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| Main Authors: | , , , , , , , , , , , |
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| Format: | Artículo científico |
| Language: | en |
| Published: |
Glycobiology
2025
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| Subjects: | |
| Online Access: | https://pubmed.ncbi.nlm.nih.gov/41025518/ |
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Table of Contents:
- Production and inhibition of human Heparan 6-O-Endosulfatase SULF1. Aljuhani, Reem Benicky, Julius Panigrahi, Aswini Mukherjee, Pritha Sanda, Miloslav Zhu, Shiya Nováková, Zora Pomin, Vitor H Liu, Jian Davidson, Bruce Bařinka, Cyril Goldman, Radoslav Humans Sulfotransferases HEK293 Cells Animals Enzyme Inhibitors Recombinant Proteins 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.