Apolipoprotein A-II-mediated Conformational Changes of Apolipoprotein A-I in Discoidal High Density Lipoproteins

Kekulawalage Gauthamadasa,Nataraja Sarma Vaitinadin,James L Dressman,Stephen Macha,Reyn Homan,Kenneth D Greis,R A Gangani D Silva

doi:10.1074/jbc.m111.291070

Kekulawalage Gauthamadasa, Nataraja Sarma Vaitinadin + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m111.291070

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Abstract

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.

Highlights

Role of apolipoprotein A-II on metabolism of high density lipoproteins (HDLs) is unknown
We used 1 mg/ml of particle concentration and a 20-min incubation step upon apoA-II introduction, as was reported before [11]. These conditions generated particles that appeared homogeneous by native PAGE, but were found to be heterogeneous in terms of apolipoprotein stoichiometry per particle upon particle cross-linking followed by SDS-PAGE analysis
The conformational changes caused by apoA-II in apoA-I are confined to two distinct regions spanning apoA-I putative helices 3– 4 and 7–9; 3) the limited tryptic digestion demonstrated that both particle types generated identical proteolytic patterns, implying comparable overall conformation for apoA-I in both particles

Summary

Introduction

Role of apolipoprotein (apo) A-II on metabolism of high density lipoproteins (HDLs) is unknown. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3– 4 and 7–9 We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process

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