Interaction of the N-terminal domain of apolipoprotein E4 with heparin

Apolipoprotein E (apoE) is an important lipid-transport protein in human pl asma and brain. It has three common isoforms (apoE2, apoE3, and apoE4), Apo E is a major genetic risk factor in heart disease and in neurodegenerative disease, including Alzheimer's disease. The interaction of apoE with hepara n sulfate proteoglycans plays an important role in lipoprotein remnant upta ke and likely in atherogenesis and Alzheimer's disease, Here we report our studies of the interaction of the N-terminal domain of apoE4 (residues 1-19 1), which contains the major heparin-binding site, with an enzymatically pr epared heparin oligosaccharide. Identified by its high affinity for the N-t erminal domain of apoE4, this oligosaccharide was determined to be an octas accharide of the structure Delta UAp2S(1-->[4)-alpha -D-GlcNpS6S(1-->4)-alp ha -L-IdoAp2S(1-->](3)4)-alpha -D-GlcNpS6S by nuclear magnetic resonance sp ectroscopy, capillary electrophoresis, and polyacrylamide gel electrophores is. Kinetic analysis of the interaction between the N-terminal apoE4 fragme nt and immobilized heparin by surface plasmon resonance yielded a K-d of 15 0 nM. A similar binding constant (K-d = 140 nM) was observed for the intera ction between immobilized N-terminal apoE4 and the octasaccharide. Isotherm al titration calorimetry revealed a K-d of 75 nM for the interaction of the N-terminal apoE fragment and the octasaccharide with a binding stoichiomet ry of approximately 1:1. Using previous studies and molecular modeling, we propose a binding site for this octasaccharide in a basic residue-rich regi on 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 predict ion was confirmed experimentally by an observed increase in fluorescence in tensity with octasaccharide binding corresponding to a K-d of similar to1 m uM.