Multidomain Human Peroxidasin 1 Is a Highly Glycosylated and Stable Homotrimeric High Spin Ferric Peroxidase

Monika Soudi,Martina Paumann-Page,Cedric Delporte,Katharina F Pirker,Marzia Bellei,Eva Edenhofer,Gerhard Stadlmayr,Gianantonio Battistuzzi,Karim Zouaoui Boudjeltia,Paul G Furtmüller,Pierre Van Antwerpen,Christian Obinger

doi:10.1074/jbc.m114.632273

Abstract

Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Here, hsPxd01 and various truncated variants lacking nonenzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure, and unfolding pathway are reported. The homotrimeric iron protein contains a covalently bound ferric high spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of -233 ± 5 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (kcat/KM(app)) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking.

Highlights

Human peroxidasin 1 mediates the formation of sulfilimine cross-links within the collagen IV scaffold of basement membranes
Properties of Recombinant Protein and Oligomeric Structure—The average yield of recombinant hsPxd01 produced in HEK 293 cells by stable transfection was 0.5– 0.9 mg per liter of harvested media
Recombinant human peroxidasin 1 either produced by stable or transient transfection applied to SDS-PAGE had a molar mass of Ͼ500 kDa (Fig. 2A, lanes 1–3)

Summary

Background

Human peroxidasin 1 (hsPxd01) mediates the formation of sulfilimine cross-links within the collagen IV scaffold of basement membranes. Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (kcat/ KM app) of full-length hsPxd is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking. This prompted us to study the biophysical and biochemical characteristics of human peroxidasin 1 for a better understanding of its mode of function and mechanism

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION