Peroxidasin Is Secreted and Incorporated into the Extracellular Matrix of Myofibroblasts and Fibrotic Kidney

Abstract

Mammalian peroxidases are heme-containing enzymes that serve diverse biological roles, such as host defense and hormone biosynthesis. A mammalian homolog of Drosophila peroxidasin belongs to the peroxidase family; however, its function is currently unknown. In this study, we show that peroxidasin is present in the endoplasmic reticulum of human primary pulmonary and dermal fibroblasts , and the expression of this protein is increased during transforming growth factor-␤1-induced myofibroblast differentiation. Myofibroblasts secrete peroxidasin into the extracellular space where it becomes organized into a fibril-like network and colocalizes with fibronectin, thus helping to form the extracellular matrix. We also demonstrate that peroxidasin expression is increased in a murine model of kidney fibrosis and that peroxidasin localizes to the peritubular space in fibrotic kidneys. In addition, we show that this novel pathway of extracellular matrix formation is unlikely mediated by the peroxidase activity of the protein.

Structure–function analysis of peroxidasin provides insight into the mechanism of collagen IV crosslinking

“…When we immunostained PXDN in Cos7 cells that heterologously expressed the protein we observed a prominent localization within the endoplasmic reticulum (ER) (Figure 7) as described in our earlier paper (9). Besides the ER localization (Figure 7A-D) we also observed punctuate structures that were strongly positive for PXDN ( Figure 7A, B, D).…”

“…All of the examined constructs showed the characteristic appearance in the endoplasmic reticulum, which was previously described by us for both endogenouslyand heterologously expressed PXDN (9). Intriguingly, wild-type PXDN or mutants which were previously shown to retain their ability to form trimers, were also detected in separate, (22;23).…”

“…Sera of the immunized animals were depleted of GST-specific antibodies and then were affinity-purified with Affigel 10 (Bio-Rad) coupled to PXDN peptides which were previously used for the immunization. Specificity of α-mmPXDN was tested with knockout control in all the described applications while validation of α-hsPXDN has already been reported in our previous paper (9). Monoclonal α-V5 antibody used for Western blot and immunostaining experiments was purchased from AbD Serotec.…”

“…In our previous study we demonstrated the widespread expression of the pxdn gene and the presence of the protein in different types of human primary cells including pulmonary, dermal fibroblasts and HUVECs (9). To get a better understanding of the function of this complex protein first we decided to study the supramolecular organization of PXDN which was previously found to oligomerize into trimers in Drosophila (6).…”

Early-onset obesity and paternal 2pter deletion encompassing the ACP1, TMEM18, and MYT1L genes

“…It encodes for a heme-containing enzyme, and is involved in extracellular matrix formation. 13 PXDN expression was not detected in brain or peripheral blood leukocytes. 14 MYT1L (see next paragraph) has not yet been described as candidate gene in obesity but six patients (P1-4 and P5, SP6 with interstitial deletion) are deleted for MYT1L and show hyperphagia (Figure 3a).…”