RESUMEN
Bis(monoacylglycero)phosphate (monoacyl-sn-glycero-1-phospho-1'-monoacyl-sn-glycerol) is a unique lipid that represents greater than 15% of the total phospholipid of the resident alveolar macrophage. Bis(monoacylglycero)phosphate is not synthesized de novo but rather is derived from phosphatidylglycerol of the lung surfactant. There are two enantiomers of bis(monoacylglycero)phosphate synthesized, but only the sn-1, and not the sn-3, enantiomer accumulates in vivo. We recently published a scheme in which the sn-3 enantiomer was an intermediate in the synthesis of the final sn-1 bis(monoacylglycero)-phosphate. Here we further expand the understanding of the biosynthesis of bis(monoacylglycero)phosphate by examining the proposed first two steps of the pathway. A phospholipase A and a transacylase activity are partially separated by gel permeation chromatography. Both are optimally active in the acid pH range that supports the concept that they function in the lysosome-endosome compartment of the cell. Independently, these two enzyme systems are incapable of converting phosphatidylglycerol into sn-3 bis(monoacylglycero)phosphate. However, combination of the two partially purified enzymes reestablishes the synthesis of sn-3 bis(monoacylglycero)phosphate from phosphatidylglycerol. The results presented here support our hypothesis that the phospholipase and transacylase are separate enzymes essential to the synthesis of bis(monoacylglycero)phosphate.
Asunto(s)
Aciltransferasas/metabolismo , Lisofosfolípidos/biosíntesis , Fosfolipasas A/metabolismo , Aciltransferasas/aislamiento & purificación , Animales , Línea Celular , Cromatografía en Gel , Concentración de Iones de Hidrógeno , Cinética , Lisofosfolípidos/química , Macrófagos Alveolares/metabolismo , Ratones , Monoglicéridos , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Fosfolipasas A/aislamiento & purificación , Especificidad por SustratoRESUMEN
1,2-Dioleoyl-sn-[3-3H]glycero-3-phospho(1-rac-glycerol) was synthesized from 1,2-dioleoyl-sn-glycerol using a new radiosynthetic procedure. 1,2-Dioleoyl-sn-glycerol was oxidized to the corresponding aldehyde using pyridinium dichromate and pyridine. The aldehyde was reduced to the radiolabeled alcohol using tritiated sodium borohydride and crown ether. This material was then converted to the phosphocholine derivative using 2-chloro-2-oxo-1,3,2-dioxaphospholane, followed by displacement with trimethylamine. In the last step, the 1,2-dioleoyl-sn-[3-3H]glycero-3-phosphocholine was converted to 1,2-dioleoyl-sn-[3-3H]glycero-3-phospho-(1-rac-glycerol) via a classic transphosphatidylation reaction using glycerol and cabbage phospholipase D. A theoretical explanation of unusual chemical behavior of the primary alcohol of diglycerides is also given, based on semi-empirical calculations.
Asunto(s)
Diglicéridos/química , Fosfatidilgliceroles/síntesis química , Glicerol/química , Oxidación-Reducción , Fosfolipasa D/metabolismo , EstereoisomerismoRESUMEN
Bis(monoacylglycerol) phosphate has a unique stereoconfiguration of sn-glycero-1-phospho-1'-sn-glycerol and is synthesized from exogenous phosphatidylglycerol by macrophages. Previous work by our laboratory showed that the macrophage-like cell line RAW 264.7 synthesizes sn-glycero-1-phospho-1'-sn-glycerol bis(monoacylglycerol) phosphate. Here we describe studies using RAW 264.7 cells that examine the biosynthetic pathway by which bis(monoacylglycerol) phosphate is formed. Experiments were conducted using precursors that were specifically radiolabeled on the glycerol backbone in order to examine the stereoconfiguration of the intermediates and products formed in intact RAW 264.7 cells. The results of our studies indicate that a complex series of reactions are involved in the synthesis of bis(monoacylglycerol) phosphate. In this proposed pathway phosphatidylglycerol is hydrolyzed to form 1-acyllysophosphatidylglycerol which is then acylated on the headgroup glycerol to form the sn-glycero-1-phospho-1'-sn-glycerol enantiomer of bis(monoacylglycerol) phosphate. The sn-glycero-1-phospho-1'-sn-glycerol enantiomer of bis(monoacylglycerol) phosphate is then thought to undergo a stereoconversion that proceeds via the required removal of the acyl group at the sn-1 position. The resulting sn-glycero-1-phospho-1'-sn-glycerol enantiomer of lysophosphatidylglycerol with the acyl moiety on the original headgroup glycerol is then acylated to form sn-glycero-1-phospho-1'-sn-glycerol bis(monoacylglycerol) phosphate.