Abstract
The reaction of highly purified lecithin:cholesterol acyltransferase (LCAT) with defined reconstituted discoidal apoA-I-containing lipoproteins (LpA-I) with 2, 3, or 4 apoA-I molecules/particle (Lp2, 3, or 4A-I) has been studied in the presence of a number of specific anti apoA-I antibodies. Among nine anti-apoA-I monoclonal antibodies (mAbs) reacting with epitopes distributed over 80% of the sequence, three significantly inhibit the LCAT reaction with all particles. The position of their epitopes located in the middle to COOH-terminal region between residues 96-121 (3G10), 135-148 (A03), and 149-186 (A44) is compatible with an inhibition by steric hindrance over a central domain. Antibody 4H1 binding to the NH2 terminus (residues 2-8) profoundly increases (5-fold) the LCAT reaction with Lp2A-I (7.8 nm), but not with other particles. Other mAbs, A11 and 5F6, binding to epitopes (residues 99-139 and 118-141) enhance LCAT reactivity with the small Lp2A-I (7.8 nm) and Lp3A-I (10.8 nm) but not with their larger counterparts. Most mAbs have similar effects on LCAT reaction with native high density lipoprotein3 as with LpA-I. The inhibitory or enhancing effects of these mAbs are also observed with Fab fragments and not related to their binding affinity for apoA-I containing reconstituted lipoprotein particles. The intercalation of epitopes for mAbs that inhibit or enhance LCAT reaction with small LpA-I is compatible not with steric hindrance but with conformational modifications of apoA-I and indirectly of the lipids in small particles. We propose that enhancing mAbs act by stabilization of an apoA-I conformation which is not favored in small LpA-I, i.e. by increasing binding of amphipathic helices to lipids or by interfering with the mobility of a hinged domain. The epitopes for the inhibitory mAbs can be shown to overlap on several LpA-I models, indicating that steric hindrance over a single site is a possible mechanism of inhibition.
Highlights
From the Lipoproteins and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, OntarioKI Y 4E9, Canada and the §Service d’Etudeet de Recherche Sur Les Lipides et I’Atheroscldrose,Znstitut Pasteur, 59019 Lille Cedex, France
Been studied in the presence of a number of specific This process of cholesterol esterification takes place princianti apoA-I antibodies.Among nine anti-apoA-Imono- pally on high density lipoprotein (HDL) and is believed to clonal antibodies reacting with epitopes dis- play a key role in reverse cholesterol transport [1].ApoA-I is tributed over 80% of the sequence, three significantly the major apolipoprotein component in HDL and the most inhibit the Lecithin cholesterol acyltransferase (LCAT) reaction with all particles
Several other sition of their epitopes located in thmeiddle to COOHterminalregionbetweenresidues96-121(3G10), 135-148 (A03), and 149-186 (A4is4c)ompatible with an inhibition by steric hindrance over a central doapolipoproteins as well as fragments of apoA-I can activate thereactionto variousdegrees[3, 4]
Summary
Vol 268, No 23, Issue of August 15, pp. 16966-16973,1993 Printed in lJ S.A. Apolipoprotein A-I DomainsInvolved in the Activation of Lecithin:Cholesterol Acyltransferase. Several other sition of their epitopes located in thmeiddle to COOHterminalregionbetweenresidues96-121(3G10), 135-148 (A03), and 149-186 (A4is4c)ompatible with an inhibition by steric hindrance over a central doapolipoproteins as well as fragments of apoA-I can activate thereactionto variousdegrees[3, 4]. It is clearthatthe presence in theseproteins of amphipathica-helices which allow lipid binding is important, but it haaslso been proposed thattheactivatingapolipoproteinfacilitatesthe access of main. Fab fragments of these mAbs were prepared by papain digestion, purified byprotein A-conjugated SepharoseCL-4Baffinitychromatography,andproven free of IgG by SDS-gel electrophoresis. IgG-bound LpA-I was precipitated with rabbit anti-murineIgG conjugated to Staphylococcus aureus cell (Pansorbin, Calbiochem) as described earlier [18, 22]
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