Development of a Novel In Situ Activity Assay for Lysyl Oxidase Like‐2 (LOXL2)

Abstract

Lysyl oxidase like‐2 (LOXL2) is a member of the lysyl oxidase (LOX) super family, which are copper‐dependent 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.Current methods to detect LOXL2 enzymatic activity are inefficient and limited in scope. The most widely used assay is hydrogen peroxide based: oxidation of the LOX active site occurs when substrate is added to samples, and releases hydrogen peroxide. The level of hydrogen peroxide is detected using an HRP‐coupled system, that utilizes the conversion of Amplex Red to the fluorescent end product, resorufin. While the results are semi‐quantitative, this assay has limited dynamic range and cannot distinguish hydrogen peroxide arising from other pathways. Moreover, the assay requires LOXL2 in solution. The harsh conditions required to obtain a solubilized ECM sample, where the pertinent LOXL2 activity occurs, cause LOXL2 unfolding and loss of activity, rendering this assay unfeasible. Thus, there is a need to develop a sensitive, specific, and practicable in situ activity assay that overcomes these limitations.This study provides a proof of concept for a novel in situ activity assay for LOXL2. Here, we developed an assay to fluorescently label the products of LOXL2 catalysis. Specifically, LOXL2 catalyzes an oxidative de‐amination of lysine and hydroxylysine residues (found on collagen and elastin) to highly reactive semi‐aldehydes called allysines. In this assay, we labeled the newly generated allysine residues with biotin hydrazide using 1‐ethyl‐3‐(3‐dimethyl‐aminopropyl) carbodiimide hydrochloride (EDC) crosslinker. This biotinylation allows for a robust method of measuring LOXL2 activity through western blotting, immunoprecipitation and immunofluorescence imaging using biotin‐streptavidin interactions.Support or Funding InformationFunded by MedImmune research funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.