Structural and Biochemical Characterization of the ANGPTL3/8 Inhibitor Complex

Lipoprotein lipase (LPL) is an enzyme that hydrolyzes large triglyceride-containing lipoprotein particles into free fatty acids that can be taken up by adipose or muscle cells for energy storage or use. LPL activity is inhibited by members of the angiopoietin-like (ANGPTL) protein family, which includes ANGPTL3 and ANGPTL4, both of which can form a multimeric complex with ANGPTL8. While it is known that the ANGPTL3/8 complex is a more potent inhibitor than ANGPTL3 alone, we lack macromolecular information on the ANGPTL3/8 complex that can be useful in understanding its interaction with LPL. In this study, we used a novel method to co-purify N-terminal ANGPTL3 and ANGPTL8 from mammalian cells. Our purification strategy allowed us to obtain ANGPTL3/8 complex products rather than the individual proteins. We characterized our complex with mass photometry and size-exclusion chromatography with multi-angle light scattering. Our study showed that the oligomeric state of the ANGPTL3/8 complex is in a 3 to 1 ratio of ANGPTL3 to ANGPTL8, respectively. The ANGPTL3/8 complex purified from Expi293 cells also inhibits LPL potently in activity assays using native LPL substrate, with IC50 values in the low nanomolar range. This data can be used to further understand the regulation of triglyceride levels in the blood plasma by LPL and ANGPTL3 and ANGPTL8 and allow for the development of small molecule inhibitors of ANGPTL3/8 to treat patients with hypertriglyceridemia.