Biomedical subjects

S Chahrokh-Zadeh

Publications and source records attributed to S Chahrokh-Zadeh.

4 recordsLinked to original sources

PubMed1997

The acid lipase gene family: three enzymes, one highly conserved gene structure.

Human gastric lipase (HGL; triacylglycerol lipase; EC 3.1.1.3) plays an important role in the digestion of dietary triglycerides in the gastrointestinal tract, especially in patients suffering from pancreatic lipase deficiencies. The enzyme is secreted by the fundic mucosa of the stomach and hydrolyzes the ester bonds of triglycerides under acidic pH conditions, while cholesteryl esters are not attacked. The 379-amino acid protein is highly homologous to two other acidic lipases, rat lingual lipase (RLL; triacylglycerol lipase; EC 3.1.1.3) and human lysosomal acid lipase (HLAL; cholesteryl esterase; EC 3.1.1.13). To determine whether this remarkable similarity is also present at the genomic level, we have elucidated the respective gene structures by screening three bacteriophage lambda libraries and by polymerase chain reaction-based intron amplification. The genes encoding HGL, RLL, and HLAL are composed of 10 exons interrupted by nine introns and span about 14 kb, 18.7 kb, and 38.8 kb of genomic DNA, respectively. The HGL and RLL gene organizations are identical, suggesting that RLL is the rat gastric lipase expressed in the serous von Ebner glands of the tongue. The positions of the HLAL intervening sequences are also absolutely conserved, except for the location of intron 1. Our results support the concept that HLAL and HGL/RLL are members of a gene family of lipases that most likely have evolved by duplication of an ancestral gene and subsequently assumed distinct roles in neutral lipid metabolism due to sequence divergence and different expression patterns.

Animals↗

PubMed1997

Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: identification of the catalytically active serine, aspartic acid, and histidine residues.

Human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) constitute a family of mammalian lipases characterized by an acidic pH optimum. HGL and RLL are secreted by the chief cells of the stomach and by the serous von Ebner's glands of the tongue, respectively, and hydrolyze dietary longchain triglycerides in the gastrointestinal tract. HLAL, in contrast, catalyzes the intralysosomal degradation of both triglycerides and cholesteryl esters in virtually all cells except erythrocytes. All three enzymes are proposed to be serine esterases with a catalytic Ser-Asp-His triad similar to other lipases, despite their sensitivity towards sulfhydryl modifying reagents. To investigate the role of conserved serine, aspartic acid, and histidine residues in HLAL and HGL, we constructed 24 mutant lipases with single amino acid substitutions using the site-directed mutagenesis approach. Our combined data strongly support the conclusion that Ser153, Asp324, and His355 are components of the catalytic triad of HLAL and HGL. Structural integrity of the conserved His-Gly dipeptide of lipases also appears to be important for neutral lipid hydrolysis, as replacement of His65 by glutamine abolished HLAL and HGL enzymic activity. Substitution of HLAL residues Asp93, Asp130, and Asp328 with glycine, in contrast, had a more pronounced impact on cholesteryl oleate hydrolysis than on triglyceride hydrolysis. These results provide new insights into the structural basis of HLAL and HGL function.

Animals↗

PubMed1997

Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: site-directed mutagenesis of Cys227 and Cys236 results in substrate-dependent reduction of enzymatic activity.

Chemical modification studies and site-directed mutagenesis experiments have provided evidence that human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) are serine esterases. Loss of HLAL and HGL activity was also observed in the presence of sulfhydryl-reactive substances, suggesting that cysteines are likewise essential for substrate hydrolysis. To study the functional role of the HLAL and HGL cysteine residues, we replaced these amino acids with alanine by site-directed mutagenesis. Substitutions at positions 227 and 236, alone or together, drastically reduced hydrolytic activity in a substrate-dependent manner while the other mutants were not affected to any great extent. HLAL(Cys227-->Ala), HLAL(Cys236-->Ala), and HLAL(Cys227-->Ala, Cys236-->Ala) were essentially inactive against cholesteryl oleate, but retained about 23-39%, 28-37%, and 13-17% of catalytic activity for both triolein and tributyrin, respectively. The data obtained with the corresponding HGL mutants confirmed the importance of residues 227 and 236 in maintaining enzymatic activity towards long- and short-chain triglycerides. In order to assess the contribution of the eight amino acids delimited by Cys227 and Cys236 to lipolysis, we generated HLAL replacement mutants containing the corresponding residues 228-235 of HGL or RLL. Both HLAL chimeras were catalytically active towards all three substrates analyzed, indicating that these amino acids do not determine HLAL substrate specificity. Deletion of the eight-amino acid alpha-helix as well as disruption of its hydrophobic surface, in contrast, abolished enzymatic activity. Our studies suggest that Cys227, Cys236, and the amphipathic helix formed by residues 228-235 are essential for HLAL- and HGL-mediated neutral lipid catabolism.

Amino Acid Sequence↗

PubMed1996

FRAXE testing.

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Female↗