Cholesteryl ester transfer proteins from different species do not have equivalent activities

Abstract

This article is available online at http://www.jlr.org homologs in more than 20 species. In species where CETP activity has been experimentally verifi ed, this is typically based solely on the presence of CE transfer activity in plasma. Mutagenesis studies have shown that minor changes in the structure of human CETP can have profound effects on its ability to bind lipoproteins or selectively alter its ability to utilize TG and/or CE as a transfer substrate ( 5, 6 ). Given this, it seems likely that CETP from common experimental models such as hamster and rabbit, which have only ‫ف‬ 80% identity to human CETP at the amino acid level, may have unique lipid transfer properties.The purpose of this study was to characterize the lipid transfer properties of CETP from three species (monkey, rabbit, and hamster) and compare them to human CETP. We have determined the relative preference of each CETP for CE and TG as a substrate and assessed their abilities to promote net TG and CE transfer between lipoproteins. Such processes are central to CETP's role in facilitating lipoprotein metabolism. Additionally, because there is growing evidence that intracellular CETP infl uences cellular lipid metabolism ( 7-9 ), perhaps by facilitating the formation of lipid droplets ( 10 ), we investigated the capacity of all four CETP species to mediate net unidirectional lipid transfer. We show species-specifi c differences in the lipid transfer properties of CETPs in terms of their substrate preference and their ability to promote net lipid transfer between lipoproteins, and between lipoproteins and model membrane surfaces. METHODS MaterialsMouse monoclonal antibody to human CETP (TP2) was purchased from the Ottawa Heart Institute (Ottawa, Ontario, Canada). Cholesteryl ester (CE) transfer activity was fi rst observed in human plasma by Nichols and Smith ( 1 ). Zilversmit, Hughes, and Balmer ( 2 ) were the fi rst to identify a plasma protein responsible for this activity, and subsequent study showed that this protein, now called cholesteryl ester transfer protein (CETP), is responsible for all CE and triglyceride (TG) transfer activity in human plasma ( 3,4 ). Although absent in the plasma of common laboratory animals such as mice and rats, genome sequencing has identifi ed CETP

Modification of CETP function by changing its substrate preference: a new paradigm for CETP drug design

“…By this mechanism CETP facilitates either the exchange of like lipid molecules (homoexchange) or the exchange of different lipids (heteroexchange). However, we previously reported that under nonphysiologic conditions where the acceptor particle lacks neutral lipid substrate, that CETP-mediated lipid transfer is greatly reduced, but there remains a low level of lipid transfer to these neutral lipid-null particles ( 5,10 ). These results show that CETP is capable of delivering CE or TG to an acceptor particle and leaving the particle surface without picking-up any TG or CE, thus mediating a one way transfer.…”

“…The x-axis units are TG/(TG + CE). When expressed this way, the substrate preference ratio for human CETP is 0.33 instead of the ‫ف‬ 0.5 value shown in Table 2 and also previously reported ( 10 ). Results are the mean ± SD of multiple experiments as follows: R201S (two), M433A (one), H232A (fi ve), wild-type human (seven), monkey (two), rabbit (two), Q199A (six), hamster (two).…”

“…We previously observed that most lipid movement by hamster CETP can be explained by a mechanism that promotes the equimolar exchange of CE for CE, TG for TG, or CE for TG ( 10 ). However, hamster CETP also promotes the transfer of TG from VLDL to HDL by a process that is lipoprotein, releases this lipid, and then picks up another lipid prior to dissociating ( 11,18,26,27 ).…”

“…In view of our observation above, we measured the TG versus CE substrate preference for two common CETP variants, D442G and R451Q. Whereas the For example, the Q199A CETP mutant strongly prefers TG as a substrate, much like that observed for hamster CETP ( 10 ). We have hypothesized that the unique ability of hamster CETP to promote the nonreciprocal transfer of TG from VLDL to HDL ( 10 ) does not arise from an underlying different transfer mechanism in this species of CETP, but is due to its unusually high preference for TG versus CE as a substrate.…”

“…cDNAs for wild-type human CETP (M30185.1) and single amino acid mutants (Q199A, R201S, H232A, M433A, D442G, and R451Q) were synthesized (GenScript, Piscataway, NJ), subcloned into pCDNA3, and sequence verifi ed ( 10 ). The cDNAs for rabbit ( Oryctolagus cuniculus , XM_002711536), monkey ( Macaca fascicularis , M86343), and hamster ( Cricetulus griseus , XM_003503614) CETPs were synthesized as previously described ( 10 ). All constructs contained native start and stop codons including the coding sequence for a 17 amino acid signal peptide.…”