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.

• Klíčová slova
• HNSCC, LC-MS/MS, Sulf-2, affinity purification, enzyme, mass spectrometry, protein-protein interaction, spheroid, sulfatase,
• MeSH
• antigeny nádorové MeSH
• chondroitinsulfáty metabolismus   MeSH
• dlaždicobuněčné karcinomy hlavy a krku metabolismus   patologie   MeSH
• heparansulfát proteoglykany metabolismus   MeSH
• lidé MeSH
• nádorové biomarkery MeSH
• nádorové buněčné linie MeSH
• nádorové mikroprostředí MeSH
• nádory hlavy a krku metabolismus   patologie   MeSH
• pohyb buněk účinky léků   MeSH
• spinocelulární karcinom metabolismus   patologie   MeSH
• sulfatasy metabolismus   MeSH
• sulfotransferasy * metabolismus   MeSH
• vazba proteinů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny nádorové MeSH
• chondroitinsulfáty MeSH
• heparansulfát proteoglykany MeSH
• LGALS3BP protein, human MeSH Prohlížeč
• nádorové biomarkery MeSH
• SULF2 protein, human MeSH Prohlížeč
• sulfatasy MeSH
• sulfotransferasy * MeSH

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). In spite of 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 secretome 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 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 invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP has functional relevance, and may regulate physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH