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Structural basis of endothelial lipase tropism for HDL

Department of Medicine and Center for Experimental Therapeutics and
^* Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, USA

² Correspondence: University of Pennsylvania Medical Center, 654 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160, USA. E-mail: rader{at}mail.med.upenn.edu

SPECIFIC AIMS

Lipoprotein lipase (LPL) and endothelial lipase (EL), related members of the triglyceride lipase gene family, have distinct preferences for lipids along a spectrum of lipolytic activity, with LPL being a better triglyceride hydrolase and EL a better phospholipase. They have discrete preferences for specific types of lipoproteins, with LPL preferring triglyceride-rich lipoproteins and EL preferring HDL. In contrast to extensive studies on lipid preferences of enzymes of the triglyceride lipase gene family, surprisingly little work has been done to elucidate the structural basis of their lipoprotein preferences. In the present study, we tested the hypothesis that the difference between LPL and EL in tropism for HDL resides in the carboxy-terminal (C-terminal) domain of the enzymes.

PRINCIPAL FINDINGS

We generated a human LPL-EL chimeric enzyme by exchanging the C-terminal domain and tested its ability to bind and hydrolyze native lipoproteins in vitro and in vivo. Surprisingly, the EL C-terminal domain converted LPL to an enzyme highly capable of binding and hydrolyzing HDL in vitro and in vivo. These studies indicate the importance of the C-terminal domain in lipoprotein preference and specifically tropism for HDL.

1. The C-terminal lipase domain does not substantially influence lipid substrate specificity in vitro
Cos-7 cells were infected with adenoviral constructs encoding parental lipases, LPL-EL, or control (GFP). Lipases were secreted and had the expected apparent molecular weight. The heterologous C-terminal domain did not change lipid substrate preference (triglyceride- vs. phospholipase) as determined using artificial substrates.

2. The C-terminal domain is a major determinant of lipoprotein binding in vitro
To compare the relative abilities of parental lipases and LPL-EL to mediate bridging between the cell surface heparan sulfate proteoglycans and lipoproteins in vitro, a cell binding assay was performed using Cos-7 cells. Western blot analysis confirmed protein expression of all enzymes. EL was shown to bridge VLDL and HDL whereas LPL had no effect on bridging HDL particles. We demonstrate here that substitution of the C-terminal domain of LPL with that of EL results in a chimeric enzyme with HDL binding properties almost identical to EL, establishing the importance of the EL C-terminal domain for HDL binding.

3. The C-terminal domain is a major determinant of lipoprotein substrate specificity in vitro and in vivo
To evaluate the relative capacity of parental enzymes and LPL-EL to hydrolyze lipids in native lipoproteins in vitro, conditioned media containing each enzyme were incubated with VLDL and HDL at equivalent phospholipid concentrations and free fatty acids were assayed. Unlike wild-type LPL, LPL-EL had substantial ability to hydrolyze HDL lipids similar to that of wild-type EL, indicating that the C-terminal of EL conferred on LPL the ability to hydrolyze HDL lipids.

To investigate the effects of lipases on lipid metabolism in vivo, C57BL/6 mice were injected with 1 x 10¹¹ particles of adenoviral constructs encoding parental lipases and LPL-EL. Overexpression of LPL-EL in vivo resulted in significantly reduced levels of HDL cholesterol and phospholipids by 93% and 85%, respectively, similar to the extent seen in EL-expressing mice, whereas no reduction of these parameters was observed in LPL-expressing mice (Fig. 1 a–c). Analysis of the lipoprotein distribution by FPLC on day 28 after virus injection confirmed that HDL-C levels in EL- and LPL-EL-expressing, but not LPL-expressing, mice were markedly reduced (Fig. 1d) . Overexpression of LPL-EL in apoE knockout mice demonstrated that the effect of the LPL-EL chimera on HDL was maintained even in the presence of significant levels of apo-B-containing lipoproteins that may compete for lipid binding andcatalysis. Overexpression of EL and LPL-EL in apoE-deficient mice resulted in a significant reduction of total cholesterol, HDL cholesterol, phospholipids, and triglycerides whereas only triglyceride levels were significantly reduced in LPL-expressing mice.

View larger version (24K): [in this window] [in a new window]   Figure 1. Plasma total cholesterol (a), HDL cholesterol (b), and phospholipid (c) levels in mice after injection of 1 x 1011 particles of adenoviral constructs encoding control (diamond), EL (square), LPL (circle), or LPL-EL (triangle). Data are presented as the mean ± SD. FPLC analysis of the plasma lipoproteins in mice 28 days after virus injection (d).

CONCLUSIONS AND SIGNIFICANCE

The triglyceride lipase gene family plays a critical role in energy and lipoprotein metabolism. Several new members of this gene family, including EL, have been discovered in the past few years. The structural basis for the preference of lipases of the triglyceride lipase gene family for specific lipoproteins is poorly understood. Because EL and LPL are at opposite ends of the spectrum of lipoprotein preferences and all members of this gene family are proposed to have a two domain structure based on X-ray crystallography data on pancreatic lipase, we generated a domain chimeric enzyme in an attempt to determine the domain responsible for lipoprotein preference.

We demonstrate that the C-terminal domain does not substantially influence lipid substrate specificity as assessed using artificial substrates consistent with the hypothesis that the lipid substrate preference of the members of this gene family is mediated primarily by the structure of the "lid," which is located in the amino-terminal domain and thought to control access of substrate to the hydrolytic pocket.

Hydrolysis of lipoprotein lipids requires initial binding of the lipoprotein to the enzyme. Our study demonstrates that the C-terminal domain of EL is crucial for the ability of EL to bind and to hydrolyze HDL and converts LPL to an enzyme fully capable of hydrolyzing HDL in vitro and in vivo, highlighting the importance of the C-terminal lipase domain in lipoprotein substrate preference (Fig. 2 ). To our knowledge, this is the first study to identify the C-terminal lipase domain as a structural basis for lipoprotein preference—in this case, specifically for HDL tropism.

View larger version (16K): [in this window] [in a new window]   Figure 2. Lipoprotein hydrolysis requires binding of the lipoprotein to the C-terminal lipase domain. The C-terminal domain of EL is crucial for the ability of EL to bind and to hydrolyze HDL and converts LPL (LPL-EL) to an enzyme fully capable of hydrolyzing HDL. LPL is not capable of hydrolyzing HDL because the C-terminal domain of LPL cannot bind HDL. HDL lipids are hydrolyzed by EL and LPL-EL bound to the endothelial cell surface by heparan sulfate proteoglycans (HSPG) generating free fatty acids (FFA) and HDL with decreased particle size.

FOOTNOTES

¹ These authors contributed equally to the manuscript.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1307fje;

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