Polymeric fluorescent heparin as one-step FRET substrate of human heparanase.

Autor: Sistla JC; Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA 23219, USA; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23219, USA., Morla S; Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA 23219, USA; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23219, USA., Alabbas AB; Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA 23219, USA; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23219, USA., Kalathur RC; Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA., Sharon C; Hunter Holmes McGuire VA Medical Center, Richmond, VA 23249, USA., Patel BB; Hunter Holmes McGuire VA Medical Center, Richmond, VA 23249, USA; Division of Hematology, Oncology, and Palliative Care, Department of Internal Medicine and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA., Desai UR; Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA 23219, USA; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23219, USA. Electronic address: urdesai@vcu.edu.
Jazyk: angličtina
Zdroj: Carbohydrate polymers [Carbohydr Polym] 2019 Feb 01; Vol. 205, pp. 385-391. Date of Electronic Publication: 2018 Oct 28.
DOI: 10.1016/j.carbpol.2018.10.071
Abstrakt: Heparanase, an endo-β-D-glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains and plays important roles in cellular growth and metastasis. Heparanase assays reported to-date are labor intensive, complex and/or expensive. A simpler assay is critically needed to understand the myriad roles of heparanase. We reasoned that fluorescent heparin could serve as an effective probe of heparanase levels. Following synthesis and screening, a heparin preparation labeled with DABCYL and EDANS was identified, which exhibited a characteristic increase in signal following cleavage by human heparanase. This work describes the synthesis of this heparin substrate, its kinetic and spectrofluorometric properties, optimization of the heparanase assay, use of the assay in inhibitor screening, and elucidation of the state of heparanase in different cell lines. Our FRET-based assay is much simpler and more robust than all assays reported in the literature as well as a commercially available kit.
(Copyright © 2018 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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