Structural Characterization of PTX3 Disulfide Bond Network and Its Multimeric Status in Cumulus Matrix Organization

Abstract

PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys 317 and Cys 318 are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3 ؊/؊ mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3 ؊/؊ mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.

Binding of the Long Pentraxin PTX3 to Factor H: Interacting Domains and Function in the Regulation of Complement Activation

“…Our findings indicate that the SCR19 -20 interacts with the N-terminal domain of PTX3, while the SCR7 binds the C-terminal domain, consistently with a previous report of CRP interacting with SCR7 (36). Furthermore, the lower level of binding of single PTX3 fragments in comparison to the full-length protein suggests the importance of structural organization of PTX3 into a full-size molecule and/or into polymers (51).…”

“…structures (51), it is possible that PTX3 polymers can bind multiple FH molecules. Importantly, the multimeric nature of PTX3 could allow it to interact with more than one ligand at the same time.…”

Selective up‐regulation of the soluble pattern‐recognition receptor pentraxin 3 and of vascular endothelial growth factor in giant cell arteritis: Relevance for recent optic nerve ischemia

“…Moreover, it is involved in the modulation of the innate response associated with ongoing cell death (9,10) and interacts with the extracellular matrix, contributing to the assembly of the cumulus oophorus (11). PTX3 synthesis is a hallmark of vascular injury (12).…”

Prototypic Long Pentraxin PTX3 Is Present in Breast Milk, Spreads in Tissues, and Protects Neonate Mice from Pseudomonas aeruginosa Lung Infection

“…5A). PTX3 is a multimeric protein composed of identical subunits linked by a network of disulfide bonds and high molecular masses (.50 kDa) correspond to not fully reduced PTX3 (39). In addition, we recently demonstrated that PTX3 is degraded by endogenous proteases (e.g., elastase), creating low molecular mass breakdown products (48).…”