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Clusterin, the human apolipoprotein and complement inhibitor, binds to complement C7, C8 beta, and the b domain of C9
1Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.
Insights
Clusterin protein inhibits the complement system by binding to C7, C8 alpha, and C9b components of the terminal complement complex (TCC, C5b-9). This interaction prevents TCC-mediated cell lysis and C9 polymerization.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Clusterin is a multifunctional protein involved in various physiological processes.
- It plays a role in regulating the terminal complement complex (TCC, C5b-9) activity.
- Clusterin, along with vitronectin, neutralizes the lytic potential of C5b-9.
Purpose of the Study:
- To elucidate the specific binding interactions of clusterin with TCC components.
- To understand the molecular mechanisms underlying clusterin's complement inhibitory function.
Main Methods:
- Ligand blotting using radiolabeled clusterin ([125I]clusterin).
- Analysis of binding interactions with isolated TCC components (C5b-6, C7, C8, C9) and vitronectin.
- Competition assays to identify specific binding sites.
Main Results:
- Clusterin specifically binds to C7, the beta-subunit of C8, and C9.
- Binding to C9 is mediated by a site exposed during its conformational transition, specifically the C9b fragment.
- Clusterin subunits inhibit both C5b-9-mediated hemolysis and Zn2+-induced C9 polymerization.
Conclusions:
- Clusterin inhibits TCC by interacting with a shared structural motif on C7, C8 alpha, and C9b.
- These interactions prevent the formation of a functional membrane attack complex and subsequent cell lysis.
Abstract:
Clusterin is a heterodimeric multifunctional protein expressed in a variety of tissues and cells. It forms high density lipid complexes in plasma and participates in the control of the lytic activity of the late complement complex (TCC, C5b-9). Together with vitronectin, clusterin binds to the nascent amphiphilic C5b-9 complex, rendering it water soluble and lytically inactive. To define the interactions that underlie the complement-inhibitory function of clusterin, we have examined the binding interactions between [125I]clusterin and the isolated components of the complex, C5b-6, C7, C8, and C9 and vitronectin. By using ligand blotting in the presence of Tween, specific binding of the labeled clusterin with C7, the beta-subunit of C8 and C9 was detected. Binding to C9 was competed by polymerized C9, but not by C8, C7, C6, and CD59, suggesting that the conformational change occurring during the hydrophilic-amphiphilic transition of C9 exposes the interaction site for clusterin. When thrombin-treated C9 was analyzed, clusterin was found to recognize the C9b fragment containing the hydrophobic membrane interaction segment. Both subunits of clusterin interact with C9 and are similarly potent in inhibiting C5b-9-mediated hemolysis and Zn+(+)-induced C9 polymerization. These results show that clusterin exerts its inhibitory effect by interacting with a structural motif common to C7, C8 alpha, and C9b.