The Proteomic Profile of Circulating Pentraxin 3 (PTX3) Complex in Sepsis Demonstrates the Interaction with Azurocidin 1 and Other Components of Neutrophil Extracellular Traps

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.

Atypical Hemolytic Uremic Syndrome-Associated Variants and Autoantibodies Impair Binding of Factor H and Factor H-Related Protein 1 to Pentraxin 3

“…Direct binding of native CFH, CFHL1, and CFHR1 was detected from human plasma. A recent study that analyzed circulating PTX3 complexes in sepsis using a proteomics approach also detected CFHR1 (46). These data further support the presence of two PTX3 binding sites within CFH: .…”

Efficacy of PTX3 and Posaconazole Combination in a Rat Model of Invasive Pulmonary Aspergillosis

“…1d). These data are in agreement with the reported binding of PTX3 to MPO (16) and suggest that trapping of MPO by PTX3 in the lung could enhance fungal clearance. The plasma concentration of endogenous PTX3 in rat is in the range of 20 to 30 ng/ml (data not shown).…”

Identification of Wilms' Tumor 1-associating Protein Complex and Its Role in Alternative Splicing and the Cell Cycle

“…Liquid Chromatography-Tandem Mass Spectrometry (LC/ MS/MS)-The immunopurified (IP) samples were subjected to in-solution trypsin digestion followed by LC/MS/MS using an LTQ Orbitrap XL ETD mass spectrometer (Thermo Scientific), as described previously (25). All of the MS/MS samples were analyzed using Mascot (version 2.4.0; Matrix Science, London, UK).…”