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.
Article: Heparan sulfate heterogeneity in skeletal muscle basal lamina: demonstration by phage display-derived antibodies.[show abstract] [hide abstract]
ABSTRACT: The basal lamina (BL) enveloping skeletal muscle fibers contains different glycoproteins, including proteoglycans. To obtain more information on the glycosaminoglycan moiety of proteoglycans, we have selected a panel of anti-heparan sulfate (HS) antibodies from a semisynthetic antibody phage display library by panning against glycosaminoglycan preparations derived from skeletal muscle. Epitope recognition by the antibodies is strongly dependent on O- and N-sulfation of the heparan sulfate. Immunostaining with these antibodies showed a distinct distribution of heparan sulfate epitopes in muscle basal lamina of various species. Clear differences in staining intensity were observed between neural, synaptic, and extrasynaptic basal laminae. Moreover, temporal and regional changes in abundancy of heparan sulfate epitopes were observed during muscle development both in vitro and in vivo. Taken together, these data suggest a role for specific heparan sulfate domains/species in myogenesis and synaptogenesis. Detailed analysis of the functions of heparan sulfate epitopes in muscle morphogenesis has now become feasible with the isolation of antibodies specific for distinct heparan sulfate epitopes.Journal of Neuroscience 07/2000; 20(11):4099-111. · 7.11 Impact Factor
Article: Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme.[show abstract] [hide abstract]
ABSTRACT: C(alpha)-formylglycine (FGly) is the catalytic residue in the active site of eukaryotic sulfatases. It is posttranslationally generated from a cysteine in the endoplasmic reticulum. The genetic defect of FGly formation causes multiple sulfatase deficiency (MSD), a lysosomal storage disorder. We purified the FGly generating enzyme (FGE) and identified its gene and nine mutations in seven MSD patients. In patient fibroblasts, the activity of sulfatases is partially restored by transduction of FGE encoding cDNA, but not by cDNA carrying an MSD mutation. The gene encoding FGE is highly conserved among pro- and eukaryotes and has a paralog of unknown function in vertebrates. FGE is localized in the endoplasmic reticulum and is predicted to have a tripartite domain structure.Cell 06/2003; 113(4):435-44. · 32.40 Impact Factor