Proprotein Convertase Processing Enhances Peroxidasin Activity to Reinforce Collagen IV

Abstract

Edited by Amanda FosangThe 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.

Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1

“…This was confirmed in a recent study that showed the cleavage of trimeric hsPxd01 at Arg 1336 C-terminal of the peroxidase domain by a proprotein convertase (8). The truncation eliminates the von Willebrand factor and renders the peroxidase more active.…”

“…However, elimination of the LRR and VWC domains increased the activity of the respective recombinant construct (5). Recently, Colon and Bhave (8) demonstrated that proprotein convertase processing enhances peroxidasin 1 activity by elimination of the VWC domains and proposed that this event represents a key step in the biosynthesis and function of hsPxd01 to support basic membrane and tissue integrity. These findings motivated us to design several constructs, including the POX domain only to search for a functional and well folded protein for first comprehensive pre-steady-state kinetic studies.…”

“…The truncation eliminates the von Willebrand factor and renders the peroxidase more active. This proteolytic maturation seems to represent a key regulatory event in hsPxd01 biosynthesis and function because the C-terminal proprotein convertase recognition sequence is evolutionarily conserved throughout the animal kingdom (8). …”

Proteolytic processing of lysyl oxidase–like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV

“…Inhibition experiments with aprotinin, PMSF, and Dec-RVKR-CMK indicated that a serine protease is likely to be responsible for the proteolytic processing of LOXL2. Interestingly, it has recently been shown that a proprotein convertase (PC) activates peroxidasin, the enzyme responsible the forma-tion of sulfilimine crosslinks in the C-terminal noncollagenous domain (NC1) of collagen IV (36). Although the blockade of LOXL2 proteolytic processing by Dec-RVKR-CMK inhibitor suggested the involvement of a PC, this family of proteases has a strong preference for Arg in P1 in the motif R-X-R/K-R.…”

Role of Hypohalous Acids in Basement Membrane Homeostasis

“…The catalytic efficiency is increased with a truncated PXDN construct containing only the Ig and heme peroxidase domains (94,126), suggesting that the LRR and/ or the vWFC domains may ''auto-inhibit'' catalytic activity. Indeed, proprotein convertase processing of PXDN with resulting loss of the vWFC domain enhances PXDN-mediated HOBr generation (20). Whether proteolytic processing or conformational changes involving the LRR domain also activate PXDN is unknown.…”