Model systems to study assembly, trafficking and secretion of apoB-lipoproteins using fluorescent fusion proteins
- Walsh, M.T., Celestin, O.M., Thierer, J.H., Rajan, S., Farber, S.A., Hussain, M.M.
- Journal of Lipid Research 61(3): 316-327 (Journal)
- Registered Authors
- Farber, Steven, Walsh, Meghan
- Apolipoproteins, Chylomicrons, LDL, Lipoproteins, Lipoproteins/Assembly, Secretion, VLDL, Zebrafish models, apoB100, apob48
- MeSH Terms
- Apolipoproteins B/metabolism*
- COS Cells
- Chlorocebus aethiops
- Green Fluorescent Proteins/metabolism*
- Models, Biological*
- Recombinant Fusion Proteins/metabolism*
- 31888978 Full text @ J. Lipid Res.
Walsh, M.T., Celestin, O.M., Thierer, J.H., Rajan, S., Farber, S.A., Hussain, M.M. (2019) Model systems to study assembly, trafficking and secretion of apoB-lipoproteins using fluorescent fusion proteins. Journal of Lipid Research. 61(3):316-327.
ApoB exists as apoB100 and apoB48, mainly found in hepatic VLDL, and intestinal chylomicrons, respectively. Elevated plasma levels of apoB-containing lipoproteins (Blps) contribute to coronary artery disease, diabetes, and other cardiometabolic conditions. Studying the mechanisms that drive assembly, intracellular trafficking, secretion and function of Blps remains challenging. Our understanding of intracellular and intra-organism trafficking of Blps can be greatly enhanced, however, with the availability of fusion proteins that can help visualize Blp transport within cells and between tissues. To visualize intracellular apoB in mammalian cells, we designed three plasmids expressing human apoB fluorescent fusion proteins: apoB48-GFP, apoB100-GFP, and apoB48-mCherry. In Cos-7 cells, transiently expressed fluorescent apoB proteins co-localized with calnexin and were only secreted if cells were co-transfected with microsomal triglyceride transfer protein. The secreted apoB-fusion proteins retained the fluorescent protein and were secreted as lipoproteins with flotation densities similar to plasma HDL and LDL. In a rat hepatoma McA-RH7777 cell line, the human apoB100 fusion protein was secreted as VLDL- and LDL-sized particles, and the apoB48 fusion proteins were secreted as LDL- and HDL-sized particles. To monitor lipoprotein trafficking in vivo, the apoB48-mCherry construct was transiently expressed in zebrafish larvae and was detected throughout the liver. These experiments show that the addition of fluorescent proteins to the C-terminus of apoB does not disrupt their assembly, localization, secretion, or endocytosis. Availability of fluorescently labeled apoB proteins will facilitate exploration of assembly, degradation, and transport of Blps and help identify novel compounds that interfere with these processes via high throughput screening.
Genes / Markers
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes