Apolipoprotein-B (ApoB) is the structural component of atherogenic lipoproteins, lipid-rich particles that drive atherosclerosis by accumulating in the vascular wall. As atherosclerotic cardiovascular disease is the leading cause of death worldwide, there is an urgent need to develop new strategies to prevent lipoproteins from causing vascular damage. Here we report the LipoGlo system, which uses a luciferase enzyme (NanoLuc) fused to ApoB to monitor several key determinants of lipoprotein atherogenicity including particle abundance, size, and localization. Using LipoGlo, we comprehensively characterize the lipoprotein profile of individual larval zebrafish and collect images of atherogenic lipoprotein localization in an intact organism. We report multiple extravascular lipoprotein localization patterns, as well as identify Pla2g12b as a potent regulator of lipoprotein size. ApoB-fusion proteins thus represent a sensitive and specific approach to study atherogenic lipoproteins and their genetic and small molecule modifiers.
Bibliographical noteFunding Information:
We thank Promega Corp. for providing the NanoLuc plasmid, as well as sample reagents and technical advice that were essential to assay development, as well as serving as a co-sponsor for a local lipid research conference where this work was presented. We would like to thank Michael Sepanski for collecting the electron micrographs, Dr. Michael McCaffery for technical support with negative-staining electron microscopy, Dr. Marnie Halpern for providing the unpublished Tg(Xla. Tubb2:mapple-CAAX) pan-neuronal marker line and valuable advice on the manuscript, Dr. Yury Miller for providing the apoC2 mutant line, and the Sanger Institute Zebrafish Mutation project for providing the pla2g12b mutant line (sa659). We would also like to thank Robert Waddail for technical advice in developing the gel quantification template (Supplementary Software 1). Support was also provided by the National Institutes of Health (R01DK093399 [S.A.F.] and R01DK116079 [S.A.F.]), National Heart, Lung, and Blood Institute (F31HL139338 [J.H.T]), and National Institute of General Medical Sciences (R01GM63904 [S.C.E] and [S.A.F] and P30DK084567 [S.C.E.]). This content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. Additional support for this work was provided by the Carnegie Institution for Science endowment and the G. Harold and Leila Y. Mathers Charitable Foundation (S.A.F).
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't