °C with 50 μM YVAD-AFC and 50 μl of reaction buffer (0.2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.2 M 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, 20% sucrose, 29 mM dithiothreitol, pH 7.5). After the incubation, the fluorescence of the AFC released from the Z-YVAD-AFC substrate was measured with a FLUOstart spectofluorometer (BGM, LabTechnologies) at an excitation wavelength of 405 nm and an emission wavelength of 492 nm (Fig. 2b). A representative caspase-1 activity assay out of three is shown accompanying each survival assay.
Pla2 activity was determined in 50 μg protein extracts obtained from whole-zebrafish larvae using the EnzChek Phospholipase A2 Assay Kit (ThermoFisher Scientific) following the manufacturer's recommendations. The data were normalized using extracts treated with 0.5 μM ACA.

Lipids used were PC 16:0/18:0 and PG 16:0/18:1 PG at 10 mg/mL in chloroform (Avanti Polar Lipids). All experiments were performed protected from light. On day one, PC was either used alone or mixed with PG in a 90/10 ratio for a final volume of 50 μL. The resulting lipid mixes were vortexed for 10 s and centrifuged for 1 min at 16,100 × g. Lipids were then dried using a SpeedVac vacuum concentrator. The resulting dried lipids were resuspended in 100 μL of buffer A (HEPES 30 mM, KCl 200 mM, pH 7.4) or buffer B (MES 30 mM, KCl 200 mM, pH 5.5) and sonicated in a water bath sonicator for 10 min to help with the formation of multilamellar vesicles/liposomes (crude liposomes). The resulting solutions were stored overnight at 4 °C. The next day, liposomes were sonicated for 10 min and placed on ice. They were then diluted 1/100 in buffer A or B to prepare the working solution. For t0 conditions, lipids were extracted right away. For t60 conditions, lipids were extracted after incubation for 1 h at 37 °C with shaking. For t60 + LPLA2 conditions, lipids were extracted after incubation for 1 h at 37 °C with shaking in the presence of recombinant active human lysosomal phospholipase A2 (Echelon Biosciences, with a LPLA2: PG molar ratio of 1/10). All buffers and tubes used for lipid extraction were pre-chilled at 4 °C. Lipids were extracted from each initial 50 μL reaction volume using 900 μL chloroform:methanol (5:4). The resulting solution was vortexed for 10 sec and supplemented with 200 μL KCl 1 M. The resulting solution was vortexed for 30 s and incubated on ice for 1 min. This step was repeated twice. The solution was then centrifuged for 2 min at 16,100 × g. The organic layer was then transferred to a new tube, dried using a SpeedVac vacuum concentrator and stored at −80 °C.

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme phospholipase A2 from a genetically modified S. violaceoruber (strain AS‐10). The dossier was updated on 14 April 2016.
Additional information was requested from the applicant during the assessment process on 19 May 2020 and was consequently provided (see ‘Documentation provided to EFSA’).
Following the reception of additional data by EFSA on 7 December 2020, EFSA requested a clarification teleconference on 17 March 2021, after which the applicant provided additional data on 27 September 2021.

Phospholipase A2 Activity Assay Kit (Fluorometric) was purchased from BioVision. To make tissue lysate for PLA2 activity measurement, around 10 mg of mouse jejunum was lysed on ice using sonicator probe for 30 seconds in 200 ul of Tris-HCl buffer (50 mM, pH 7.5) containing Pierce protease and phosphatase inhibitors (Thermo Fisher Scientific), followed by centrifugation at 10,000 x g for 10 min at 4 °C. The supernatant was transferred to a new Eppendorf tube. Samples were diluted to 0.2 mg/ml based on protein concentrations determined using BCA assay. 5 ul of each sample was used for PLA2 activity measurement following the manufacturer’s instructions. To measure the activity of low molecular weight secretory PLA2, tissue lysate were filtered using 30 kDa MWCO spin columns to remove any high molecular PLA2.