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

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, Cα-formylglycine, which is generated post-translationally 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 suppression, depending on either activating or inhibitory effects upon cell signaling (13–16).To investigate the physiological role of Sulf1 and Sulf2, single and double knock-out mice were generated (17–21). Both Sulf1 and Sulf2 knock-out mice are characterized by increased embryonic lethality, impaired neurite outgrowth, and other neurological abnormalities in the developing and adult nervous system (22). The corresponding double knock-out mice display an obvious reduction in body weight and developmental malformations, including skeletal and renal defects (18, 19, 23). Together with biochemical analyses on the impact of Sulf loss on HS sulfation, the phenotypic observations suggest a functional cooperativity between Sulf1 and Sulf2 in modulating the 6O-sulfation of UA(2S)-GlcNS(6S) disaccharide units within the S-domains of HS chains (17, 24). Moreover, analyses of heparan sulfate disaccharide compositions from Sulf1 and Sulf2 knock-out mice cell lines have indicated dynamic influences of Sulf loss also on non-substrate N-, 2O-, and 6O-sulfate groups via modulation of sulfotransferase expression, which may contribute to the developmental defects associated with the Sulf knock-out mice (24).From the biochemical perspective, it is an important question how the Sulfs are able to recognize their HSPG substrates and how cell surface localization is achieved, despite a lack of transmembrane domains or lipid anchors. Classical GAG-binding proteins, such as antithrombin III (25) or FGF1 (26), interact with their negatively charged GAG partners via small clusters of positively charged amino acid residues. Although some consensus sequences for heparin binding have been identified (XBBXBX, XBBBXXBX, and XBBXXBBBXXBBX, where B is a basic residue and X a hydropathic) (27–29), they are neither required nor sufficient. Unlike these classical GAG-binding proteins, Sulf1 and Sulf2 contain a large hydrophilic domain (HD), located between the N-terminal catalytic domain and the C-terminal domain. The HD is a unique feature of the extracellular sulfatases that is neither found in other sulfatases nor shows any homology with other known protein domains. According to sequence alignments, the HD of human Sulf1 has a size of ∼320 amino acid residues, 27% of which are basic and 14% acidic, resulting in a strong positive charge at neutral pH and a high theoretical pI of 9.8. Remarkably, the C-terminal end of the HD is composed of a cluster of 12 basic amino acid residues. Whereas the outer regions of the HD are highly conserved between Sulf1 and Sulf2 as well as between human, murine, and avian orthologs, the inner region, encoded by exons 13 and 14 in the case of human Sulf1 (6), is significantly less conserved.The role of the HD has previously been investigated for the avian ortholog QSulf2 (30). Results from this study indicated that the HD binds to negatively charged ligands and might serve to anchor the enzyme on the cell surface. Sulfate release assays indicated the necessity of the avian HD for enzymatic activity. Moreover, a very recent analysis of the HD of human Sulf1/Sulf2 revealed the presence of two furin-type proteinase cleavage sites within the inner region, explaining their partial processing into disulfide-linked subunits of 75 and 50 kDa (31). Sulf1/2 mutants, in which these sites were deleted, retained enzymatic activity but failed to potentiate Wnt signaling when overexpressed in human embryonic kidney 293 cells.Due to the observed differences in enzyme secretion and detergent solubility between the human and avian orthologs (24, 30) and the likely importance of this domain for mammalian Sulf localization and activity, we analyzed the function of the HD of human Sulf1 in mediating enzyme activity, cell surface targeting, secretion, and substrate recognition. Using different Sulf1 deletion mutants and glutathione S-transferase (GST)-HD fusion proteins, this study demonstrates that specific regions of the HD, especially at the conserved N and C termini, are responsible for heparin/HS binding, cell surface localization, and enzymatic activity of human Sulf1. Interaction analyses show that binding of the HD to heparin is significantly stronger compared with other typical heparin-binding proteins, suggesting a new mode of GAG binding. The deletion of the inner region of the HD leads to significantly increased secretion of the enzyme, allowing the purification of an active variant that is able to modulate FGF signaling in cell culture experiments.