Domain-structure analysis of recombinant rat hormone-sensitive lipase.
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.
Osterlund
T
T
Section for Molecular Signalling, Lund University, Sweden.
Danielsson
B
B
Degerman
E
E
Contreras
J A
JA
Edgren
G
G
Davis
R C
RC
Schotz
M C
MC
Holm
C
C
D000595:Amino Acid Sequence; D000818:Animals; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D002787:Sterol Esterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D002787:Sterol Esterase