A Liquid Chromatography-Mass Spectrometry-based Approach to Characterize the Substrate Specificity of Mammalian Heparanase

Mao, Yang; Huang, Yu; Buczek‐Thomas, Jo Ann; Ethen, Cheryl M.; Nugent, Matthew A.; Wu, Zhengliang L.; Zaia, Joseph

doi:10.1074/jbc.m114.589630

Cited by 31 publications

(33 citation statements)

References 46 publications

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“…4B), confirming that the staining is indeed on HS. On the other hand, HPSE digestion on HS results in uniform N-sulfated GlcNAc residue (GlcNS) at the ends of HS chains (Mao, Y., Huang, Y., et al 2014). As a result, pretreatment with HPSE significantly increased the labeling (Fig.…”

Section: Strict Specificity Of Hs Stainingmentioning

confidence: 99%

“…HS is synthesized by exostosins (EXTs), dual enzymes that have both GlcA and GlcNAc transferase activities (Senay, C., Lind, T., et al 2000). HS is degraded by heparanase (HPSE), an endoglucuronidase that specifically hydrolyzes the βGlcA-1,4-GlcNAc bond in highly sulfated HS domains (Mao, Y., Huang, Y., et al 2014, Vlodavsky, I., Friedmann, Y., et al 1999. HS can also be digested with bacterial lyases such as heparinase III, which leaves an unsaturated GlcA residue (ΔGlcA) at its non-reducing end (Hovingh, P. and Linker, A.…”

Section: Introductionmentioning

confidence: 99%

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Imaging specific cellular glycan structures using glycosyltransferases via click chemistry

Person

Anderson

et al. 2017

Preprint

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Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells.Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for Oglycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans.

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Section: Strict Specificity Of Hs Stainingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Imaging specific cellular glycan structures using glycosyltransferases via click chemistry

Person

Anderson

et al. 2017

Preprint

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“…Because heparanase is strongly implicated in the breakdown of matrix in tumor metastasis, these modified heparin compounds have potential as antimetastatic agents (Casu et al, 2008). The detailed substrate specificity of heparanase was studied recently (Mao et al, 2014) using a mass spectrometric approach. Heparanase was found to cleave at the nonreducing side of highly sulfated domains, exposing potential FGF-2 binding motifs and thereby increasing the FGF-2 potentiating capacity of treated HS.…”

Section: G Interactions Between Heparin and Pathogensmentioning

confidence: 99%

Pharmacology of Heparin and Related Drugs

et al. 2015

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“…The challenge in the development of structurally defined heparanase probes with fluorescence readout largely stems from its elusive substrate recognition mode, despite the extensive investigations being made [23][24][25] . One known fact is that heparanase is an endoglycosidase that can cleave the internal glycosidic bond between a glucuronic acid (GlcUA) residue and an Nsulfoglucosamine (GlcN(NS)) residue bearing either a 3-O-sulfo or a 6-O-sulfo group 26 .…”

Section: Structurally Defined Fluorogenic Heparanase Probesmentioning

confidence: 99%

Ultrasensitive small molecule fluorogenic probe for human heparanase

Schleyer

Bryan

et al. 2020

Preprint

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Heparanase is a critical enzyme involved in the remodeling of the extracellular matrix (ECM), and its elevated expression has been linked with diseases such as cancer and inflammation. The detection of heparanase enzymatic activity holds tremendous value in the study of the cellular microenvironment, and search of molecular therapeutics targeting heparanase, however, assays developed for this enzyme so far have suffered prohibitive drawbacks. Here we present an ultrasensitive fluorogenic small-molecule probe for heparanase enzymatic activity. The probe exhibits a 756-fold fluorescence turn-on response in the presence of human heparanase, allowing one-step detection of heparanase activity in real-time with a picomolar detection limit. The high sensitivity and robustness of the probe are exemplified in a high-throughput screening assay for heparanase inhibitors.Heparanase, an endo-β-glucuronidase of the glycoside hydrolase 79 (GH79) family 1,2 , is responsible for the cleavage of heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPG) 3 . These protein-polysaccharide conjugated macromolecules, abundantly expressed in the extracellular matrix (ECM), play an essential structural role in maintaining the ECM integrity.Moreover, the HS side chains bind to an array of biological effector molecules, such as growth factors, chemokines, and cytokines, thereby serving as their reservoir that can liberate the desired signaling molecules when needed.

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A Liquid Chromatography-Mass Spectrometry-based Approach to Characterize the Substrate Specificity of Mammalian Heparanase

Cited by 31 publications

References 46 publications

Imaging specific cellular glycan structures using glycosyltransferases via click chemistry

Imaging specific cellular glycan structures using glycosyltransferases via click chemistry

Pharmacology of Heparin and Related Drugs

Ultrasensitive small molecule fluorogenic probe for human heparanase

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