Human alpha 2M-macroglobulin (alpha 2M) is an abundant plasma protein capable of inhibiting a very broad spectrum of proteinases through a unique trapping mechanism, in which the proteinase is physically sequestered through a massive conformational change within a2M. This conformational change also exposes the receptor binding domain (RBD) of a2M, thus allowing binding of a2M-proteinase complexes to their specific cell-surface receptor, LRP (the low density lipoprotein receptor-related protein), and consequent clearance and degradation of the complexes. The a2M-proteinase complex is only one of a relatively large number of structurally diverse protein ligands, including certain lipoproteins, that can bind to LRP and be internalized. LRP may therefore be involved in regulation of both proteinase and lipoprotein metabolism, and in cases of natural variants, be involved in pathological processes. The long term goals are to understand at a molecular level the interactions between a2M and LRP and hence the role of a2M and LRP in pathological processes that result from such mutations that lead to aberrations in metabolism of proteinases and other protein ligands. Three hypotheses related to a2M and LRP will be tested. (i) That binding of a2M-proteinase complexes and other protein ligands to LRP involves relatively weak combinatorial interactions between the ligands and structural repeats within LRP that together result in high affinity. (ii) That there is a specific interaction between beta-amyloid peptide and a2M that enhances a2M binding to LRP and hence peptide clearance and thus plays an ameliorating role in Alzheimer's disease. (iii) That bait region truncations could lead to a2M with altered proteinase inhibitor properties that could contribute to the etiology of diseases such as Alzheimer's disease.
Five specific aims are proposed. (i) To determine the full binding site within LRP for RBD and to determine its structure in solution by NMR spectroscopy. (ii) To determine the basis for specific binding of RBD to LRP by mutagenesis and biophysical characterization. (iii) To determine the specificity of binding of RAP (receptor-associated protein) to LRP by NMR spectroscopy. (iv) To determine the nature and consequences of binding of beta-amyloid peptide to a2M by calorimetry and NMR spectroscopy. (v) To characterize the bait region variants of a2M that could be products of alternative splicing of the a2M gene, to determine any alteration in proteinase inhibitory properties. The presence of such variants in plasma from Alzheimer's patients will be determined.
