Structure and activity of human clusterin.

Abstract

Clusterin is a heterodimeric disulfide linked glycoprotein secreted by many epithelia in adult and developing tissues. Clusterin is also induced in response to diverse injury or cell death stimuli. The protein interacts with, and is suggested to have, a relatively non specific affinity for markedly hydrophobic/amphipathic ligands including terminal complement components, Alzheimer's disease amyloid beta (Abeta) peptides, and apo AI-HDL in addition to immunoglobulins, TGFbeta receptors, heparin and the endocytic receptor LRP2. Little is known of clusterin structure and function. Sequences modeled as amphipathic alpha helices have been proposed as promiscous binding sites(s) for hydrophobic ligands. A reanalysis of these sequences using the COIL algorithm predicts a single conserved coiled-coil helix in each of the alpha and beta chains. These overlap, with heptads aligned, when each chain is aligned (antiparallel) with respect to the pattern of interchain disulfide bonds, and may reflect formation of an antiparallel coiled-coil with a cationic face that is proposed as an LRP2\heparin binding site. Distinct alpha and beta chain amphipathic helices encoded by exon 5 may constitute the hydrophobic ligand binding site. Partial proteolysis predominantly within or near these latter helices accompanies expression of human clusterin in Pichia pastoris and is associated with significant decreases in specific binding activity to IgG and Abeta 1-40 but not to LRP2 overexpressing differentiated F9 cells, in agreement with these binding site assignments. Although Zn+2 polymerized C9 (poly C9) and Abeta1-40 are mutual heterologous competitive clusterin-binding inhibitors, the specific binding of the recombinant protein to poly C9 is not as significantly diminished, suggesting a distinct but nearby binding site for this presumptive hydrophobic ligand. The C9 binding site is likely to be distinct from that of LRP2 since like IgG and Abeta 1-40 but unlike F9 cell surface binding poly C9 binding is not inhibited by anti-human clusterin monoclonal antibody G7. Distinct interactions may also mediate clusterin recognition of IgG and Abeta1-40 since mild acidification and diethylpyrocarbonate (DEPC) modification of clusterin histidine side chains had markedly different effects on each interaction. Binding to Abeta1-40 is singularly and dramatically enhanced by mild acidification and diminished by DEPC modification suggesting that a salt bridge between a clusterin imidazole and an acidic side chain of Abeta1-40, reminiscent of electrostatic interactions postulated to stabilize Abeta peptide monomers in polymeric fibrils, in addition to hydrophobic interactions may be important in the stability of the clusterin-Abeta 1-40 complex. These findings support the hypothesis that the interaction of clusterin with some hydrophobic ligands is highly specific and suggest conversely a physiological function specifically related to these interactions.