Interaction of the N-Terminal Domain of Apolipoprotein E4 with Heparin
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- 作者：Jun Dong ; Clare A. Peters-Libeu ; Karl H. Weisgraber ; Brent W. Segelke ; Bernhard Rupp ; Ishan Capila ; Maria J. Herná ; iz ; Laurie A. LeBrun ; and Robert J. Linhardt
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：March 6, 2001
- 年：2001
- 卷：40
- 期：9
- 页码：2826 - 2834
- 全文大小：256K
- 年卷期：v.40,no.9(March 6, 2001)
- ISSN：1520-4995
文摘
Apolipoprotein E (apoE) is an important lipid-transport protein in human plasma and brain. Ithas three common isoforms (apoE2, apoE3, and apoE4). ApoE is a major genetic risk factor in heartdisease and in neurodegenerative disease, including Alzheimer's disease. The interaction of apoE withheparan sulfate proteoglycans plays an important role in lipoprotein remnant uptake and likely inatherogenesis and Alzheimer's disease. Here we report our studies of the interaction of the N-terminaldomain of apoE4 (residues 1-191), which contains the major heparin-binding site, with an enzymaticallyprepared heparin oligosaccharide. Identified by its high affinity for the N-terminal domain of apoE4, thisoligosaccharide was determined to be an octasaccharide of the structure 
UAp2S(1
[4)-
-D-GlcNpS6S(14)--L-IdoAp2S(1]34)--D-GlcNpS6S by nuclear magnetic resonance spectroscopy, capillaryelectrophoresis, and polyacrylamide gel electrophoresis. Kinetic analysis of the interaction between theN-terminal apoE4 fragment and immobilized heparin by surface plasmon resonance yielded a Kd of 150nM. A similar binding constant (Kd = 140 nM) was observed for the interaction between immobilizedN-terminal apoE4 and the octasaccharide. Isothermal titration calorimetry revealed a Kd of 75 nM for theinteraction of the N-terminal apoE fragment and the octasaccharide with a binding stoichiometry ofapproximately 1:1. Using previous studies and molecular modeling, we propose a binding site for thisoctasaccharide in a basic residue-rich region of helix 4 of the N-terminal fragment. From the X-ray crystalstructure of the N-terminal apoE4, we predicted that binding of the octasaccharide at this site wouldresult in a change in intrinsic fluorescence. This prediction was confirmed experimentally by an observedincrease in fluorescence intensity with octasaccharide binding corresponding to a Kd of ~1 
M.
UAp2S(1[4)--D-GlcNpS6S(14)--L-IdoAp2S(1]34)--D-GlcNpS6S by nuclear magnetic resonance spectroscopy, capillaryelectrophoresis, and polyacrylamide gel electrophoresis. Kinetic analysis of the interaction between theN-terminal apoE4 fragment and immobilized heparin by surface plasmon resonance yielded a Kd of 150nM. A similar binding constant (Kd = 140 nM) was observed for the interaction between immobilizedN-terminal apoE4 and the octasaccharide. Isothermal titration calorimetry revealed a Kd of 75 nM for theinteraction of the N-terminal apoE fragment and the octasaccharide with a binding stoichiometry ofapproximately 1:1. Using previous studies and molecular modeling, we propose a binding site for thisoctasaccharide in a basic residue-rich region of helix 4 of the N-terminal fragment. From the X-ray crystalstructure of the N-terminal apoE4, we predicted that binding of the octasaccharide at this site wouldresult in a change in intrinsic fluorescence. This prediction was confirmed experimentally by an observedincrease in fluorescence intensity with octasaccharide binding corresponding to a Kd of ~1 M.