Distinct binding sites in the structure of α2-macroglobulin mediate the interaction with β-amyloid peptide and growth factors

Abstract

α2-Macroglobulin (α2M) and its receptor, low density lipoprotein receptor-related protein (LRP), function together to facilitate the cellular uptake and degradation of β-amyloid peptide (Aβ). In this study, we demonstrate that Aβ binds selectively to α2M that has been induced to undergo conformational change by reaction with methylamine. Denatured α2M subunits, which were immobilized on polyvinylidene difluoride membranes, bound Aβ, suggesting that α2M tertiary and quaternary structure are not necessary. To determine whether a specific sequence in α2M is responsible for Aβ binding, we prepared and analyzed defined α2M fragments and glutathione S-transferase-α2M peptide fusion proteins. A single sequence, centered at amino acids (aa) 1314-1365, was identified as the only major Aβ-binding site. Importantly, Aβ did not bind to the previously characterized growth factor-binding site (aa 718-734). Although the Aβ binding sequence is adjacent to the binding site for LRP, the results of experiments with mutated fusion proteins indicate that the two sites are distinct. Furthermore, a saturating concentration of Aβ did not inhibit LRP-mediated clearance of α2M-MA in mice. Using various methods, we determined that the KD for the interaction of Aβ with its binding site in the individual α2M subunit is 0.7-2.4 μM. The capacity of α2M to bind Aβ and deliver it to LRP may be greater than that predicted by the KD, because each α2M subunit may bind Aβ and the bound Aβ may multimerize. These studies suggest a model in which α2M has three protein interaction sites with distinct specificities, mediating the interaction with Aβ, growth factors, and LRP.