Abstract
Pentraxin 3 (PTX3), a long pentraxin subfamily member in the pentraxin family, plays an important role in innate immunity as a soluble pattern recognition receptor. Plasma PTX3 is elevated in sepsis (∼200 ng/ml) and correlates with mortality. The roles of PTX3 in sepsis, however, are not well understood. To investigate the ligands of PTX3 in sepsis, we performed a targeted proteomic study of circulating PTX3 complexes using magnetic bead-based immunopurification and shotgun proteomics for label-free relative quantitation via spectral counting. From septic patient fluids, we successfully identified 104 candidate proteins, including the known PTX3-interacting proteins involved in complement activation, pathogen opsonization, inflammation regulation, and extracellular matrix deposition. Notably, the proteomic profile additionally showed that PTX3 formed a complex with some of the components of neutrophil extracellular traps. Subsequent biochemical analyses revealed a direct interaction of bactericidal proteins azurocidin 1 (AZU1) and myeloperoxidase with PTX3. AZU1 exhibited high affinity binding (KD = 22 ± 7.6 nm) to full-length PTX3 in a calcium ion-dependent manner and bound specifically to an oligomer of the PTX3 N-terminal domain. Immunohistochemistry with a specific monoclonal antibody generated against AZU1 revealed a partial co-localization of AZU1 with PTX3 in neutrophil extracellular traps. The association of circulating PTX3 with components of the neutrophil extracellular traps in sepsis suggests a role for PTX3 in host defense and as a potential diagnostic target.
Highlights
From the ‡Department of Molecular Biology and Medicine and the **Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan, the Departments of §Emergency and Critical Care Medicine and ‡‡Cardiology, Juntendo University Nerima Hospital, Tokyo 177-0033, Japan, the ¶Department of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan, and Perseus Proteomics Inc., Tokyo 153-0041, Japan
The stored Pentraxin 3 (PTX3) in neutrophils is released into the extracellular space and localizes to neutrophil extracellular traps (NETs) [4], which are extracellular fibers consisting of DNA, histones, and antimicrobial proteins that capture and kill pathogens [5]
Further investigation revealed that azurocidin 1 [21], a bactericidal protein that localizes to NETs, was one of these direct PTX3 interacting partners that act through the oligomer N-terminal domain of PTX3 in a calcium ion-dependent manner
Summary
From the ‡Department of Molecular Biology and Medicine and the **Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan, the Departments of §Emergency and Critical Care Medicine and ‡‡Cardiology, Juntendo University Nerima Hospital, Tokyo 177-0033, Japan, the ¶Department of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan, and Perseus Proteomics Inc., Tokyo 153-0041, Japan. We found novel interactants, including some of the components of NETs, as well as known PTX3 ligands such as complement and extracellular matrix proteins. The proteomic profile of the PTX3 complexes in these septic patients included protein components of NETs, as well as the known PTX3 ligands, such as complement and extracellular matrix component proteins (Fig. 3C).
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