Acidic Domain of ApoB-100 and Other Short Negatively Charged Peptides Anchored to Lipoproteins Cause Hypertriglyceridemia in Mice

Human Apolipoprotein B-100 (ApoB-100) is the main protein of VLDL and LDL particles in plasma. It is a large 4536 amino acid protein with various structural domains, such as the LDL-receptor binding motif and multiple proteoglycan binding sites, all of which have a high positive charge. A search of the primary amino acid sequence of ApoB-100 revealed the presence of the following acidic domain: DMDEDDD (AA 3985-3991). We hypothesized that this motif could affect the interaction of ApoB-containing lipoproteins with liver and or possibly GPIHBP1, which also contains an acidic domain that interacts with the positive charged motifs on VLDL. We synthesized a fluorescent tagged DMDEDDD peptide and conjugated it at the N-terminus with two molecules of alpha-tocopherol to serve as an anchor to lipoprotein particles. After adding the peptide to human plasma, it was found to readily bind to all lipoproteins, as determined by FPLC analysis. VLDL incubated with the peptide showed an increased negative charge and lost its ability to bind to heparin as determined by chromatography on a HiTrap TM heparin column. The peptide injected in LDLr-KO mice (30 mg/kg) caused a rapid 3-fold increase in plasma triglycerides within 1 h and the triglyceride elevation persisted for at least 24 h. A similarly designed negative charged peptide attached to tocopherol (EEEEEEEE) also raised triglycerides in mice but a neutral charged control peptide (KEKEKEKE) did not. To understand the mechanism, we tested the above peptides for their ability to inhibit LPL, using an in vitro assay with intralipid as a substrate and found that negative charged peptides inhibited lipolysis (IC 50= 50 uM), but the neutral control peptide did not. We are now investigating other potential mechanisms for how acidic peptides may cause hypertriglyceridemia. In summary, a peptide based on an acidic domain on ApoB-100 appears to cause hypertriglyceridemia in mice by inhibition of LPL and possibly by interfering with proteoglycan binding of lipoproteins. These results may be relevant in understanding VLDL metabolism and for the design of therapeutic apolipoprotein mimetic peptides.