IN VITRO AND IN VIVO STRUCTURE-FUNCTION ANALYSIS OF LPL AND HL

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Hepatic lipase (HL) and lipoprotein lipase (LPL) are lipolytic enzymes which play a major role in normal triglyceride metabolism. As members of the lipase family, both enzymes share a high degree of homology; however, LPL and HL have different cofactor requirements and substrate specificities. Although both LPL and HL are triacylglycerol hydrolases, HL also functions as a phospholipase. We have investigated the structural basis for the different substrate specificities of LPL and HL both in vitro and in vivo by generating mutant lipases using site-directed mutagenesis as well as recombinant adenoviral vectors. Previously, we have demonstrated that the LPL and HL lids, which modulate access of the substrate to the catalytic site, play a crucial role in mediating lipid-substrate interaction. Disruption of the 2 amphipathic helices present in the lipase lids results in loss of activity using either triolein or phospholipid (PL) substrates while esterase activity against the short-chained watersoluble substrate tributyrin is preserved. Chimeric lipases containing the LPL backbone with the HL lid, the HL backbone with the LPL lid and the amino terminus of LPL with the carboxyl-terminus of HL had a markedly different ability to hydrolyze PL substrates. The presence of the HL lid augmented PL hydrolysis whereas the LPL lid enhanced triglyceride hydrolysis. These in vitro studies have now been extended in vivo using recombinant adenovirus expressing either native HL or HL with the LPL lid. These constructs were injected in HL-deficient mice who have elevated plasma levels of TC and PL resulting in post-heparin plasma activities (day 4 post-infusion) of 8,953 and 9,133 nmoles/min/ml. Animals injected with the native HL virus had a 30% reduction in plasma PL and a marked change in lipoprotein profile; however, those injected with HL with LPL lid had no change in plasma PL levels. These studies indicate that exchange of the HL with the LPL lid modifies the ability of the lipase, in vivo, to hydrolyze PL and thus, establish an important role of the lipase lid in mediating lipase substrate specificity.