An in situ activity assay for lysyl oxidases

Current methods for detecting activity of lysyl oxidases lack specificity and do not provide information on how much activity is occurring in situ. Hydrazide reagents can be used to measure their in situ activity levels in a practicable, sensitive, and specific manner.

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Motivation for the Project

Lysyl oxidase (LOX) family of enzymes are responsible for crosslinking extracellular matrix (ECM) proteins like collagen and elastin. Beyond prototypical LOX, there are four other lysyl oxidase like (LOXL 1-4) enzymes. Increased expression of LOX family enzymes is highly associated with diverse disease processes with a known element of ECM remodeling and accumulation such as fibrosis, pulmonary hypertension, cancer metastasis, and vascular stiffening. Our lab focuses on one example - vascular stiffening - a process that occurs with aging; as we get older, our arteries become stiffer, resulting in high blood pressure. In our previous study, we showed LOXL2 plays a central role in aging associated deterioration of vascular mechanics and that LOXL2 depletion is protective against age-related vascular stiffening. Since dysregulation of this family of enzymes is involved in many diseases, they – and LOXL2 especially – are attractive targets for therapeutic interventions. Unfortunately, there are, to date, no sensitive, specific assays that can detect the amount of LOX family catalytic activity in situ in the extracellular matrix (ECM) of cells and tissue. Without the proper tools to understand their activities and how to regulate them, we cannot fully determine the role of these crosslinking enzymes in pathobiological mechanisms and develop targeted therapies.

The Starting Point

Currently, there are two methods commonly used to determine the activity level of LOX family enzymes. The first is an H2O2 based assay that uses Amplex Red in horseradish peroxidase coupled reactions in homogenized/ solubilized preparations. The second is a Western blotting approach to quantify collagen IV 7S dodecamer crosslinking from the ECM after a time-consuming solubilization process. Neither provides comprehensive information about how much LOXs catalytic activity is actually occurring in situ, and both suffer from technical limitations and challenges. Not to mention, watching lab members gruel over the involved and time-consuming protocols added to our motivation to develop a new approach that not only overcomes the limitations of prior methods but is also easy and convenient to implement.

Development of Concept

LOX family enzymes all perform the same primary reaction: converting lysine and hydroxylysine residues into reactive semialdehydes called allysines. From an organic chemistry perspective, this is an oxidative deamination that produces aldehydes capable of aldol condensation or other nucleophilic addition reactions. Our goal was to find a reagent that will react readily with these allysine residues before it has the chance to perform its natural crosslinking reactions. Upon further research, we postulated that hydrazides represent a functional group that can readily react with the intermediates generated by LOXs catalytic function. Specifically, we chose biotin-hydrazide (BHZ), which can be easily detected using biotin-avidin interactions.

Proof-of-concept experiments using gain-of-function and loss-of-function assays showed that the assay indeed detects LOXL2 generated intermediates in the ECM. We show that this approach can specifically and robustly label the LOX activity in situ for both cellular and tissue samples. An added benefit is that it also displays where the activity is occurring rather than just providing a footprint of catalysis that has previously occurred.

Future Steps

Currently, our lab members are using this protocol to explore how LOXL2 is regulated in vivo and how these mechanisms modulate its activity level. They are also optimizing the assay for high throughput drug screening purposes to expand our ability to develop new, targeted therapeutics against LOX family enzymes. The first step to solving the issue of matrix accumulation diseases is to develop the tools needed to accurately measure how much activity the responsible enzymes are performing. We are excited to see the next steps taken using this new technique.

You can access our Communications Biology paper here.

Alan Poe

Research Assistant, Johns Hopkins University