Inactivation of NPC1L1 Causes Multiple Lipid Transport Defects and Protects against Diet-induced Hypercholesterolemia

Joanna P. Davies, Catherine Scott, Kimihiko Oishi, Anastasia Liapis, Yiannis A. Ioannou
2005 Journal of Biological Chemistry  
NPC1L1, a recently identified relative of Niemann-Pick C1, was characterized to determine its subcellular location and potential function(s). NPC1L1 was highly expressed in HepG2 cells and localized in a subcellular vesicular compartment rich in the small GTPase Rab5. mRNA expression profiling revealed significant differences between mouse and man with highest expression found in human liver and significant expression in the small intestine. In contrast, liver expression in mouse was extremely
more » ... ow with mouse small intestine exhibiting the highest NPC1L1 expression. A mouse knock-out model of NPC1L1 was generated and revealed that mice lacking a functional NPC1L1 have multiple lipid transport defects. Surprisingly, lack of NPC1L1 exerts a protective effect against diet-induced hyperlipidemia. Further characterization of cell lines generated from wildtype and knock-out mice revealed that in contrast to wild-type cells, NPC1L1 cells exhibit aberrant plasma membrane uptake and subsequent transport of various lipids, including cholesterol and sphingolipids. Furthermore, lack of NPC1L1 activity causes a deregulation of caveolin transport and localization, suggesting that the observed lipid transport defects may be the indirect result of an inability of NPC1L1 null cells to properly target and/or regulate caveolin expression. Niemann Pick C1-like 1 protein (NPC1L1) 1 was previously identified based on its high degree of similarity, 42% amino acid identity and 51% similarity to the polytopic, late endosome-resident protein, NPC1. Both possess a putative sterolsensing domain, suggesting roles in sterol/lipid transport (1), and they also have an amino-terminal "NPC1 domain" (2). Based on this homology and preliminary data, we hypothesized that NPC1L1 has a lipid permease function similar to that of NPC1 (3). In contrast, however, the two proteins have variant targeting signals and thus are predicted to function similarly but at different intracellular locations. To gain a further understanding of the function of this family of proteins, we have carried out cell and molecular studies to determine the location and tissue expression of NPC1L1. In addition, since no known disorders map at 7p13, the chromosomal location of human NPC1L1 (1), we have generated a mouse knock-out of NPC1L1. Our results are in contrast to a published report suggesting that NPC1L1 resides at the plasma membrane (4) and indicate that this protein is predominantly intracellular and colocalizes with the small GTPase Rab5. In addition, the expression profile of human NPC1L1 shows this protein to be highly enriched in liver. Finally, inactivation of NPC1L1 leads to multiple lipid transport defects including cholesterol and sphingolipids, suggesting that NPC1L1 plays a critical role in lipid homeostasis and transport in support of our original hypothesis. MATERIALS AND METHODS Tissue Culture, Transfection, and Immunofluorescence Studies-All cells, including COS7, HepG2, and Caco-2 cells, were obtained from ATCC (Manassas, VA). Cells were maintained at 37°C in a humidified environment with 5% CO 2 in Dulbecco's modified Eagle's medium containing 2 mM glutamine, 10% fetal bovine serum, and 10 g/ml gentamicin. HepG2 cells and Caco-2 cells were grown on glass coverslips for 2 days before staining. All other cells were grown overnight before transfection using Lipofectamine and Plus reagents (Invitrogen), according to the manufacturer's instructions. Unless indicated otherwise, cells were fixed using methanol at 4°C for 6 min and then processed for indirect immunofluorescence staining using the appropriate anti-IgG secondary antibodies that were tagged with either Alexa 488 or Alexa 594 (Molecular Probes). For coimmunofluorescence with transferrin-Alexa 568, the cells were incubated with 50 g/ml transferrin for 25 min followed by a 15-min chase period. They were subsequently washed, fixed with methanol, and stained as above. Cells were photographed using a Nikon Eclipse microscope equipped with a CCD camera. Images were deconvoluted using the MetaMorph Software package (Universal Imaging). Antibodies-Monoclonal antibodies for the Golgi markers GS15, GS28, GM130, Vti1b, GS27, and p230 and those for Rab8, calnexin, clathrin, and EEA1 were purchased from Pharmingen. Goat polyclonal antibodies for ABCD3, Rab11, and calnexin and rabbit polyclonal for Rab5A were obtained from Santa Cruz Biotechnology. Transferrin-Alexa 568 and Golgin 97 were from Molecular Probes. The Alexa Fluor 594 labeling kit (Molecular Probes) was used to directly label the affinity-purified NPC1L1 rabbit polyclonal antibody with Alexa 594, enabling it to be used for colocalization of NPC1L1 with other rabbit polyclonal antibody markers. The MLN64 rabbit polyclonal antibodies were generated against the carboxyl terminus region of MLN64, spanning amino acids 239 -444. The antibodies were purified by affinity chromatography using Affi-Gel resin (Bio-Rad) coupled to the MLN64 polypeptide. The anti-Rab9 polyclonal antibodies were generated and purified in a similar manner. Generation of Anti-NPC1L1 Polyclonal Antibodies-Histidine-tagged fragments of human NPC1L1 (accession number AY515256) amino acids 416 -635 and amino acids 1276 -1332 were expressed in Esche-
doi:10.1074/jbc.m409110200 pmid:15671032 fatcat:defn6g6ikfe5bddiycnezrmycm