![]() ![]() Single binding experiments and concentrations of ligands used are specified in the respective figure legends. For binding, the ligands were diluted in PBS/T, except for the calcium-dependency study, where the absence of calcium was assured by the use of PBS without divalent cations and the addition of 10 mM EGTA. All reaction volumes were 100 μl and plates were washed after each step with PBS ++ containing 0.05% v/v Tween 20 (PBS/T). Nonspecific binding to the plates was blocked with 5% dry milk in PBS ++ for 2 h at room temperature. Microtiter plates (NUNC) were coated with FH (5 μg/ml), PTX3 (10 μg/ml), or C3b (10 μg/ml) in PBS with Ca 2+ and Mg 2+ (PBS ++ contains 1.17 mM CaCl 2 and 1.05 mM MgCl 2 Lonza) by overnight incubation at 4☌. Recent studies in gene-modified mice have shown that PTX3 plays complex nonredundant functions in vivo, ranging from innate immunity against diverse microorganisms to the assembly of a hyaluronic acid-rich extracellular matrix and female fertility ( 2, 7, 14, 16, 17, 20, 21, 22). PTX3 binds to the complement component C1q, apoptotic cells, the extracellular matrix component TNF-stimulated gene 6 and selected microorganisms, including Aspergillus fumigatus and Pseudomonas aeruginosa ( 13, 14, 15, 16, 17, 18, 19). PTX3 is rapidly produced and released by several cell types in response to primary inflammatory signals (e.g., TLR engagement, TNF-α, IL-1β) ( 2, 7, 8, 9, 10, 11) and its production is amplified by IL-10 ( 12). However, it has an unrelated long N-terminal domain coupled to the C-terminal pentraxin domain, and differs in gene organization, cellular source, inducing stimuli and ligands recognized ( 2, 6). The prototypic long pentraxin PTX3 shares similarities with the classical short pentraxins. ![]() ![]() Thus, our findings identify PTX3 as a unique FH ligand in that it can bind both of the two hot-spots of FH, namely SCR7 and SCR19–20 and indicate that PTX3 participates in the localization of functionally active FH. Surface-bound PTX3 enhances FH recruitment and iC3b deposition and PTX3-bound FH retains its activity as a cofactor for factor I-mediated C3b cleavage. The FH Y402H polymorphism, which affects binding to the short pentraxin CRP, did not affect binding to PTX3. ![]() The primary binding site is located on FH domains 19–20, which interact with the N-terminal domain of PTX3, while a secondary binding site on domain 7 binds the glycosylated PTX3 pentraxin domain. We report that PTX3 binds FH with an apparent K d of 1.1 × 10 −7 M, and define two binding sites for PTX3 on FH. This study was designed to investigate the interaction of PTX3 with factor H (FH), the main soluble alternative pathway regulatory protein. As an acute phase protein, PTX3 binds to the classical pathway complement protein C1q, limits tissue damage in inflammatory conditions by regulating apoptotic cell clearance, and plays a role in the phagocytosis of selected pathogens. The long pentraxin PTX3 is a multifunctional soluble molecule involved in inflammation and innate immunity. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |