首页> 外文期刊>Biochimica et Biophysica Acta. Molecular and cell biology of Lipids >Difference in substrate specificity between human and mouse lysosomal acid lipase: low affinity for cholesteryl ester in mouse lysosomal acid lipase
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Difference in substrate specificity between human and mouse lysosomal acid lipase: low affinity for cholesteryl ester in mouse lysosomal acid lipase

机译:人类和小鼠溶酶体酸性脂肪酶之间底物特异性的差异:对小鼠溶酶体酸性脂肪酶中胆固醇酯的亲和力低

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Lysosomal acid lipase (LAL) is essential for the intracellular degradation of cholesteryl esters (CE) and triacylglycerols (TG) that are delivered to lysosomes by low density lipoprotein (LDL) receptor mediated endocytosis. We have analysed the difference in the catalytic properties and substrate specificity of human and mouse LALs. LAL activities were measured in human and mouse fibroblasts and in HeLa cells transiently expressing wild-type or site-directed mutant LALs of the two species using the T7 vaccinia system. Cholesteryl esterase and triacylglycerol lipase activities were determined in cellular homogenates with a phospholipid/detergent vesicle assay, an assay frequently used to diagnose human LAL deficiency syndromes, and with LDL particles, a more physiological substrate. Characterisation of human and mouse LAL using these two assays demonstrated marked differences in their TG and CE hydrolysing activities. Compared to human LAL mouse LAL showed a much lower cholesteryl esterase activity in both assays used. The differences was more pronounced in the vesicle assay. The lower cholesteryl esterase activity of mouse LAL did not affect the LDL-CE degradation in intact fibroblasts. The analysis of site-directed mutants suggests a role of the non-conserved cysteine residue to position 240 in cholesteryl esterase activity in human LAL. Our results show a significant difference between human and mouse LAL in their specificity toward cholesteryl esters. The low cholesteryl esterase activity does not result in reduced LDL-cholesterol ester degradation in mouse fibroblasts in situ. In addition, this work emphasises the importance of the physical state of substrates in studies of the specificity and properties of lipolytic enzymes.
机译:溶酶体酸性脂肪酶(LAL)对于胆固醇酯(CE)和三酰基甘油(TG)的细胞内降解至关重要,这些胆固醇酯通过低密度脂蛋白(LDL)受体介导的内吞作用传递至溶酶体。我们已经分析了人类和小鼠LAL的催化特性和底物特异性的差异。使用T7痘苗系统,在人和小鼠的成纤维细胞和瞬时表达两种物种的野生型或定点突变LAL的HeLa细胞中测量LAL活性。使用磷脂/洗涤剂囊泡测定法(一种常用于诊断人LAL缺乏综合症的测定法)和LDL颗粒(一种更具生理性的底物)测定细胞匀浆中的胆固醇酯酶和三酰基甘油脂酶活性。使用这两种测定法对人和小鼠LAL的表征证明其TG和CE水解活性存在明显差异。与人LAL相比,小鼠LAL在使用的两种测定中均显示出低得多的胆固醇酯酶活性。在囊泡测定中差异更加明显。小鼠LAL较低的胆固醇酯酶活性不影响完整成纤维细胞中LDL-CE的降解。定点突变体的分析表明非保守的半胱氨酸残基在人LAL的胆固醇酯酶活性中位于240位的作用。我们的结果表明,人和小鼠LAL对胆固醇酯的特异性存在显着差异。胆固醇酯酶的低活性不会降低小鼠成纤维细胞中LDL-胆固醇酯的降解。此外,这项工作强调了底物物理状态在脂解酶特异性和特性研究中的重要性。

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