Analysis of Protein Structure-Function in Vivo: ADENOVIRUS-MEDIATED TRANSFER OF LIPASE LID MUTANTS IN HEPATIC LIPASE-DEFICIENT MICE

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

Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster.

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5-ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter