Glycobiology Advance Access published online on October 12, 2009
Glycobiology, doi:10.1093/glycob/cwp159
Direct Detection of HSulf-1 and HSulf-2 Activities on Extracellular Heparan Sulfate and their Inhibition by PI-88
1 Section of Pathophysiology and Neurobiology, National Institute for Longevity Sciences, Japan
2 Department of Anatomy, Program in Immunology, University of California, San Francisco, CA, USA
3 Research Complex for the Medicine Frontiers, Aichi Medical University, Japan
4 Department of Biochemistry 280, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Center, The Netherlands
Address correspondence: Kenji Uchimura, Section of Pathophysiology and Neurobiology, National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, Aichi 474-8522 Japan Phone: +81-562-46-2311, Fax: +81-562-46-3157, e-mail: arumihcu{at}nils.go.jp
Received on February 8, 2009; accepted on September 30, 2009
Heparan sulfates (HS) bind a diversity of protein ligands on the cell surface and in the extracellular matrix and thus can modulate cell signaling. The state of sulfation in glucosamines and uronic acids within the chains strongly influences their binding. We have previously cloned and characterized two human extracellular endoglucosamine 6-sulfatases, HSulf-1 and HSulf-2, which selectively liberate the 6-O sulfate groups on glucosamines present in N, 6-O and 2-O trisulfated disaccharides of intact HS and heparins. These enzymes serve important roles in development and are upregulated in a number of cancers. To determine whether the Sulfs act on the trisulfated disaccharides that exist on the cell surface, we expressed HSulfs in cultured cells and performed a flow cytometric analysis with the RB4CD12, an anti-HS antibody that recognizes N- and O- sulfated HS saccharides. The endogenously expressed level of the cell surface RB4CD12 epitope was greatly diminished in CHO, HEK293 and HeLa cells transfected with HSulf-1 or HSulf-2 cDNA. In correspondence with the RB4CD12 finding, the N, 6-O and 2-O trisulfated disaccharides of the HS isolated from the cell surface/extracellular matrix were dramatically reduced in the Sulf-expressed HEK 293 cells. We then developed an ELISA and confirmed that the RB4CD12 epitope in immobilized heparin was degraded by purified recombinant HSulf-1 and HSulf-2, and conditioned medium (CM) of MCF-7 breast carcinoma cells, which contain a native form of HSulf-2. Furthermore, HSulf-1 and HSulf-2 exerted activity against the epitope expressed on microvessels of mouse brains. Both HSulf activities were potently inhibited by PI-88, a sulfated heparin mimetic with anti-cancer activities. These findings provide new strategies for monitoring the extracellular remodeling of HS by Sulfs during normal and pathophysiological processes.
Key words: confocal microscopy/extracellular sulfatase/flow cytometry/heparan sulfate/PI-88