双(单酰基甘油)磷酸盐(BMP)是位于晚期内体和溶酶体的酸性甘油磷脂。然而,对BMP的代谢知之甚少。由于许多引起磷脂症的药物抑制溶酶体磷脂酶A2(LPLA2,PLA2G15,LYPLA3)的活性,我们研究了该酶是否在BMP分解代谢中起作用。重组人LPLA2(hLPLA2)和含有天然存在的BMP(sn-(2-油酰基-3-羟基)-甘油-1-磷酸-sn-1\'-(2\'-油酰基-3\'-羟基)-甘油(S,S-(2,2\',C18:1)-BMP)导致该BMP异构体的脱酰化。脱酰速率比二油酰磷脂酰甘油(DOPG)低70倍,BMP的异构体和前体。LPLA2从DOPG和四种BMP立体异构体中释放油酸的速率不同。水解速率的等级顺序为DOPG>S,S-(3,3\',C18:1)-BMP>R,S-(3,1\',C18:1)-BMP>R,R-(1,1\',C18:1)>S,S-(2,2\')-BMP。阳离子两亲性药物胺碘酮(AMD)以浓度依赖性方式抑制hLPLA2对DOPG和BMP异构体的脱酰化。在这些实验条件下,胺碘酮诱导的四种BMP异构体和DOPG的IC50小于20μM和约30μM,分别。在AMD处理的RAW264.7细胞中观察到BMP积累。通过用活性重组hLPLA2而不是用二异丙基氟磷酸盐灭活的重组hLPLA2外源处理细胞,积累的BMP显著减少。最后,筛选了一系列已知引起磷脂症的阳离子两亲性药物对LPLA2活性的抑制,如通过BMP或磷脂酰胆碱作为底物的转酰化或脂肪酸水解所测量的。15种化合物表现出显著的抑制作用,IC50为6.8至63.3μM。这些结果表明,LPLA2在酸性条件下降解具有不同底物特异性的BMP异构体,并且可能是与药物诱导的磷脂症中BMP积累相关的关键酶。
Bis(monoacylglycerol)phosphate (BMP) is an acidic glycerophospholipid localized to late endosomes and lysosomes. However, the metabolism of BMP is poorly understood. Because many drugs that cause phospholipidosis inhibit lysosomal phospholipase A2 (LPLA2, PLA2G15, LYPLA3) activity, we investigated whether this enzyme has a role in BMPcatabolism. The incubation of recombinant human LPLA2 (hLPLA2) and liposomes containing the naturally occurring BMP (sn-(2-oleoyl-3-hydroxy)-glycerol-1-phospho-sn-1\'-(2\'-oleoyl-3\'-hydroxy)-glycerol (S,S-(2,2\',C18:1)-BMP) resulted in the deacylation of this BMP isomer. The deacylation rate was 70 times lower than that of dioleoyl phosphatidylglycerol (DOPG), an isomer and precursor of BMP. The release rates of oleic acid from DOPG and four BMP stereoisomers by LPLA2 differed. The rank order of the rates of hydrolysis were DOPG>S,S-(3,3\',C18:1)-BMP>R,S-(3,1\',C18:1)-BMP>R,R-(1,1\',C18:1)>S,S-(2,2\')-BMP. The cationic amphiphilic drug amiodarone (AMD) inhibited the deacylation of DOPG and BMP isomers by hLPLA2 in a concentration-dependent manner. Under these experimental conditions, the IC50s of amiodarone-induced inhibition of the four BMP isomers and DOPG were less than 20 μM and approximately 30 μM, respectively. BMP accumulation was observed in AMD-treated RAW 264.7 cells. The accumulated BMP was significantly reduced by exogenous treatment of cells with active recombinant hLPLA2 but not with diisopropylfluorophosphate-inactivated recombinant hLPLA2. Finally, a series of cationic amphiphilic drugs known to cause phospholipidosis were screened for inhibition of LPLA2 activity as measured by either the transacylation or fatty acid hydrolysis of BMP or phosphatidylcholine as substrates. Fifteen compounds demonstrated significant inhibition with IC50s ranging from 6.8 to 63.3 μM. These results indicate that LPLA2 degrades BMP isomers with different substrate specificities under acidic conditions and may be the key enzyme associated with BMP accumulation in drug-induced phospholipidosis.