LPL is a complex enzyme containing multiple functional domains which are required for normal activity. These domains include a site for dimer interaction, a catalytic domain as well as a site for heparin, lipid and apoCII-binding. As a member of the lipase family, LPL shares a high degree of homology with pancreatic lipase and hepatic lipase. However, despite their similarity, these enzymes have major differences in substrate specificity, cofactor requirement and heparin affinity. These functional differences have a major impact on the individual role these lipases play in normal lipid metabolism. We have used the techniques of site directed mutagenesis and chimera analysis to gain further insight into these important functional domains of LPL. LPL residues Ser-132, Asp-156 and His-241 have been tentatively identified as the catalytic triad based on primary sequence homology to pancreatic lipase. Using site-directed mutagenesis, constructs containing these 3 residues replaced by Gly were expressed in human embryonic kidney-293 cells. The LPL mutants, secreted at levels similar to the control, were inactive against triolein, tributyrin and paranitrophenylbutyrate subtrate. Control mutations in LPL maintained > 70% of the hydrolytic activity against all substrates. Analysis by heparin-Sepharose chromatography and Intralipid binding established that the LPL mutants had normal heparin and lipid affinity. These studies provide new evidence for the role of Ser-132, Asp-156 and His-241 as the active residues in the catalytic triad of LPL. We have also initiated studies involving the synthesis of chimeric proteins between LPL and hepatic lipase. The cDNA encoding the amino terminal residues of LPL has been ligated to cDNA encoding amino acids 352 to 499 of hepatic lipase and subcloned into an expression vector for transfection into human embryonal kidney-293 cells. The converse chimera, containing the amino terminal residues of hepatic lipase and the carboxyl terminal residues of LPL has also been generated. Transient transfection studies are currently underway. The expressed chimeric proteins will be analyzed for hydrolytic activity, lipid binding and heparin affinity.
