Domain-structure analysis of recombinant rat hormone-sensitive lipase

Abstract

Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains.

A Novel Hormone-sensitive Lipase Isoform Expressed in Pancreatic β-Cells

“…recombinant HSL adi (18)) and the affinity purified rabbit antibody directed toward the GST-HSL-A fusion protein (1:100), described above. A horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences) was used as secondary antibody (1: 2000) and the blots were developed by enhanced chemiluminescence.…”

Inhibition of Lipase Activity and Lipolysis in Rat Islets Reduces Insulin Secretion

“…Enzyme activity and acylglyceride assays. The white adipose cell suspension was homogenized and islets were sonicated in buffer (0.25 mol/l sucrose, 1 mmol/l EDTA [pH 7.0], 1 mmol/l dithioerythritol, 20 g/ml leupeptin, 20 g/ml antipain, and 1 g/ml pepstatin A) to allow assay of diglyceride lipase activity, as described elsewhere (20); the diacylglycerol analogue monooleoyl-2-O-mono-oleylglycerol (MOME) was used as substrate (21). ATP and ADP determinations.…”

Interaction of Hormone-sensitive Lipase with Steroidogeneic Acute Regulatory Protein

“…The C-terminal portion of HSL displays secondary structural homology with that of acetylcholinesterase and several fungal lipases (5) and bacterial brefeldin A esterase (6), consisting of parallel ␤-sheets flanked by ␣-helical connections, which has allowed these proteins to be classified as ␣/␤-hydrolases (7). Using limited proteolysis, it has been suggested that HSL is composed of two major structural domains (8,9). Based on sequence alignment, structural homology with fungal lipases, and mutational analyses, the C-terminal domain has been shown to contain the catalytic triad and other residues important in hydrolytic activity, as well as a 150-amino acid insert that has been termed the regulatory module because several serines located within this region have been shown to be phosphorylated (1,10).…”

“…Based on sequence alignment, structural homology with fungal lipases, and mutational analyses, the C-terminal domain has been shown to contain the catalytic triad and other residues important in hydrolytic activity, as well as a 150-amino acid insert that has been termed the regulatory module because several serines located within this region have been shown to be phosphorylated (1,10). The N-terminal domain in rat HSL constitutes the first 323 amino acids, which are encoded by exons 1-4, and displays no sequence or structural similarity with any other known proteins (8,9).…”