Lysosomal Acid Lipase (LAL) is one of the key enzymes involved in the catabolism of triglycerides and cholesteryl esters entering the cells during the receptor mediated uptake of lipoprotein particles. The deficiency of LAL activity leads to variable levels of accumulation of cholesteryl esters and triglycerides in various tissues of the body leading to pathological conditions such as Wolman's Disease (WD) and Cholesteryl Ester Storage Disease (CESD). Unlike most of the well characterised mammalian lipases such as pancreatic, hepatic and lipoprotein lipases, LAL is capable of hydrolysing both triglycerides and cholesteryl esters. Moreover, primary amino acid sequence of LAL shows negligible homology with the members of lipase super family. The present study was designed to investigate structure-function relationships of LAL by analysing a variety of natural and site-directed mutants. The molecular basis of Cholesteryl Ester Storage Disease (CESD) has been characterised in three Italian patients and these patients were found to be compound heterozygotes for mutations in the LAL gene. Altogether, three different missense mutations (P181L, G66V, L273S) and two splicing defects resulting in exon skipping due to mutations at the 3' or 5' splice site consensus (Delta205-253, Delta254-277 respectively) were detected. The effect of these mutations on the triglyceride and cholesteryl ester hydrolase activities of the enzyme has been analysed by in vitro expression of the mutant alleles in HeLa cells and the results confirmed that these mutations were detrimental for both catalytic activities. Moreover, some of these mutations altered the glycosylation pattern of the enzyme. In this context, glycosylation pattern of the recombinant LAL protein expressed in HeLa cells was studied by endoglycosidase H treatment followed by immunoblotting. The effect of deglycosylation on the catalytic activities of LAL has also been verified. These studies suggested that N-linked glycosylation is not essential for the catalytic activity of the enzyme but could be necessary for optimal rate of catalysis. For further investigation of the functional aspects of LAL catalysis, site directed mutagenesis studies were performed in order to identify amino acid residues forming part of the putative Ser- Asp-His type of catalytic triad of LAL. Convincing evidence is provided for the importance of Ser^153, His^274 and D^130 residues for both tri acyl glycerol lipase and cholesteryl ester hydrolase activities of LAL. Site directed mutagenesis of the residues in the vicinity of H274 showed that this region of the protein is of critical importance for optimal catalytic activity.
|Autori:||Pariyarathuparambil, Rajalakshmi G.|
|Titolo:||Human Lysosomal Acid Lipase: Functional Characterisation by Molecular Genetic Analysis and Site-Directed Mutagenesis Studies|
|Relatore/i esterni:||Baralle, Francisco Ernesto|
|Data di pubblicazione:||4-nov-1996|
|Appare nelle tipologie:||8.1 PhD thesis|