Human LOXL2 adenoviral particles

Lysyl oxidase-like 2 (LOXL2) has been identified as an important mediator of extracellular matrix (ECM) remodeling in a variety of disease processes, including cardiovascular disease. As a result, there is growing interest in understanding the regulatory mechanisms of LOXL2 in cells and tissues. Although LOXL2 is present in cells and tissues in both full-length and processed forms, the exact identity of the proteases that process LOXL2 and the impact of processing on LOXL2 function remain incompletely understood. Here, researchers show that factor Xa (FXa) is a protease that processes LOXL2 at Arg-338. Processing by FXa does not affect the enzymatic activity of soluble LOXL2. However, in vascular smooth muscle cells, FXa treatment of LOXL2 results in reduced cross-linking activity in the ECM and shifts the substrate preference of LOXL2 from type IV to type I collagen. Furthermore, FXa treatment increases the interaction between LOXL2 and prototypic LOX, suggesting a potential compensatory mechanism to maintain the activity of total LOX in the vascular ECM. FXa expression is ubiquitous in various organ systems and plays a role similar to LOXL2 in the progression of fibrotic diseases. Therefore, processing of LOXL2 by FXa may have a significant impact on pathologies involving LOXL2.

Here, researchers exposed A7r5 cells to FXa for 48 h with or without rivaroxaban. Western blotting revealed loss of full-length LOXL2 in the ECM (Figure 1a, b). In the presence of FXa, prototypic LOX expression remained unchanged in the ECM and was only detected in precursors, suggesting that prototypic LOX contributes little to overall LOX activity. Next, total LOX activity in cell-derived ECM was measured to better capture enzymatic activity in situ to deposit cell-derived matrices. Intermediates generated by the catalytic activity of LOX on ECM proteins were biotinylated with biotin hydrazide (BHZ), labeled with fluorescein-conjugated streptavidin, and analyzed by confocal microscopy (Figure 1c). A7r5 cells were transduced with adenovirus to overexpress WT-LOXL2 (AdLOXL2). Overexpression of LOXL2 (AdLOXL2) resulted in an activity signal higher than that of the untransduced condition (control); in the presence of FXa, the activity signal was attenuated and restored by rivaroxaban (Figure 1d, e). Overexpression of the inactive LOXL2-DM and inhibition of LOXL2 using PAT-1251 both showed significantly lower activity signals compared to full-length wild-type LOXL2.

Figure 1. LOXL2 processing by FXa reduces total LOX activity in the cell-derived ECM. (Wang H, et al., 2023)