Difference in substrate specificity between human and mouse lysosomal acidlipase: low affinity for cholesteryl ester in mouse lysosomal acid lipase

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. W e have analysed the difference in the catalytic properties and substrate sp ecificity of human and mouse LALs. LAL activities were measured in human an d mouse fibroblasts and in HeLa cells transiently expressing wild-type or s ite-directed mutant LALs of the two species using the T7 vaccinia system. C holesteryl esterase and triacylglycerol lipase activities were determined i n cellular homogenates with a phospholipid/detergent vesicle assay, an assa y frequently used to diagnose human LAL deficiency syndromes, and with LDL particles, a more physiological substrate. Characterisation of human and mo use LAL using these two assays demonstrated marked differences in their TG and CE hydrolysing activities. Compared to human LAL mouse LAL showed a muc h lower cholesteryl esterase activity in both assays used. The difference w as more pronounced in the vesicle assay. The lower cholesteryl esterase act ivity of mouse LAL did not affect the LDL-CE degradation in intact fibrobla sts. The analysis of site-directed mutants suggests a role of the non-conse rved cysteine residue at position 240 in cholesteryl esterase activity in h uman LAL. Our results show a significant difference between human and mouse LAL in their specificity toward cholesteryl esters. The low cholesteryl es terase activity does not result in reduced LDL-cholesterol ester degradatio n in mouse fibroblasts in situ. In addition, this work emphasises the impor tance of the physical state of substrates in studies of the specificity and properties of lipolytic enzymes. (C) 2000 Elsevier Science B.V. All rights reserved.