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      Non-reducing end labeling of heparan sulfate via click chemistry and a high throughput ELISA assay for heparanase

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          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.

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          Author and article information

          Journal
          Glycobiology
          Glycobiology
          glycob
          Glycobiology
          Oxford University Press
          0959-6658
          1460-2423
          June 2017
          06 February 2017
          : 27
          : 6
          : 518-524
          Affiliations
          [2 ]Department of Enzyme, Bio-Techne, R&D Systems, Minneapolis, MN 55413, USA
          [3 ] Department of Protein Purification, Bio-Techne, R&D Systems, Inc. 614 McKinley Place N.E., Minneapolis, MN 55413, USA
          [4 ] Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University School of Medicine , 670 Albany Street, Boston, MA 02118, USA
          Author notes
          [1 ]To whom correspondence should be addressed: Tel: +1-612-656-4544; Fax: +1-612-379-6580; e-mail: Leon.wu@ 123456bio-techne.com
          Article
          PMC5421504 PMC5421504 5421504 cww130
          10.1093/glycob/cww130
          5421504
          28025251
          23326090-394e-45f4-b1b4-354af802e78a
          © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
          History
          : 23 July 2016
          : 11 December 2016
          : 14 December 2016
          Page count
          Pages: 7
          Funding
          Funded by: National Institutes of Health 10.13039/100000002
          Award ID: P4 1GM 104603 to J.Z.
          Categories
          Original Articles

          carbohydrate ELISA,click chemistry,heparan sulfate,heparin,HPSE

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