Radoslav, GoldmanAljuhani, Reem2025-09-072025https://hdl.handle.net/20.500.14154/76351SULF1 is an extracellular sulfatase that modifies heparan sulfate proteoglycans (HSPGs) by selectively removing 6-O-sulfate groups from glucosamine residues. This enzymatic activity regulates the binding of growth factors, morphogens, and many other ligands to HSPGs, thereby modulating pathways such as FGF, WNT, and TGF-β. Despite its potential role in extracellular matrix remodeling and stromal regulation, SULF1 remains under-characterized at the biochemical level, due to difficulty in producing active full-length protein. In this study, we optimized a mammalian expression system using HEK293 cells in serum-free, suspension-adapted culture. Lentiviral transduction, combined with co-expression of the formylglycine- generating enzyme SUMF1, yielded over 2 mg/L of active full-length human SULF1, representing a significant improvement over previously reported expression systems. Using two in vitro assays, a general arylsulfatase substrate (4-MUS) and a defined synthetic heparan sulfate oligosaccharide (2S2-6S4) 6-O- endosulfatase assays, we verified that SULF1 has catalytic properties consistent with the activity of type I sulfatases. Specifically, we demonstrated that SULF1 activity in vitro requires the SUMF1-dependent formylglycine modification; Ca2+ but not Mg2+, as a divalent cofactor, and intact N-glycosylation. Partial enzymatic deglycosylation led to an 85–90% reduction in activity, indicating that N-glycosylation contributes significantly to enzyme activity. AlphaFold-based structural modeling, revealed conserved residues predicted to coordinate Ca2+ binding at the active site and enabled visualization of electrostatic potential and N-glycosylation site distribution across the protein surface, supporting the biochemical findings. The improved expression system also facilitated the development and validation of monoclonal antibodies targeting human SULF1. Among the panel screened, three clones (5D1, 6E12, 4D2) demonstrated high specificity and affinity, supporting their utility in detection and quantification assays. Lastly, we evaluated the marine-derived fucosylated chondroitin sulfate (HfFucCS) as a candidate glycan-based SULF1 inhibitor. In vitro assays revealed dose-dependent, non-competitive inhibition. In a head and neck squamous cell carcinoma (HNSCC) xenograft model, high-dose HfFucCS treatment modestly reduced stromal content without significantly affecting tumor growth. Collectively, this work overcomes key technical limitations in the biochemical study of SULF1, introduces validated reagents for its detection and inhibition, and provides a robust starting point for future investigations of its functional roles in the extracellular matrix and tumor stroma.101en-USHeparan sulfate 6-O-endosulfatase 1 (SULF1)Protein purificationEnzyme kinetics and specificityCalcium-dependent enzymatic activityN-glycosylation analysisLC-MS/MS proteomicsMarine-derived glycosaminoglycan inhibitor (HfFucCS)AlphaFold structural modelingProtein electrostaticsHead and neck squamous cell carcinoma (HNSCC)Ca²⁺ binding site modelingAntibody-based inhibitionThesis