title: The effect of apolipoprotein A-II on the structure and function of apolipoprotein A-I in a homogeneous reconstituted high density lipoprotein particle.
abstract: In this study we examined the effects of apoA-II on the structure and function of apoA-I in homogeneous reconstituted HDL (rHDL). First, we measured the binding of apoA-II to apoA-I-rHDL, containing dipalmitoylphosphatidylcholine or palmitoyloleoylphosphatidylcholine, and the degree of apoA-I displacement at various ratios of apolipoproteins. Using fluorescence methods, we determined that apoA-II binding is rapid, irreversible, and associated with apoA-I displacement only when the molar ratio of apoA-II/apoA-I is greater than 1:2. Next, we used the stable apoA-II/apoA-I-rHDL complex at the apoA-II/apoA-I ratio of 1:2 to examine its physical properties, apoA-I structure, and reactivity with lecithin:cholesterol acyltransferase (LCAT). Using chemical cross-linking in conjunction with fluorescence and electrophoretic methods, we demonstrated that the conformation of apoA-I must be flexible to allow apoA-II binding to the apoA-I-rHDL particles and showed that the hybrid particles have an unchanged Stokes diameter. Fluorescence and circular dichroism measurements revealed little or no change in the secondary structure or in the N-terminal domain of apoA-I, but showed a marked destabilization of apoA-I to denaturation by guanidine hydrochloride. Limited tryptic digestion indicated that the central region of apoA-I becomes accessible to proteolysis in the hybrid particles. Together, these results suggest that amphipathic alpha-helices of apoA-II replace four central helices of one apoA-I molecule (residues approximately 99-187) in the complex and in the process destabilize apoA-I. Thus, apoA-II binding at physiologic ratios may not completely displace apoA-I from HDL, but may provide a reservoir of easily exchangeable apoA-I. Finally, we showed that the reaction of the hybrid HDL with LCAT was inhibited 2-5-fold, relative to apoA-I-rHDL, due to a corresponding increase in the apparent Km value. This suggests that LCAT binding to the hybrid particles is sterically hindered by the excess protein (portions of apoA-I and apoA-II not bound to lipid). Therefore, apoA-II can modulate the reaction of HDL with LCAT by decreasing LCAT binding to hybrid particles and making the enzyme available for reaction with other substrates.
authors.affiliation: Department of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois, Urbana, Illinois 61801, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Durbin
authors.forename: A
authors.initials: A
authors.lastname: Jonas
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism; D016633:Apolipoprotein A-II / Q000494:pharmacology*; D002942:Circular Dichroism; D006801:Humans; D066298:In Vitro Techniques; D008075:Lipoproteins, HDL / Q000235:genetics / Q000378:metabolism*; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
chemical_list: D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D008075:Lipoproteins, HDL; D011994:Recombinant Proteins; D015060:1,2-Dipalmitoylphosphatidylcholine; D007862:Phosphatidylcholine-Sterol O-Acyltransferase