ELUCIDATING THE STRUCTURE AND METABOLISM OF AN ENDOSOME-SPECIFIC LIPID, BIS(MONOACYLGLYCERO)PHOSPHATE
| dc.contributor.author | HUI HUI, TAN | |
| dc.date.accessioned | 2016-01-12T03:42:00Z | |
| dc.date.available | 2016-01-12T03:42:00Z | |
| dc.date.issued | 2014-09 | |
| dc.description.abstract | Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid (LBPA) is a unique lipid enriched in the inner membranes of late endosomes. The involvement of BMP in both structural and functional aspects of late endosomes particularly in the trafficking of lipids and proteins has been reported in numerous studies. To date, no direct evidence for the stereochemical configuration of natural BMP has been reported despite its important role in cholesterol homeostasis and lysosomal storage diseases. This study outlined a novel chemical approach to determine the stereochemical configuration of the diglycerophosphate (DGP) backbone of BMP by NMR spectroscopy. First, enantiopure synthetic BMP analogues, namely sn-1-glycerophospho-sn-1’-glycerol, sn-3-glycerophospho-sn-3’-glycerol and sn-1-glycerophospho-sn-3’-glycerol backbones, were synthesized as reference materials utilizing H-phosphonate chemistry. Enantiomeric discrimination of the DGP backbones was achieved by introduction of D-camphor ketal as chemical shift reagent. Direct comparison of the 1H-NMR spectra revealed that natural BMP exclusively exhibits the unusual sn-1-glycerophospho-sn-1’-glycerol backbone. Moreover, fatty acids esterification position in native BMP was reported to be 2,2'-BMP or 3,3'-BMP in previous studies. These disputative reports are yet to be resolved and re-evaluation of acylation pattern in BMP is necessary since it can influence the biophysical properties of BMP and thus, its biological functions. In this study, a feasible and efficient method using liquid chromatography-tandem mass xxii spectrometry (LC-MS/MS) was developed to investigate acyl migration kinetic of DOBMP, as well as to verify the acylation pattern of native BMP in culture cells. The acyl migration rates of DOBMP in water-saturated methanol and buffered liposomal system were determined in this study. The knowledge of acyl migration rates of DOBMP under various conditions is valuable for biochemical and biophysical studies on BMP. Concomitantly, the MS chromatograms revealed that the fatty acyl position of native BMP is exclusively 2,2'-BMP in cultured cells. After determination of the complete structure of BMP, the focus was shifted towards the elucidation of BMP metabolism. To elucidate the biosynthetic pathway of BMP in RAW cell, novel phosphatidylglycerol analogues were designed and chemically synthesized to serve as exogenous BMP precursors. Detailed synthetic approaches and strategies using H-phosphonate chemistry were illustrated in this study. The synthetic PG analogues (NPG) were less susceptible to phospholipase A2 (PLA2) hydrolysis and tagged with a stable isotope labelled oleic acid (dO) at the sn-2 position to facilitate the study via LC-MS/MS analysis. Supplementation with OdO-NPG reduced BMP content while PdO-NPG and SdO-NPG induced BMP accumulation with an enhanced proportion of palmitate and stearate species respectively. The development of novel and specific antagonists in BMP biosynthetic pathway will accelerate the clarification of the involved mechanism, enabling a better understanding of BMP implication in pathology. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/1440 | |
| dc.subject | ELUCIDATING THE STRUCTURE AND METABOLISM | en_US |
| dc.title | ELUCIDATING THE STRUCTURE AND METABOLISM OF AN ENDOSOME-SPECIFIC LIPID, BIS(MONOACYLGLYCERO)PHOSPHATE | en_US |
| dc.type | Thesis | en_US |
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