Abstract
Association of hepatic lipase (HL) with pure heparan sulfate proteoglycans (HSPG) has little effect on hydrolysis of high density lipoprotein (HDL) particles, but significantly inhibits (>80%) the hydrolysis of low (LDL) and very low density lipoproteins (VLDL). Lipolytic inhibition is associated with a differential ability of the lipoproteins to remove HL from the HSPG. LDL and VLDL are unable to displace HL, whereas HDL readily displaces HL from the HSPG. These data show that HSPG-bound HL is inactive. Purified apolipoprotein (apo) A-I is more efficient than HDL at liberating HL from HSPG, and HL displacement is associated with the direct binding of apoA-I to HSPG. However, displacement of HL by apoA-I does not enhance hydrolysis of VLDL particles. This appears due to the direct inhibition of HL by apoA-I. Both apoA-I and HDL are able to inhibit VLDL lipid hydrolysis by up to 60%. Inhibition of VLDL hydrolysis is associated with the binding of apoA-I to the surface of the VLDL particle and a concomitant decreased affinity for HL. These data show that apoA-I can regulate lipid hydrolysis by HL by liberating/activating the enzyme from cell surface proteoglycans and by directly modulating lipoprotein binding and hydrolysis.
Highlights
Human hepatic lipase (HL)1 is a 64-kDa, 476-amino acid glycoprotein that is synthesized and secreted primarily by the liver [1]
Activity of heparan sulfate proteoglycans (HSPG)-bound Hepatic Lipase—Removawells were coated with pure HSPG (5 g/Removawell), incubated with fatty acid-free bovine serum albumin (FAF-BSA) to block any nonspecific binding of HL, and incubated with excess purified human HL (120 units) for 2 h
To evaluate whether the association of HL to HSPG alters the catalytic activity of the enzyme, Removawells were coated with HSPG, blocked with FAF-BSA, incubated with excess HL for 2 h, and washed
Summary
The free fatty acid diagnostic kits were purchased from Roche Diagnostics (Laval, Quebec (PQ), Canada). The anti-mouse IgG, horseradish peroxidase (HRP)-linked whole antibody (isolated from sheep) and the broad range molecular weight markers were obtained from Amersham Pharmacia Biotech (Baie d’Urfe, PQ, Canada). The anti-HL monoclonal antibody (mAb) [3,4,5,6] was obtained from Dr A. The protein content of the lipoprotein fractions was determined by the Lowry method as modified by Markwell et al [16]. PL, cholesterol, and TG contents were determined enzymatically using diagnostic kits (Roche Diagnostics, Laval, PQ, Canada). Post-heparin human plasma was collected from normolipidemic subjects and a 20% TG emulsion (Intralipid 20%, Pharmacia & Upjohn Inc., Missassauga, Ontario, Canada) was added to the plasma. The resuspended, aqueous solution of crude HL was loaded onto a heparinSepharose CL-6B column and eluted with a linear salt gradient of 0.15–1.5 M NaCl in 5 mM sodium barbital, pH 7.4, 20% glycerol and the pooled fractions stored at Ϫ80 °C until use
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