Ayyash Mariam, Algahmi Amal, Gillespie John, Oelkers Peter
University of Michigan - Dearborn, Department of Natural Sciences, 4901 Evergreen Rd., Dearborn, MI 48128, USA.
University of Michigan - Dearborn, Department of Mathematics, 4901 Evergreen Rd., Dearborn, MI 48128, USA.
Biochim Biophys Acta. 2014 Apr 4;1841(4):505-13. doi: 10.1016/j.bbalip.2013.12.015. Epub 2014 Jan 7.
Phospholipid remodeling involves phospholipase activity to remove acyl chains and acyltransferases to replace acyl chains. We here describe the characterization of a lysophospholipid acyltransferase in the opportunistic fungal pathogen, Candida albicans. Expression of this gene, C.a. LPT1, complemented the lysophospholipid acyltransferase defect in Saccharomyces cerevisiae strains lacking the homologous LPT1 gene. In vitro, lysophospholipid acyltransferase activity in these strains showed acyl-CoA substrate specificity, as measured by apparent Vmax/Km ratios, to be linolenoyl-CoA>oleoyl-CoA>linoleoyl-CoA>stearoyl-CoA. To address the physiological importance of C.a. LPT1, homozygous deletion strains were generated. Lysophospholipid acyltransferase activity with amine containing lysophospholipids was dramatically reduced while lysophosphatidylinositol and lysophosphatidic acid esterification was not significantly lowered. However, C.a. LPT1 over-expression yielded an increased amount of lysophosphatidic acyltransferase activity, suggesting a role in de novo phospholipid synthesis. LPT1 deletion strains showed slightly slowed growth in standard liquid media but no phenotype in media containing three antifungals that target sterols. To assess the role of C.a. Lpt1 in phospholipid remodeling, an in vivo, pulse-chase assay utilizing polysorbitan palmitate and mass spectrometry was developed. Cellular phospholipid composition became atypical with the provision of palmitate and gradually returned to the typical distribution when palmitate was removed. Deletion of C.a. LPT1 showed a modest yet significant effect on remodeling under these conditions.
磷脂重塑涉及磷脂酶活性以去除酰基链以及酰基转移酶以取代酰基链。我们在此描述了机会性真菌病原体白色念珠菌中一种溶血磷脂酰基转移酶的特性。该基因（白色念珠菌LPT1）的表达弥补了酿酒酵母中缺乏同源LPT1基因的菌株的溶血磷脂酰基转移酶缺陷。在体外，通过表观Vmax/Km比值测定，这些菌株中的溶血磷脂酰基转移酶活性显示出对酰基辅酶A底物的特异性，即亚麻酸辅酶A>油酰辅酶A>亚油酸辅酶A>硬脂酰辅酶A。为了探究白色念珠菌LPT1的生理重要性，构建了纯合缺失菌株。含胺溶血磷脂的溶血磷脂酰基转移酶活性显著降低，而溶血磷脂酰肌醇和溶血磷脂酸的酯化作用并未显著降低。然而，白色念珠菌LPT1的过表达导致溶血磷脂酰基转移酶活性增加，表明其在磷脂从头合成中发挥作用。LPT1缺失菌株在标准液体培养基中的生长略有减缓，但在含有三种靶向甾醇的抗真菌药物的培养基中没有表型。为了评估白色念珠菌Lpt1在磷脂重塑中的作用，开发了一种利用聚山梨醇酯棕榈酸酯和质谱的体内脉冲追踪试验。当提供棕榈酸时，细胞磷脂组成变得不典型，而当去除棕榈酸时，逐渐恢复到典型分布。在这些条件下，白色念珠菌LPT1的缺失对重塑有适度但显著的影响。
