Characterization of the Human Sulfatase Sulf1 and Its High Affinity Heparin/Heparan Sulfate Interaction Domain

Abstract

The extracellular sulfatases Sulf1 and Sulf2 remodel the 6O-sulfation state of heparan sulfate proteoglycans on the cell surface, thereby modulating growth factor signaling. Different from all other sulfatases, the Sulfs contain a unique, positively charged hydrophilic domain (HD) of about 320 amino acid residues. Using various HD deletion mutants and glutathione S-transferase (GST)-HD fusion proteins, this study demonstrates that the HD is required for enzymatic activity and acts as a high affinity heparin/heparan sulfate interaction domain. Association of the HD with the cell surface is sensitive to heparinase treatment, underlining specificity toward heparan sulfate chains. Correspondingly, isolated GST-HD binds strongly to both heparin and heparan sulfate in vitro and also to living cells. Surface plasmon resonance studies indicate nanomolar affinity of GST-HD toward immobilized heparin. The comparison of different mutants reveals that especially the outer regions of the HD mediate heparan sulfate binding, probably involving "tandem" interactions. Interestingly, binding to heparan sulfate depends on the presence of 6O-sulfate substrate groups, suggesting that substrate turnover facilitates release of the enzyme from its substrate. Deletion of the inner, less conserved region of the HD drastically increases Sulf1 secretion without affecting enzymatic activity or substrate specificity, thus providing a tool for the in vitro modulation of HS-dependent signaling as demonstrated here for the signal transduction of fibroblast growth factor 2. Taken together, the present study shows that specific regions of the HD influence different aspects of HS binding, cellular localization, and enzyme function.The human sulfatases represent a family of 17 enzymes responsible for the turnover and remodeling of sulfate esters and sulfamates. Their reaction mechanism relies on a special amino acid residue, C␣-formylglycine, which is generated posttranslationally via oxidation of a conserved cysteine residue in the active site (1-3). Besides the lysosomal sulfatases involved in the cellular degradation of various sulfated substrates (4), two extracellular sulfatases, Sulf1 and Sulf2 (the Sulfs), have been described (5, 6). The Sulfs are endosulfatases with restricted substrate specificity toward 6O-sulfate groups of heparan sulfate (HS), 2 an information-rich glycosaminoglycan (GAG) polymer attached to proteoglycans at the cell surface and in the extracellular matrix (6 -8). HS proteoglycans (HSPGs) act as co-receptors in cell signaling pathways and provide binding sites for growth factors and morphogens via specific sulfation patterns on their HS chains. By enzymatically removing 6O-sulfate groups from HSPGs on the cell surface, Sulf1 and Sulf2 differentially regulate the activity of FGF, vascular endothelial growth factor, Wnt, and other HS ligands, thereby modulating important processes such as development, cell growth, and differentiation (9 -12). Misregulation of the Sulfs has been linked with both tumor progression and suppr...

Organ-specific Sulfation Patterns of Heparan Sulfate Generated by Extracellular Sulfatases Sulf1 and Sulf2 in Mice

“…Given that Sulf1/2 can hydrolyze 6-Osulfate in UA-GlcNS6S disaccharide units in HS in vitro (11,24,25), these findings suggest the possibility that Sulfs act on UA-GlcNS6S disaccharide units in some organs. Because the Sulf1 expression is highest in the lung, high endosulfatase activity may lead to desulfation of UA-GlcNS6S disaccharide units.…”

“…These increases may simply mean that Sulfs have 6-O-endosulfatase activities toward these disaccharide units. However, given that HS 6-O-endosulfatase activity toward UA-GlcNAc6S units has never been detected in vitro (9,11,13,24,25,34) and that Sulfs show no endosulfatase activity toward CS (this study; see also Refs. 9, 13), these changes seem to have occurred as a secondary consequence of alteration of the HS sulfation patterns, although the possibility cannot be formally excluded that Sulfs acquire such activity in collaboration with an unknown factor(s) in vivo.…”

“…Therefore, Sulf1 and Sulf2 are thought to be key regulators of cell proliferation, differentiation, and migration and are also implicated in cancer progression and metastasis (22,23). Biochemical studies have demonstrated that Sulf1 and Sulf2 are most active in the UA2S-GlcNS6S trisulfated disaccharide unit, which is present in the NS domain (9,11,13,16,24,25). In addition, weaker 6-O-desulfation activity is also detectable in the UA-GlcNS6S-disulfated disaccharide unit (11,24,25).…”

“…Biochemical studies have demonstrated that Sulf1 and Sulf2 are most active in the UA2S-GlcNS6S trisulfated disaccharide unit, which is present in the NS domain (9,11,13,16,24,25). In addition, weaker 6-O-desulfation activity is also detectable in the UA-GlcNS6S-disulfated disaccharide unit (11,24,25). Subsequent disaccharide analysis of embryonic fibroblasts from Sulf1 and Sulf2 knock-out mice revealed changes suggestive of the specificity of Sulfs toward UA2S-GlcNS6S and UA-GlcNS6S disaccharide units (26).…”

Dynamic expression patterns of 6‐O endosulfatases during zebrafish development suggest a subfunctionalisation event for sulf2

“…Studies on human and quail Sulf HD demonstrate its importance in heparin binding, cell surface anchoring, and enzymatic activity. In particular, the predicted 14 basic amino acid sequence residing in the HD domain marginally contributes to these functional roles Frese et al, 2009;Tang and Rosen, 2009). Although these sequences are fairly conserved in the zebrafish Sulfs, the additional stretch of sequences in the C-terminal region of HD of Sulf2 and Sulf2a may confer different function, such that it may alter heparin binding affinity or its localisation to the cell surface.…”