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Mapping the heparin binding domain of human hepatic lipase Rosenke, Karla

Abstract

Hepatic lipase (HL) is an enzyme that plays many roles in lipid metabolism. HL is involved in the catabolism of lipoprotein particles and the hydrolysis of intermediate density lipoprotein (IDL) with the formation of low density lipoprotein (LDL). HL facilitates the remodeling and recycling of anti-atherogenic high density lipoprotein (HDL), an important step in the putative process of reverse cholesterol transport. HL is also involved in the selective hydrolysis and uptake of cholesterol ester (CE) contained within HDL. However, little is known about the mechanisms of action of HL responsible for its functional roles. What is known, is that HL is found bound primarily to hepatocyte cell surface heparan sulfate proteoglycans (HSPG). Previous studies have suggested the heparin-binding domain of HL is located in the terminal 60 amino acids of the HL protein. It was our hypothesis that by the application of multiple strategies we could further define specific amino acid residues responsible for the heparin binding of HL. Regions 294-315 and 453-475 are rich in reported heparin binding consensus sequences but homology modeling implicated only the former region as the latter could not be modeled. A synthetic peptide corresponding to amino acids 304-323, containing R306, R310, K312, K314, R315 and R321, displayed moderate heparin affinity eluting from a heparin-Sepharose column at 0.42M and 0.35M NaCl, in the absence or presence of a secondary structure inducer (TFE), respectively. A synthetic peptide representing residues 455-471, containing K459, K463, K467, K469, displayed no affinity for heparin in the absence of TFE (0.15M), but eluted in two peaks (0.15M and 0.72M NaCl) in the presence of TFE. Molecular identification of the species responsible for the two peaks could not be determined. Peptides of regions 355-377, 419-442, and 452-471 did not display affinity for the heparin-Sepharose column. However, none of the peptide displaced HL bound to the surface of transfected Chinese hamster ovary cells. Additionally, a panel of monoclonal antibodies against the 452-471 failed to associate with HL or inhibit the binding of HL to heparin. These results indicate that regions 304-323 and 455-471 may contribute to HL heparin binding domain.

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