The specific amino acid sequence between putative helices 7 and 8 of human apolipoprotein A-I influences HDL subclass association and generation

HDL is generally regarded as being atheroprotective, operating through a number of possible mechanisms. Primate HDL is distinguished from that of other mammalian species in having subclasses of HDL, with the two major subclasses being HDL2 and HDL3. Epidemiological evidence suggests that a high HDL2 level is more cardioprotective than high HDL 3, while others contest small dense HDL3 is the better. Evidence from apoA-I transgenic mice, adenoviral gene transfer in mice, and transfected hepatocytes indicates that a major determinant of HDL subclasses is the amino acid sequence of apoA-I. The sequence of apoA-I is made up of a set of repeating 11 or 22 amino acid amphipathic alpha-helical repeats, with the majority of the repeats in human apoA-I being proline punctuated. A notable exception is the boundary between putative helices 7 and 8, which is located in the transitional segment between the stable N-terminal domain (NTD) and the C-terminal domain (CTD).;In this study, I ask whether the substitution of a proline containing sequence (PCS) separating other potential helices in human apoA-I for the non-proline containing sequence (NPCS) between putative helices 7 and 8 (residues 184-190) influences HDL association and generation. The human apoA-I mutant with PCS2 replacing NPCS preferentially bound to HDL2. In contrast, the mutant where PCS3 replaced NPCS preferentially associated with HDL 3. The HDL profile of McArdle rat hepatoma (McA74) cells stably expressing wild-type or human apoA-I proline containing sequence (PCS) mutants that I previously showed to preferentially associate with human HDL subclasses in vitro largely parallel their HDL generation in vitro. Wild-type human apoA-I (HuA-I) generates two major HDL particles, with diameters of 10.6 (within HDL2 size range) and 8.6nm (within HDL3 size range). HuA-I (NPCS→PCS2) generates HDL with a diameter of 10.6nm. HuA-I (NPCS→PCS3) generates HDL with a diameter of 10.6 and 8.6nm, with 80% being 8.6nm. I observed no major differences in the HDL particles generated by the exogenous incubation of these apoA-I proteins with cholesterol loaded macrophages. However, modest differences in several physical and biological properties were observed. Both modified forms of human apoA-I in question here (i.e. HuA-I (NPCS→PCS2) and HuA-I (NPCS→PCS3)) displayed increased lipid-associated stability versus wild-type apoA-I. The increased stability of the apoA-I proteins could also result in reduced clearance of both apoA-I and HDL, yielding higher apoA-I/HDL levels and in turn additional beneficial action. This is the first evidence that the specific amino acid sequence between putative helices 7 and 8 influences apoA-I stability and HDL particle generation by hepatocytes in culture and could potentially lead to preferential generation of HDL subclasses or improvement of apoA-I and/or HDL atheroprotection in vivo.