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...

Sulfs are extracellular sulfatases that have emerged recently as critical regulators of heparan sulfate (HS) activities through their ability to catalyze specific 6-O-desulfation of the polysaccharide. Consequently, Sulfs have been involved in many physiological and pathological processes, and notably for Sulf-2, in the development of cancers with poor prognosis. Despite growing interest, little is known about the structure and activity of these enzymes and the way they induce dynamic remodeling of HS 6-O-sulfation status. Here, we have combined an array of analytical approaches, including mass spectrometry, NMR, HS oligosaccharide sequencing, and FACS, to dissect HSulf-2 sulfatase activity, either on a purified octasaccharide used as a mimic of HS functional domains, or on intact cell-surface HS chains. In parallel, we have studied the functional consequences of HSulf-2 activity on fibroblast growth factor (FGF)-induced mitogenesis and found that the enzyme could differentially regulate FGF1 and FGF2 activities. Notably, these data supported the existence of precise 6-O-sulfation patterns for FGF activation and provided new insights into the saccharide structures involved. Altogether, our data bring to light an original processive enzymatic mechanism, by which HSulfs catalyze oriented alteration of HS 6-O-desulfation patterns and direct fine and differential regulation of HS functions.

In biological adhesion, the biophysical mechanism of specific biomolecular interaction can be divided in slip and catch bonds, respectively. Conceptually, slip bonds exhibit a reduced bond lifetime under increased external force and catch bonds, in contrast, exhibit an increased lifetime (for a certain force interval). Since 2003, a handful of biological systems have been identified to display catch bond properties. Upon investigating the specific interaction between the unique hydrophilic domain (HD) of the human cell-surface sulfatase Sulf1 against its physiological glycosaminoglycan (GAG) target heparan sulfate (HS) by single molecule force spectroscopy (SMFS), we found clear evidence of catch bond behavior in this system. The HD, ∼320 amino acids long with dominant positive charge, and its interaction with sulfated GAG-polymers were quantitatively investigated using atomic force microscopy (AFM) based force clamp spectroscopy (FCS) and dynamic force spectroscopy (DFS). In FCS experiments, we found that the catch bond character of HD against GAGs could be attributed to the GAG 6-O-sulfation site whereas only slip bond interaction can be observed in a GAG system where this site is explicitly lacking. We interpreted the binding data within the theoretical framework of a two state two path model, where two slip bonds are coupled forming a double-well interaction potential with an energy difference of ΔE ≈ 9 kBT and a compliance length of Δx ≈ 3.2 nm. Additional DFS experiments support this assumption and allow identification of these two coupled slip-bond states that behave consistently within the Kramers-Bell-Evans model of force-mediated dissociation.

Background: Cell surface heparan sulfate is an essential regulator of cell signaling. Results: Sulf 6-O-endosulfatase deficiency results in degenerative phenotypes, and HSPG sulfation status induces Wnt10a-mediated activation of odontoblast differentiation. Conclusion: Sulf-mediated desulfation is an important modification for the activation of the Wnt signaling in odontoblasts. Significance: This is the first molecular evidence for the functional roles of HSPG sulfation in dentin formation.

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