Lipid-free apolipoproteins A-I and A-II promote remodeling of reconstituted high density lipoproteins and alter their reactivity with lecithin:cholesterol acyltransferase.
We examined the effect of lipid-free apolipoprotein A-I (apoA-I) and apoA-II on the structure of reconstituted high density lipoproteins (rHDL) and on their reactivity as substrates for lecithin:cholesterol acyltransferase (LCAT). First, homogeneous rHDL were prepared with either apoA-I or apoA-II using palmitoyloleoylphosphatidylcholine (POPC) and cholesterol. Lipid-free apoA-I and apoA-II were labeled with the fluorescent probe dansyl chloride (DNS). The binding kinetics of apoA-I-DNS to A-II-POPCrHDL and of apoA-II-DNS to A-I-POPCrHDL were monitored by fluorescence polarization, adding the lipid-free apolipoproteins to the rHDL particles in a 1:1 molar ratio. For both apolipoproteins, the binding to rHDL was rapid, occurring within 5 min. Next, the effect on rHDL structure and particle size was determined after incubations of lipid-free apolipoproteins with homogeneous rHDL at 37 degrees C from 0.5 to 24 h. The products were analyzed by non-denaturing gradient gel electrophoresis followed by Western blotting. The effect of apoA-I or apoA-II on 103 A A-II-POPCrHDL was a rearrangement into 78 A particles containing apoA-I and/or apoA-II, and 90 A particles containing only apoA-II. The effect of apoA-I or apoA-II on 98 A A-I-POPCrHDL was a rearrangement into complexes ranging in size from 78 A to 105 A containing apoA-I and/or apoA-II, with main particles of 78 A, 88 A, and 98 A. Finally, the effect of lipid-free apoA-I and apoA-II on rHDL as substrates for LCAT was determined. The addition of apoA-I to A-II-POPCrHDL increased its reactivity with LCAT 24-fold, reflected by a 4-fold increase in apparent V(m)ax and a 6-fold decrease in apparent K(m), while the addition of apoA-II to A-II-POPCrHDL had no effect on its minimal reactivity with LCAT. In contrast, the addition of apoA-II to A-I-POPCrHDL decreased the reaction with LCAT by about one-half. The inhibition was due to a 2-fold increase in apparent K(m); there was no significant change in apparent V(m)ax. Likewise, the addition of apoA-I to A-I-POPCrHDL inhibited the reaction with LCAT to about two-thirds that of A-I-POPCrHDL without added apoA-I. In summary, both lipid-free apoA-I and apoA-II can promote the remodeling of rHDL into hybrid particles of primarily smaller size. Both apoA-I and apoA-II affect the reactivity of rHDL with LCAT, when added to the reaction in lipid-free form. These results have important implications for the roles of lipid-free apoA-I and apoA-II in HDL maturation and metabolism.
Durbin
D M
DM
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Jonas
A
A
D016632:Apolipoprotein A-I / Q000378:metabolism* / Q000494:pharmacology; D016633:Apolipoprotein A-II / Q000378:metabolism* / Q000494:pharmacology; D003619:Dansyl Compounds / Q000378:metabolism; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000187:drug effects / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure
D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D003619:Dansyl Compounds; D008075:Lipoproteins, HDL; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; C029038:dansyl chloride; C028694:1-palmitoyl-2-oleoylphosphatidylcholine