Proprotein Convertase Processing Enhances Peroxidasin Activity to Reinforce Collagen IV

Abstract

The basement membrane (BM) is a form of extracellular matrix that underlies cell layers in nearly all animal tissues. Type IV collagen, a major constituent of BMs, is critical for tissue development and architecture. The enzyme peroxidasin (Pxdn), an extracellular matrix-associated protein, catalyzes the formation of structurally reinforcing sulfilimine cross-links within the collagen IV network, an event essential to basement membrane integrity. Although the catalytic function of Pxdn is known, the regulation of its activity remains unclear. In this work we show through N-terminal sequencing, pharmacologic studies, and mutational analysis that proprotein convertases (PCs) proteolytically process human Pxdn at Arg-1336, a location relatively close to its C terminus. PC processing enhances the enzymatic activity of Pxdn and facilitates the formation of sulfilimine cross-links in collagen IV. Thus, PC processing of Pxdn is a key regulatory step that contributes to its function and, therefore, supports BM integrity and homeostasis.

Keywords: basement membrane; collagen; collagen IV; extracellular matrix; furin; peroxidase; peroxidasin; proprotein convertase; sulfilimine bond.

FIGURE 1.

Heterologously expressed human peroxidasin is proteolyzed. A, schematic depiction of recombinant hPxdn with leucine repeat-rich (LRR), immunoglobulin (Ig), peroxidase, and vWFC domains. His₆ and V5 epitope tags were added to the C terminus. Validated intramolecular disulfide bridges are shown as connected lines, while suggested intermolecular disulfides are labeled S (8, 18). B, immunoblot of purified, recombinant hPxdn. C, immunoblot of ammonium sulfate-precipitated media under reducing conditions from HEK293 cells stably transfected with Pxdn and cultured without (control) or with indicated protease inhibitors. AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride) at 1 mm was cytotoxic. Blots were probed with anti-V5 antibody and are representative of three independent experiments.

FIGURE 2.

Pxdn undergoes PC processing. A, proprotein convertase cleavage site prediction results for Pxdn using the ProP 1.0 server (14). B, immunoblot of ammonium sulfate precipitated media under reducing conditions from HEK293 cells stably producing Pxdn that were either cultured without (−) or with (+) 100 μm decanoyl-RVKR-chloromethyl ketone (Dec-RVKR-CMK) proprotein convertase inhibitor. C, immunoblot of ammonium sulfate-precipitated media under reducing conditions from HEK293T cells transiently transfected with vector only (Mock), Pxdn, the PC inhibitor α1-antitrypsin Portland variant (PDX), or co-transfected with Pxdn and PDX (Pxdn + PDX). D, immunoblot of cell lysate and ammonium sulfate-precipitated media under reducing conditions from HEK293 cells stably expressing Pxdn. Blots were probed with anti-V5 antibody and are representative of three independent experiments.

FIGURE 3.

The C-terminal fragment of peroxidasin (Pxdn-PCF) released by proprotein convertase processing is a non-covalently linked oligomer. A, immunoblot of ammonium sulfate-precipitated media from HEK293 cells stably transfected with hPxdn. The migration of the proteolytically processed C-terminal fragment is unchanged with the addition of 50 mm dithiothreitol (DTT; arrow). Blots were probed with anti-V5 antibody. B, gel filtration chromatography elution profile based on absorbance at 280 nm (mAU) of proteolytically processed C-terminal fragment of Pxdn (Pxdn-PCF; solid line) and molecular mass standards (dashed line) run successively on GE S200 gel filtration column (GE Healthcare). C, estimation of molecular weight of PXDN-PCF using gel filtration chromatography retention volume of molecular weight standards (black) and PXDN-PCF (red).

FIGURE 4.

Proprotein convertases cleave peroxidasin at Arg-1336. A, schematic representation of the predicted Pxdn fragments that result upon proprotein convertase proteolytic processing. B, N-terminal Edman degradation sequencing cycles of the C-terminal fragment of Pxdn released after PC processing (Alphalyse Inc., Palo Alto, CA). C, immunoblot of ammonium sulfate-precipitated media under reducing conditions from HEK293 cells transiently transfected with hPxdn or hPxdn-RGAA and probed with anti-V5 antibody. Shown is a blot representative of three independent experiments.

FIGURE 5.

Proprotein convertase proteolysis of peroxidasin enhances enzymatic activity. A, ratio of absorbance at 415 nm and 280 nm for both Pxdn and Pxdn-RGAA demonstrating equal heme incorporation. B, peroxidase activity as determined by TMB oxidation measured as absorbance at 650 nm (A₆₅₀). C, HOBr production expressed as nmol of HOBr generated/nmol of enzyme/min measured in 1× PBS supplemented with 100 μm NaBr for wild type hPxdn (WT) and mutant hPxdn-RGAA (RGAA). In all panels individual data points are displayed with mean (dotted line) and S.D. Data were analyzed using t test or analysis of variance followed by post hoc pairwise comparisons with Bonferroni's correction for multiple comparisons (**, p ≤ 0.01 compared with WT Pxdn).

FIGURE 6.

Proprotein convertase processing of peroxidasin enhances the formation of sulfilimine cross-links in collagen IV. A and B, CRISPR generated Pxdn knock out PFHR-9 cells (Control) were rescued by stable transfection of either wild type (WT) or the mutant hPxdn-RGAA (RGAA). The matrix containing collagen IV network was isolated 5 days post confluency. The number of cross-links per collagen IV NC1 hexamer was determined using densitometric quantitation of the dimeric and monomeric subunits from SDS-PAGE of the collagenase-digested matrix. C and D, untransfected HEK293 cells or HEK 293 cells stably transfected with either wild type (WT) or the mutant hPxdn-RGAA (RGAA) were plated on PFHR-9 uncross-linked matrix, and the underlying matrix was isolated 24 h after overlay for analysis of collagen IV sulfilimine cross-link content. The number of cross-links per collagen IV NC1 hexamer was determined using densitometric quantitation of the dimeric and monomeric subunits from SDS-PAGE of the collagenase-digested matrix. In all panels individual data points are displayed with mean (dotted line) and S.D. Data were analyzed using analysis of variance followed by post hoc pairwise comparisons with Bonferroni's correction for multiple comparisons (*, p ≤ 0.05; **, p ≤ 0.01 compared with WT Pxdn).

FIGURE 7.

The presence of a C-terminal proprotein convertase recognition sequence in peroxidasin is evolutionarily conserved. Shown is a schematic representation of peroxidasin among differing species. The presence of a predicted PC recognition sequence is found across several Eumetazoa phyla. Sequence data were gathered from National Center for Biotechnology Information Reference Sequence. PC recognition site scores were predicted by the ProP 1.0 server (14).