Interaction of the N-terminal domain of apolipoprotein E4 with heparin.
Apolipoprotein E (apoE) is an important lipid-transport protein in human plasma and brain. It has three common isoforms (apoE2, apoE3, and apoE4). ApoE is a major genetic risk factor in heart disease and in neurodegenerative disease, including Alzheimer's disease. The interaction of apoE with heparan sulfate proteoglycans plays an important role in lipoprotein remnant uptake and likely in atherogenesis and Alzheimer's disease. Here we report our studies of the interaction of the N-terminal domain of apoE4 (residues 1-191), which contains the major heparin-binding site, with an enzymatically prepared heparin oligosaccharide. Identified by its high affinity for the N-terminal domain of apoE4, this oligosaccharide was determined to be an octasaccharide of the structure DeltaUAp2S(1-->[4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->](3)4)-alpha-D-GlcNpS6S by nuclear magnetic resonance spectroscopy, capillary electrophoresis, and polyacrylamide gel electrophoresis. Kinetic analysis of the interaction between the N-terminal apoE4 fragment and immobilized heparin by surface plasmon resonance yielded a K(d) of 150 nM. A similar binding constant (K(d) = 140 nM) was observed for the interaction between immobilized N-terminal apoE4 and the octasaccharide. Isothermal titration calorimetry revealed a K(d) of 75 nM for the interaction of the N-terminal apoE fragment and the octasaccharide with a binding stoichiometry of approximately 1:1. Using previous studies and molecular modeling, we propose a binding site for this octasaccharide in a basic residue-rich region of helix 4 of the N-terminal fragment. From the X-ray crystal structure of the N-terminal apoE4, we predicted that binding of the octasaccharide at this site would result in a change in intrinsic fluorescence. This prediction was confirmed experimentally by an observed increase in fluorescence intensity with octasaccharide binding corresponding to a K(d) of approximately 1 microM.
Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, and Department of Pathology, University of California, San Francisco, California 94941-9100, USA.
J
J
Dong
C A
CA
Peters-Libeu
K H
KH
Weisgraber
B W
BW
Segelke
B
B
Rupp
I
I
Capila
M J
MJ
Hernáiz
L A
LA
LeBrun
R J
RJ
Linhardt
D000818:Animals; D053327:Apolipoprotein E4; D001057:Apolipoproteins E / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D002240:Carbohydrate Sequence; D018360:Crystallography, X-Ray; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013552:Swine
D053327:Apolipoprotein E4; D001057:Apolipoproteins E; D009844:Oligosaccharides; D010446:Peptide Fragments; D006493:Heparin