Abstract
Hepatic lipase (HL) and lipoprotein lipase (LPL) are key enzymes involved in the hydrolysis of triglycerides and phospholipids present in circulating plasma lipoproteins. Despite their similarities, the role that each of these two lipases play in the metabolism of triglyceride-rich lipoproteins and high density lipoproteins is distinct. In order to identify structural domains that may confer the different substrate specificities between HL and LPL, we have utilized a novel approach for performing structure-function analysis of a protein, in vivo, by using recombinant adenovirus vectors to express native and mutant enzymes in an animal model for a human genetic deficiency. HL-deficient mice (n = 19) characterized by increased plasma cholesterol and phospholipid concentrations were injected with adenovirus expressing luciferase (rLucif-AdV), native hepatic (rHL-AdV), and lipoprotein lipase (rLPL-AdV) or lipase mutants in which the lid covering the catalytic site of either enzyme was exchanged (rHL+LPL lid-AdV and rLPL+HL lid-AdV). Mice injected with rLucif-AdV had no changes in post-heparin HL and LPL activities (217 +/- 29 and 7 +/- 2 nmol/min/ml, respectively) as well as plasma lipids. Despite expression of similar levels of post-heparin plasma lipase activity on day 5 post-adenovirus infusion (9806 +/- 915 and 9677 +/- 2033 nmol/min/ml, respectively) mice injected with rHL-AdV or rHL+LPL lid-AdV demonstrated marked differences in the reduction of plasma phospholipids (70% and 32%, respectively, p < 0.005). Similarly, despite post-heparin plasma lipolytic activities of 4495 +/- 534 and 4844 +/- 1336 nmol/min/ml, injection of rLPL-AdV or rLPL+HL lid-AdV resulted in phospholipid reductions of 31% and 81% (p < 0.005). Exchange of the lipase lid did not significantly alter plasma triglyceride concentrations. Thus, preferential in vivo hydrolysis of phospholipids was demonstrated in animals expressing lipases containing the HL lid but not the LPL lid. These studies identify the lipase lid as a major structural motif responsible for conferring the different in vivo phospholipase activities between HL and LPL, a function which may modulate the distinct physiological roles of these two similar lipolytic enzymes in lipoprotein metabolism. The use of recombinant adenovirus to express mutant proteins in animal models for human genetic deficiencies represents a powerful, new approach for performing structure-function analysis of proteins in vivo.
Highlights
Hepatic lipase (HL)-deficient mice (n ؍19) characterized by increased plasma cholesterol and phospholipid concentrations were injected with adenovirus expressing luciferase, native hepatic, and lipoprotein lipase or lipase mutants in which the lid covering the catalytic site of either enzyme was exchanged
(panel C) as well as the mutant, chimeric lipases containing either the human HL backbone with the lipoprotein lipase (LPL) lid or the human LPL backbone with the HL lid, which were expressed in HL-deficient mice using these recombinant adenovirus vectors
Despite the similarity in the structure and hydrolytic function of HL and LPL, the role that these two enzymes play in lipoprotein metabolism is distinct
Summary
Preferential in vivo hydrolysis of phospholipids was demonstrated in animals expressing lipases containing the HL lid but not the LPL lid These studies identify the lipase lid as a major structural motif responsible for conferring the different in vivo phospholipase activities between HL and LPL, a function which may modulate the distinct physiological roles of these two similar lipolytic enzymes in lipoprotein metabolism. We describe a method that permits the identification of important functional domains of a lipolytic enzyme, by using recombinant adenovirus to express native and mutant proteins in an animal model for a human genetic deficiency This approach permits structure-function analysis of proteins in vivo, circumventing the limitations of as well as complementing the information obtained from in vitro expression studies. Both HL and LPL have similar catalytic sites [7,8,9] and, like pancreatic lipase [10], appear to be organized into functionally distinct carboxyl- and amino-terminal domains
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