Identification and regulation of the high affinity binding site of the Alzheimer's disease amyloid protein precursor (APP) to glycosaminoglycans

Abstract

The specific binding of the amyloid protein precursor (APP) to glycosaminoglycans (GAG) suggests that APP is a cell adhesion molecule (CAM) and/or substrate adhesion molecule (SAM). In order to characterize this activity of APP in the brain at the molecular level, we have purified and characterized the major APP species from rat brain. The major isoform isolated was sequenced and found to be APP 695. In a solid-phase binding assay, the specificity of this brain-specific APP isoform-GAG interaction was analysed. The binding of APP to the glycosaminoglycan heparin was found to be time-dependent and saturable. A strong heparin-binding site within a region conserved in rodent and human APP, APLP1 and APLP2, was identified. Saturable binding to heparin through this binding site was found to occur at nmol concentrations of APP. This putative high-affinity site was then located within a sequence of 22 amino acids in length corresponding to residues 316–337 of APP 695. This sequence is encoded by APP exon 9 and the first three codons of exon 10. Since all APP and L-APP isoforms so far described include these exons, the strong heparin binding site is a ubiquitous feature of all APP and L-APP isoforms strongly suggesting that the brain-specific and neuronal, as well as the non-neuronal and peripheral APPs and L-APPs do have CAM- and SAM-like activities. Certain metal ions including zinc (II) have been proposed as risk factors in Alzheimer's disease (AD). Recently we showed that APP binds zinc(II) at higher nmol concentrations. We identified this zinc binding site to be located within the sequence of APP encoded by exon 5. Because zinc ions are involved in tissue repair and because of the CAM- and SAM-activity of APP, the mechanisms by which zinc(II) may exert an influence on the pathogenesis of AD was sought by studying the binding of APP to heparin in the presence of varying zinc(II) concentrations. We found that zinc(II) is a modulating factor for the CAM- and SAM-activity of APP that is exerted through the heparin-like GAG side-chains of heparansulfate proteoglycans (HSPGs). Because the binding of APP to GAG in the presence of zinc(II) resembles an allosteric interaction, the proposed disturbance of zinc(II) homeostasis in Alzheimer's disease may have an effect on conformation and stability of APP and regulate the release of amyloid βA4 protein.