Abstract 1819 Purification and in vitro Characterization of Human Extracellular Heparan 6-O-Endo-Sulfatases Sulf1 and Sulf2

Introduction: Extracellular sulfatases (Sulf1, Sulf2) are enzymes that catalyze the removal of 6-O-sulfate group from heparan sulfate proteoglycans (HSPG) in the extracellular matrix. The resulting sulfation remodeling modifies the interactions with many proteins, which adjusts important cellular functions. Both enzymes have a highly conserved N-terminal region containing the consensus sulfatase motifs, and a unique active-site aldehyde residue, formylglycine (FGly). The recognition and binding of Sulf1 and Sulf2 to their substrates is mediated by several factors, including the overall structure and charge distribution of the HS chains, and the presence of specific sulfation patterns. Despite their biological significance, structure and function of the Sulf1 and Sulf2 enzymes remain insufficiently characterized largely due to the lack of the enzyme-sources and/or specific activity assays. Results: To understand their structure and function and to evaluate potential inhibitors, we have developed human expression systems for the production of recombinant Sulf1 and Sulf2 enzymes in high titers. We produce functional recombinant proteins in up to 3mg/L, as analyzed by SDS PAGE and Western blot. We characterize activity of the Sulf enzymes by an optimized arylsulfatase assay (4-MUS) and by a newly developed 6-O-endosulfatase assay. This assay is highly specific for Sulf1 and Sulf2 activity using a synthetic oligosaccharide (HS mimics) GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP as a substrate in an HPLC assay monitoring the para-nitrophenol (pNP) absorbance of de-sulfated products separated from the substrate by ion exchange chromatography. In our assays, Sulf2 recombinant protein displayed high enzymatic activity, while Sulf1 exhibited low enzymatic activity in both assays. To enhance the enzymatic activity of Sulf1, we co-transfected the Sulf1 HEK293T overexpression system with FGE (formylglycine generating enzyme, also known as SUMF1). This led to a significant recovery of Sulf1 enzymatic activity in both assays. Furthermore, the quantification of Sulf1 and Sulf2 enzymatic activities facilitated screening of potential inhibitors using chemical and/or biological compounds. We identified a novel marine fucosylated chondroitin sulfate inhibitor and demonstrated its ability to block the activity of Sulf1 and Sulf2 recombinant proteins in vitro. Conclusion: Our optimized expression systems and newly developed enzymatic assays provide us with the capability to comprehensively analyze Sulf1 and Sulf2 enzymatic activity. Furthermore, we can effectively evaluate inhibitors of the enzymes. It is noteworthy that the Sulf1 and Sulf2 activity have been associated with poor survival outcomes in various malignancies, which highlights their significance as essential diagnostic and therapeutic targets.