title: Non-reducing end labeling of heparan sulfate via click chemistry and a high throughput ELISA assay for heparanase.
abstract: Heparan sulfate (HS) is a linear polysaccharide found in the extracellular matrix (ECM) and on the cell membrane. It plays numerous roles in cellular events, including cell growth, migration and differentiation through binding to various growth factors, cytokines and other ECM proteins. Heparanase (HPSE) is an endoglycosidase that cleaves HS in the ECM and cell membrane. By degrading HS, HPSE not only alters the integrity of the ECM but also releases growth factors and angiogenic factors bound to HS chains, therefore, changes various cellular activities, including cell mobility that is critical for cancer metastasis. Accordingly, HPSE is an ideal drug target for cancer therapeutics. Here, we describe a method for non-reducing end labeling of HS via click chemistry (CC), and further use it in a novel HPSE assay. HS chains on a recombinant human syndecan-4 are first labeled at their non-reducing ends with GlcNAz using dimeric HS polymerase EXT1/EXT2. The labeled sample is then biotinylated through CC, immobilized on a multi-well plate and detected with ELISA. HPSE digestion of the biotinylated sample removes the label and, therefore, reduces the signal in ELISA assay. Non-reducing end labeling avoids the interference in an HPSE reaction caused by any internal labeling of HS. The assay is very sensitive with only 2.5 ng of labeled syndecan-4 needed in each reaction. The assay is also highly reproducible with a Z' > 0.6. Overall, this new method is suitable for high-throughput drug screening on HPSE.
authors.affiliation: Department of Enzyme, Bio-Techne, R&D Systems, Minneapolis, MN 55413, USA.
authors.forename: Zhengliang L
authors.initials: ZL
authors.lastname: Wu
authors.affiliation: Department of Enzyme, Bio-Techne, R&D Systems, Minneapolis, MN 55413, USA.
authors.forename: Xinyi
authors.initials: X
authors.lastname: Huang
authors.affiliation: Department of Enzyme, Bio-Techne, R&D Systems, Minneapolis, MN 55413, USA.
authors.forename: Cheryl M
authors.initials: CM
authors.lastname: Ethen
authors.affiliation: Department of Enzyme, Bio-Techne, R&D Systems, Minneapolis, MN 55413, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Tatge
authors.affiliation: Department of Protein Purification, Bio-Techne, R&D Systems, Inc. 614 McKinley Place N.E., Minneapolis, MN 55413, USA.
authors.forename: Marta
authors.initials: M
authors.lastname: Pasek
authors.affiliation: Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University School of Medicine, 670 Albany Street, Boston, MA 02118, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Zaia
mesh_terms: D057930:Click Chemistry / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005966:Glucuronidase / Q000737:chemistry* / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry*; D006801:Humans; D000099175:Heparanase
chemical_list: D005966:Glucuronidase; D006497:Heparan Sulfate; D000099175:Heparanase
keywords: HPSE; carbohydrate ELISA; click chemistry; heparan sulfate; heparin