Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88

Abstract

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 HEK293 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.

Heparan sulfate sulfatase SULF2 regulates PDGFRα signaling and growth in human and mouse malignant glioma

“…Therefore, in patients with ligand-dependent tumors, inhibition of SULF2 may be a useful therapeutic strategy in combination with other treatments in GBM, such as inhibition of PDGFRα. Interestingly, the HS mimetic, PI-88, inhibits SULF function (56) and has been tested in a number of cancer clinical trials (62). Originally designed for their ability to inhibit heparanase, PI-88 and other HS mimetics have antiproliferative and antiangiogenic properties (63)(64)(65).…”

Cellular Interaction and Cytotoxicity of the Iowa Mutation of Apolipoprotein A-I (ApoA-IIowa) Amyloid Mediated by Sulfate Moieties of Heparan Sulfate

“…An anti-actin antibody was purchased from Sigma. CHO cells that were stably transfected with cDNA encoding HSulf-1 or HSulf-2 were established as described previously (32). FITC-labeled heparin was purchased from Polysciences, Inc. (Warrington, PA).…”

HSulf‐1 inhibits cell proliferation and invasion in human gastric cancer

“…To determine the effect of Hsulf-1 expression on the sulfation status of HSPG in gastric cancer cells, we used the anti-HS antibody 10E4, which has been reported to recognize HS 6-O-sulfated glucosamine residues as well as HS the N-sulfated glucosamine moiety. (3,(25)(26)(27) Using flow cytometry, we found that the expression of the 10E4 epitope on the cell surface was significantly higher in MKN28 cells than in AGS cells (Fig. 1g,h), indicating possible differences in the HS structures between these two cell lines.…”