SULF1, a human extracellular heparan 6-O-endosulfatase isoform 1, plays a critical role in embryonic development and cancer progression by modulating the 6-O-sulfation of heparan sulfate proteoglycans. However, limited recombinant protein production has hindered structural and functional characterization. To address this issue, we optimized SULF1 expression in HEK293F and HEK293T cells. We achieved yields of 2.2 mg/L of culture media after Ni2+-affinity purification of greater than 80% purity, representing a substantial improvement compared to the reported expression systems. We demonstrated that co-expression of sulfatase-modifying factor 1 in this expression system is essential for enhancing SULF1 enzymatic activity, which depends on conversion of active site cysteine to Cα-formylglycine and the presence of a Ca2+ ion. We further showed that a marine fucosylated chondroitin sulfate polymer isolated from the sea cucumber Holothuria floridana inhibits SULF1 enzymatic activity with IC50 of 0.05 ± 0.006 μg/mL and 0.07 ± 0.008 μg/mL for the GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA and 4-methylumbelliferyl sulfate substrates, respectively. Kinetic analysis revealed a mixed-mode inhibition, characterized by alterations in Vmax at all inhibitor concentrations and Km at high inhibitor concentrations. Efficient SULF1 production also enabled us to develop specific monoclonal antibodies, which confirmed SULF1 expression in the stroma of head and neck squamous cell cancer tissues. Collectively, this study provides an efficient workflow for the production of active human SULF1, investigates SULF1 inhibitors, and characterizes anti-SULF1 monoclonal antibodies, which will support further studies of this enzyme in various pathophysiological conditions.

Clinical and Translational Glycoscience Research Center Georgetown University 3800 Reservoir Rd NW Washington DC 20057 United States

Department of Biochemistry and Molecular and Cellular Biology 3800 Reservoir Rd NW Georgetown University Washington DC 20057 United States

Department of BioMolecular Sciences University of Mississippi University Mississippi 38677 United States

Department of Oncology Lombardi Comprehensive Cancer Center 3800 Reservoir Rd NW Georgetown University Washington DC 20057 United States

Department of Otolaryngology Head and Neck Surgery MedStar Georgetown University Hospital Washington DC 20057 United States

Division of Chemical Biology and Medicinal Chemistry Eshelman School of Pharmacy University of North Carolina Chapel Hill NC 27599 United States

Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 Vestec 252 50 Czech Republic

Max Planck Institut fuer Herz und Lungenforschung Ludwigstrasse 43 Bad Nauheim 61231 Germany

Research Institute of Pharmaceutical Sciences School of Pharmacy University of Mississippi University Mississippi 38677 United States

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