Application of a Novel In Situ Activity Assay to Detect Lysyl Oxidase Like‐2 (LOXL2)

Abstract

IntroductionLysyl oxidase like‐2 (LOXL2) is an enzyme of the lysyl oxidase (LOX) super family, which are amine oxidases that initialize collagen and elastin cross‐linking to form a stable and organized matrix. Augmented LOXL2 expression is highly associated with diverse disease processes with a known element of matrix accumulation including fibrosis, pulmonary hypertension, and cancer metastasis. To date, lack of sensitive and specific assays that can detect LOXL2 catalytic function in situ in the extracellular matrix (ECM) of cells and tissue has limited the ability to fully elucidate the role of LOXL2 in pathobiological mechanisms. Existing assays are limited in dynamic range and are unfeasible to interrogate matrix‐associated LOXL2 as they require LOXL2 to be in solution. We developed a novel in situ activity assay that overcomes these limitations. Our previous proof‐of‐concept experiments showed that the assay is able to detect overexpression of LOXL2 or loss of enzymatic function in cell culture. Here, we applied the assay in biologically relevant specimens.Materials and MethodsIn our assay, we utilize the strong reaction between hydrazide and aldehyde to label the LOX catalyzed allysine (−CHO) residues with biotin hydrazide (BHZ). This allows for a robust method of visualizing all ECM protein sidechains that have been catalyzed by LOXs via biotin‐streptavidin interaction. This assay has been incorporated in a few of our studies on LOXL2. First, we tested the expression and activity level of various LOXL2 mutant proteins overexpressed in A7r5 cells via adenoviral delivery. Second, we used this assay on A7r5 cells to study the modulation of LOXL2 activity when processed by protease Factor Xa (FXa). Lastly, we tested expression of LOXL2 in aortas from wild type and LOXL2+/− mice, which were cut into 5mm rings and transected open. In all experiments, samples were incubated with 50–150nM BHZ for 24 hours to allow for extracellular matrix deposition and labeling of LOXL2 catalysis. Biotinylation of allysines and expression of LOXL2 were then visualized by confocal immunofluorescence (IF) microscopy.ResultsAs seen in Figure 1, cells overexpressing LOXL2‐wt showed significantly increased biotinylation, suggesting an increase in LOXL2 activity as expected. Fibrous structures can also be seen for longer BHZ incubation as new matrix form. Whereas cells overexpressing LOXL2DM showed no difference in biotinylation compared to control, as LOXL2DM cannot catalyze oxidative deamination to produce allysines. Additional conditions have also been tested to study the functions of different domains of LOXL2. Activity assay on cells exposed to FXa processing showed that processing disrupted LOXL2 catalysis. Lastly, we were able to clearly visualize significant difference in LOXL2 expression in Young and Old WT vs. LOXL2 +/− aorta, confirming that the assay is feasible not only in cellular experiments but also on tissue samples.ConclusionsWe were able to visualize LOXL2 reaction in the ECM by IF microscopy through the biotinylation by BHZ. This study demonstrates different applications of a novel in situ activity assay for LOXL2 in both cell culture and tissue samples, which can be impactful for future research that studies LOXL2 as a therapeutic target.Support or Funding InformationJHU ACCM StAAR grant and MedImmuneIF images of A7r5 cells overexpressing wild type LOXL2 and enzymatically inactive LOXL2DM. Cells were incubated with biotin hydrazide for 24 hours.Figure 1IF confocal Zstack maximum projection images of aortas from Young (<16 weeks) and Old (>52 weeks) WT and LOXL2+/− mice.Figure 2