Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented, and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). Despite its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the conditioned media of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed the importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration-dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited the invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP may regulate the physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

Department of Biochemistry and Molecular and Cellular Biology Georgetown University Washington District of Columbia USA

Department of Oncology Lombardi Comprehensive Cancer Center Georgetown University Washington District of Columbia USA

Department of Oncology Lombardi Comprehensive Cancer Center Georgetown University Washington District of Columbia USA; Clinical and Translational Glycoscience Research Center Georgetown University Washington District of Columbia USA

Department of Oncology Lombardi Comprehensive Cancer Center Georgetown University Washington District of Columbia USA; Clinical and Translational Glycoscience Research Center Georgetown University Washington District of Columbia USA; Department of Biochemistry and Molecular and Cellular Biology Georgetown University Washington District of Columbia USA

Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Vestec Czech Republic

bioRxiv. 2023 Dec 21:2023.12.20.572603. doi: 10.1101/2023.12.20.572603. PubMed

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