A fluorogenic phospholipid for the detection of lysosomal phospholipase A2 activity

Abstract

Lysosomal phospholipase A2 (group XV PLA2, LPLA2) is a lysosomal enzyme linked to drug induced phospholipidosis. We developed phospholipid “smart probes” based on the conversion of a quenched fluorogenic substrate to a fluorescent product. Due to the preference of LPLA2 for phosphatidylglycerol, three fluorogenic phosphatidylglycerols were synthesized. Two fluorogenic phosphatidylglycerols were conjugated with one fluorescein amidite (FAM) group and one 4-(4-dimethylaminophenylazo)-benzoyl (DABCYL) group; the third substrate consisted of two FAM groups conjugated at the sn-1 and sn-2 positions. The sn-1 ester linkage was replaced with an amide linkage. 1-FAM-2-DABCYL-PG was degraded by recombinant LPLA2 and mouse serum but not by the serum obtained from LPLA2-deficient mice when 1,2-dioleoyl-PG/1-FAM-2-DABCYL-PG liposomes were used. The formation of 1-FAM-lyso-PG generated from 1-FAM-2-DABCYL-PG in the presence of LPLA2 was quantitatively determined by fluorescent measurements. The 1-FAM-2-DABCYL-PG incorporated into 1,2-dioleoyl-phosphpatidylcholine/sulfatide liposomes was used to evaluate the effect of the cationic amphiphilic drugs amiodarone and fluoxetine on LPLA2 activity. The IC50s of amiodarone and fluoxetine estimated by fluorescent measurement were 10 and 19 µM. These results indicate that 1-FAM-2-DABCYL-PG is a specific substrate for LPLA2 and a useful reagent for the detection of LPLA2 activity from multiple sources.

Comparative analysis of lipotoxicity induced by endocrine, pharmacological, and innate immune stimuli in rat basophilic leukemia cells

“…The expanded LB population in these cells can be visualized by both Oil Red O and LipidTox Green (Figure 1A). When these insulin-treated cells were stained with LipidTox Red Phospholipidosis Detection Reagent (Muehlbacher et al, 2012; Abe et al, 2013), it was seen that phospholipidosis was not significantly induced as compared to the effect of a positive control agent, chloroquine (Figures 1B and 1C). Chloroquine also induced LB accumulation.…”

A turn-on fluorescent probe for imaging lysosomal hydrogen sulfide in living cells

“…In recent years, some uorescent probes have been reported to stain lysosomes [8][9][10][11][12][13] or image lysosomal pH, [14][15][16] Ca 2+ , 17 Zn 2+ , [18][19][20] Cu 2+ , 21,22 NO, 23 H 2 O 2 , 24 legumain, 25 viscosity, 26 and phospholipase A2 activity. 27 Hydrogen sulde (H 2 S), a well known pungent gas, is generated endogenously in mammalian tissues from the amino acids cysteine and homocysteine by three enzymes including cystathionine-b-lyase (CSE), cystathionine-g-synthetase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). 28 Nowadays, H 2 S has been considered as a crucial signal molecule in nervous system, cardiovascular system, and inammatory system.…”