An in Situ Activity Assay for Lysyl Oxidases

Overview

Journal Commun Biol

Specialty Biology

Date 2021 Jul 6

PMID 34226627

Citations 7

Authors

Huilei Wang

Alan Poe

Lydia Pak

Kavitha Nandakumar

Sandeep Jandu

Jochen Steppan

Reik Loser

Lakshmi Santhanam

Affiliations

Soon will be listed here.

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.

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