A Fluorogenic Heparan Sulfate Disaccharide for the Measurement of Heparanase Activity

26 June 2020, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

The endo-β-glucuronidase heparanase mediates mammalian heparan sulfate catabolism, and is of considerable medical interest due to its prominent role in cancer aggression and metastasis. Biochemical studies of heparanase are currently hampered by a lack of suitable chromogenic or fluorogenic assay substrates, instead relying on lengthy multistep procedures to measure activity. Herein, we demonstrate that N’,6-O’-bis-sulfated 4-methylumbelliferyl heparan sulfate disaccharide is a competent fluorogenic heparanase substrate. Despite somewhat slow turnover, the high sensitivity of 4-methylumbelliferyl fluorescence provides a wide signal window that enables both enzyme turnover and inhibition kinetics measurements. Crystal structures with heparanase also provide the first ever observation of a substrate in an activated 1S3 conformation, highlighting previously unknown interactions involved in turnover. Our results pave the way for the design of further improved HPSE substrates that may enable rapid assessment of enzyme activity, which in turn will drive development of new heparanase inhibitors for therapeutic use.

Keywords

heparanase
heparan sulfate
fluorogenic substrate
kinetics assays
Enzyme Substrate Complex
crystallography
Conformational Analysis

Supplementary materials

Title
Description
Actions
Title
Wu et al 4MU HPSE SuppInfo
Description
Actions

Comments

Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.