Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy

Lei Zhang,James Song,Giorgio Cavigiolio,Brian Y Ishida,Shengli Zhang,John P Kane,Karl H Weisgraber,Michael N Oda,Kerry-Anne Rye,Henry J Pownall,Gang Ren

doi:10.1194/jlr.d010959

Abstract

Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.

Highlights

Plasma lipoprotein levels are predictors of risk for coronary artery disease
LDL particles vary in size, shape, and composition [4, 5] and comprise large LDL (LDL1–2) and small, dense LDL (LDL3–7) subclasses [6]; the latter are more prone to Abbreviations: apo, apolipoprotein; CE, cholesteryl ester; cryoEM, cryo-electron microscopy; cryo-NS, cryo-negative-staining; Dulbecco’s PBS (DPBS), Dulbecco’s phosphate-buffered saline; EM, electron microscopy; IDL, intermediate density lipoprotein; NS, negative staining; negative staining EM (NS-EM), negativestaining electron microscopy; PTA, phosphotungstic acid; RCT, reverse cholesterol transport; rHDL, reconstituted HDL; UC, unesterified cholesterol; UF, uranyl formate
Evidence that rouleau formation is an artifact of the conventional NS-EM protocol comes from results for nondenaturing polyacrylamide gradient gel electrophoresis and cryoEM studies [2, 4, 8, 21, 42], where apoA-I rHDL, apoE4 rHDL, and LDL all appear as distinct particles without stacking

Summary

Introduction

Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. We validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.—Zhang, L., J. LDL particles vary in size, shape, and composition [4, 5] and comprise large LDL (LDL1–2) and small, dense LDL (LDL3–7) subclasses [6]; the latter are more prone to Abbreviations: apo, apolipoprotein; CE, cholesteryl ester; cryoEM, cryo-electron microscopy; cryo-NS, cryo-negative-staining; DPBS, Dulbecco’s phosphate-buffered saline; EM, electron microscopy; IDL, intermediate density lipoprotein; NS, negative staining; NS-EM, negativestaining electron microscopy; PTA, phosphotungstic acid; RCT, reverse cholesterol transport; rHDL, reconstituted HDL; UC, unesterified cholesterol; UF, uranyl formate. Each LDL particle contains one molecule of apolipoprotein B-100 (apoB-100), a ligand for hepatic clearance of plasma cholesterol via LDL receptors [7]

Methods

Results

Conclusion