An in situ activity assay for lysyl oxidases

Huilei Wang,Alan Poe,Jochen Steppan,Sandeep Jandu,Lakshmi Santhanam,Kavitha Nandakumar,Reik Löser,Lydia Pak

doi:10.1038/s42003-021-02354-0

Abstract

The lysyl oxidase family of enzymes (LOXs) catalyze oxidative deamination of lysine side chains on collagen and elastin to initialize cross-linking that is essential for the formation of the extracellular matrix (ECM). Elevated expression of LOXs is highly associated with diverse disease processes. To date, the inability to detect total LOX catalytic function in situ has limited the ability to fully elucidate the role of LOXs in pathobiological mechanisms. Using LOXL2 as a representative member of the LOX family, we developed an in situ activity assay by utilizing the strong reaction between hydrazide and aldehyde to label the LOX-catalyzed allysine (-CHO) residues with biotin-hydrazide. The biotinylated ECM proteins are then labeled via biotin-streptavidin interaction and detected by fluorescence microscopy. This assay detects the total LOX activity in situ for both overexpressed and endogenous LOXs in cells and tissue samples and can be used for studies of LOXs as therapeutic targets.

Highlights

The lysyl oxidase family of enzymes (LOXs) catalyze oxidative deamination of lysine side chains on collagen and elastin to initialize cross-linking that is essential for the formation of the extracellular matrix (ECM)
Viral transduction of catalytically active LOXL2 in A7r5 rat aortic vascular smooth muscle cells (VSMCs) resulted in a significant increase in BHZ (100 μM; 24 h) incorporation in the extracellular space when compared with control A7r5 cells (Fig. 2a)
To further verify that the catalytic activity of LOXL2 was responsible for the increase in BHZ signal, we used the following negative controls: (1) the overexpression of catalytically inactive H626/628Q LOXL2 double mutant (LOXL2DM)[39,40], (2) inhibition of LOXL2 specific activity using the small molecule inhibitor PAT-1251 (10 μM) in cells overexpressing catalytically active LOXL241, and (3) inhibition of total LOX activity using the non-selective small molecule LOX inhibitor βAminopropionitrile (BAPN; 10 μM) and LOXL2-specific inhibitor PAT-125141,42 in cells overexpressing catalytically active LOXL2

Summary

Introduction

The lysyl oxidase family of enzymes (LOXs) catalyze oxidative deamination of lysine side chains on collagen and elastin to initialize cross-linking that is essential for the formation of the extracellular matrix (ECM). Collagenase resistant 7S dodecamers are extracted and purified by ion exchange and gel filtration chromatography; (2) this method is not suitable for in situ application and does not yield information on the specific regions within a tissue where LOXL2 activity is upregulated in disease; (3) Prior studies have shown that LOXL2 can oxidize other matrix proteins including type I collagen[31] and tropoelastin[32].

Methods

Results

Conclusion