Transfer and Uptake of secreted Lysosomal Acid Lipase

Abstract

Lysosomal acid lipase (LAL) is essential for intracellular lipid metabolism, catalyzing the hydrolysis of triacylglycerols and cholesteryl esters into free fatty acids and free cholesterol. A genetic deficiency in LAL causes lysosomal lipid accumulation, clinically defined as lysosomal acid lipase deficiency (LAL-D), which ranges from severe Wolman disease to the milder cholesteryl ester storage disease. Here, the aim was to investigate the transfer and cellular uptake of recombinant LAL in vitro, focusing on the macrophage cell line RAW264.7 and primary bone marrow-derived macrophages (BMDM). The study evaluated the potential of experimental enzyme replacement therapy using recombinant LAL proteins, assessing their enzymatic activity and ability to normalize lipid metabolism. I optimized and validated a fluorescence-based activity assay utilizing 4-methylumbelliferyl-palmitate as a substrate for cells. Different transfection methods, cell lines, and protein preparations were evaluated to ensure optimal uptake and enzymatic activity of recombinant LAL. Furthermore, I analyzed intracellular localization by immunofluorescence, as well as quantitative lipid profiling upon functional enzyme uptake using thin-layer chromatography. Results demonstrated efficient uptake and lysosomal localization of recombinant LAL in macrophages, where the enzyme retained functional activity. Significant reduction in lipid accumulation was observed upon treating BMDM with recombinant LAL, validating the therapeutic potential of this approach, including the macrophages most severely affected in LAL-D. In conclusion, this study provides essential insights into the cellular uptake, activity, and therapeutic efficacy of recombinant LAL in cell models of LAL-D. These findings contribute to the understanding of the lysosomal enzyme transfer and uptake and present a base for the development of improved therapeutic strategies for LAL-D patients.

Datum der Bewilligung	2026
Originalsprache	Englisch
Gradverleihende Hochschule	Medizinische Universität Graz
Betreuer/-in	Dagmar Kratky (Betreuer*in) & Laszlo Schooltink (Mitbetreuer*in)