Interaction of the N-Terminal Domain of Apolipoprotein E4 with Heparin

Abstract

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.

Inhibition of Apolipoprotein E-Related Neurotoxicity by Glycosaminoglycans and Their Oligosaccharides

“…This higher IC 50 could be due to a conformational change of the heparin molecule induced by the excess of N-sulfo groups, thus resulting in a lower affinity binding between the N-sulfonated heparin and the apoE peptide. The importance of the N-sulfo groups for the inhibition of apoE peptide toxicity is in accordance with a recent study showing that N-sulfo groups are required for the formation of a high affinity complex between heparin and apoE4 (44). Selective 2-O-desulfonation, 6-O-desulfonation, and total O-desulfonation decreased the protective effect by 2-, 2.5-, and 8-fold, respectively.…”

Dromedary glycosaminoglycans: Molecular characterization of camel lung and liver heparan sulfate

“…The disaccharide composition of camel liver HS, however, is strikingly different from that of other liver HS samples examined in our laboratory, with low levels of both unsulfated (ΔUA-GlcNAc) and trisulfated (ΔUA2S-GlcNS6S) disaccharides. Of particular note is the exceedingly low level of trisulfated disaccharide, since a octasaccharide sequence comprised of this repeating disaccharide corresponds to the liver receptor for apoE (Dong et al, 2001) as well as a receptor for the circumsporozoite form of the malaria parasite (Rathore et al, 2001). This peculiar structural feature of camel HS may play a role in maintaining the lower level of camel LDL compared to the LDL levels reported in other mammals (Nazifi et al, 2000).…”

“…Liver is also a target for a number of pathogens and liver HS has been demonstrated in several cases to play a pivotal role in infectivity (Chen et al, 1997;Rathore et al, 2001;Barth et al, 2003). Liver HSPGs at the endothelial level act as critical receptors for apoE and is involved in lipid metabolism (Bocksch et al, 2001;Dong et al, 2001;Bazin et al, 2002).…”

Crystal structure of the OpcA integral membrane adhesin from Neisseria meningitidis

“…First, we would anticipate that the binding site is located on the outer surface of the protein; the crevice is the dominant feature in the surface structure of the external loop regions. Second, structural studies of heparin-binding sites in other proteins have shown a preponderance of basic side chains (30)(31)(32)(33)(34)(35). Third, the dimensions of the crevice could plausibly accommodate a heparin polysaccharide (36).…”

“…None of the heparin-binding sites in this structure forms a well defined crevice, but they all contain Lys and Arg residues which form specific hydrogen bonds to sulfate groups in the heparin ligand. In other cases, structures of heparin-binding proteins are available in the unliganded state, and supporting biochemical information has helped to identify the heparin-binding site (33)(34)(35). The proposed heparin-binding site on OpcA is notable for the pronounced crevice structure and also for the patch of positive charge in this region.…”