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.
Fig: Reaction of biotin-hydrazide with protein intermediates generated by LOXs-catalysis: Oxidative deamination catalyzed by lysyl oxidases (LOXs) leads to collagen crosslinking by spontaneous condensation. Intermediate semialdehyde and the imine and aldol products of the crosslinking reaction can be labeled by biotinylated hydrazide. LOXs oxidize lysine and hydroxylysine side chains on collagen and elastin to highly reactive semialdehydes called allysines, while producing hydrogen peroxide (H2O2) and ammonia (NH3) as byproducts. Allysines then spontaneously condense to form cross-linkages. In our approach to detect LOXL2 activity, adding biotin-hydrazide (BHZ) results in biotinylation of LOXL2-catalyzed peptidyl lysines.
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.
Wang, H., Poe, A., Pak, L. et al. An in situ activity assay for lysyl oxidases. Commun Biol 4, 840 (2021). https://doi.org/10.1038/s42003-021-02354-0