The effect of apolipoprotein A-II (apoA-II) on the structure and stability of HDL has been investigated in reconstituted HDL particles. Purified human apoA-II was incorporated into sonicated, spherical LpA-I particles containing apoA-I, phospholipids, and various amounts of triacylglycerol (TG), diacylglycerol (DG), and/or free cholesterol. Although the addition of PC to apoA-I reduces the thermodynamic stability (free energy of denaturation) of its ␣ -helices, PC has the opposite effect on apoA-II and significantly increases its helical stability. Similarly, substitution of apoA-I with various amounts of apoA-II significantly increases the thermodynamic stability of the particle ␣ -helical structure. ApoA-II also increases the size and net negative charge of the lipoprotein particles. ApoA-II directly affects apoA-I conformation and increases the immunoreactivity of epitopes in the N and C termini of apoA-I but decreases the exposure of central domains in the molecule (residues 98-186). ApoA-II appears to increase HL association with HDL and inhibits lipid hydrolysis. ApoA-II mildly inhibits PC hydrolysis in TG-enriched particles but significantly inhibits DG hydrolysis in DG-rich LpA-I. In addition, apoA-II enhances the ability of reconstituted LpA-I particles to inhibit VLDL-TG hydrolysis by HL. Therefore, apoA-II affects both the structure and the dynamic behavior of HDL particles and selectively modifies lipid metabolism.

We have previously shown that hepatic lipase (HL) is inactive when bound to purified heparan sulfate proteoglycans and can be liberated by HDL and apolipoprotein A-I (apoA-I), but not by LDL or VLDL. In this study, we show that HDL is also able to displace HL directly from the surface of the hepatoma cell line, HepG2, and Chinese hamster ovary cells stably overexpressing human HL. ApoA-I is more efficient at displacing cell surface HL than is HDL, and different HDL classes vary in their ability to displace HL from the cell surface. Human hepatic lipase (HL) is a 64 kDa glycoprotein anchored by heparan sulfate proteoglycans (HSPGs) to the surface of endothelial cells and hepatocytes (1). HL functions both as a cell surface ligand for lipoprotein uptake and as a lipolytic enzyme that mediates the clearance of triacylglycerol from the blood stream and the conversion of VLDL to LDL (2-9). It has been known for over five decades that displacement of lipolytic enzymes from cell surface binding sites with heparin results in rapid hydrolysis of triacylglycerol-rich lipoproteins in lipemic serum (10, 11). However, it is still commonly believed that both HL and lipoprotein lipase (LPL) are catalytically active when bound to cell surface proteoglycans and that this association may indeed enhance the lipolysis of triacylglycerol [as reviewed in ref. (12)].This common view is in fact counterintuitive to the interfacial catalytic models proposed for lipases, which have shown a clear requirement for enzyme hopping or shuttling between substrates for optimal hydrolysis (13,14). In agreement with this view, we showed that HL is active only when it is free in solution, and indeed is completely inactive when bound to pure HSPG (15). In addition, we showed that HL could be displaced from a pure HSPG matrix by HDL, and specifically by apolipoprotein A-I (apoA-I), but not by LDL or VLDL. In the present study, we have reevaluated this displacement phenomenon in HepG2 cells and in a Chinese hamster ovary cell line stably overexpressing human HL (CHO-hHL). As with our pure HSPG studies, we show that only apoA-I and HDL are able to displace cell surface HL. We further show that different HDL classes vary in their ability to displace HL, and demonstrate that the lowest density fractions (HDL 2 ) have the greatest capacity to remove HL from the cell surface and intracellular compartments.In addition to their ability to displace cell surface HL, we have previously reported that apoA-I and HDL directly affect HL-mediated triacylglycerol hydrolysis, and showed that the rate of triacylglycerol hydrolysis is regulated by the amount of HDL in plasma (15). This observation sugAbbreviations: CHO-hHL, Chinese hamster ovary cell line stably overexpressing human hepatic lipase; ECM, extracellular matrix; EMEM, Eagle's minimal essential medium; FAF-BSA, essentially fatty acid-free BSA; HL, hepatic lipase; HRP, horseradish peroxidase; HSPG, heparan sulfate proteoglycan; [ 3 H]TG, [ 3 H]triolein; MAb, monoclonal antibody; pen/strep, penicillin/...

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.

To evaluate the factors that regulate HDL catabolism in vivo, we have measured the clearance of human apoA-I from rabbit plasma by following the isotopic decay of (125)I-apoA-I and the clearance of unlabeled apoA-I using a radioimmunometric assay (RIA). We show that the clearance of unlabeled apoA-I is 3-fold slower than that of (125)I-apoA-I. The mass clearance of iodinated apoA-I, as determined by RIA, is superimposable with the isotopic clearance of (125)I-apoA-I. The data demonstrate that iodination of tyrosine residues alters the apoA-I molecule in a manner that promotes an accelerated catabolism. The clearance from rabbit plasma of unmodified apoA-I on HDL(3) and a reconstituted HDL particle (LpA-I) were very similar and about 3-4-fold slower than that for (125)I-apoA-I on the lipoproteins. Therefore, HDL turnover in the rabbit is much slower than that estimated from tracer kinetic studies. To determine the role of the kidney in HDL metabolism, the kinetics of unmodified apoA-I and LpA-I were reevaluated in animals after a unilateral nephrectomy. Removal of one kidney was associated with a 40-50% reduction in creatinine clearance rates and a 34% decrease in the clearance rate of unlabeled apoA-I and LpA-I particles. In contrast, the clearance of (125)I-labeled molecules was much less affected by the removal of a kidney; FCR for (125)I-LpA-I was reduced by <10%. The data show that the kidneys are responsible for most (70%) of the catabolism of apoA-I and HDL in vivo, while (125)I-labeled apoA-I and HDL are rapidly catabolized by different tissues. Thus, the kidney is the major site for HDL catabolism in vivo. Modification of tyrosine residues on apoA-I may increase its plasma clearance rate by enhancing extra-renal degradation pathways.