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.
Mol Cell Proteomics. 2024 Jul;23(7):100793. doi: 10.1016/j.mcpro.2024.100793. Epub 2024 Jun 1.
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.
人类细胞外 6-O-内磺基酯酶 Sulf-1 和 Sulf-2 是唯一能在后合成阶段改变硫酸乙酰肝素蛋白聚糖（HSPG）6-O 位硫酸化的酶，该修饰调节 HSPG 与许多蛋白质的相互作用。Sulf-2 在不同癌症中的致癌性已被记录在案，我们还表明 Sulf-2 与头颈部鳞状细胞癌（HNSCC）的不良生存结局相关。尽管 Sulf-2 很重要，但关于其在肿瘤微环境中的直接蛋白质-蛋白质相互作用的信息有限。在这项研究中，我们使用单克隆抗体（mAb）亲和纯化和质谱法鉴定出半乳糖凝集素-3 结合蛋白（LG3BP）是 HNSCC 细胞系条件培养基中 Sulf-2 的高度特异性结合伴侣。我们使用重组蛋白在体外验证了它们的直接相互作用，并表明共价结合到 Sulf-2 上的硫酸软骨素（CS）影响与 LG3BP 的结合。我们通过生成缺乏 CS 的突变 Sulf-2 蛋白来证实 CS 链对相互作用的重要性。重要的是，我们已经表明，LG3BP 以浓度依赖的方式在体外抑制 Sulf-2 活性。因此，向球体细胞培养物中添加 LG3BP 可抑制 HNSCC 细胞向 Matrigel 的侵袭。因此，Sulf-2 与 LG3BP 的相互作用可能调节 Sulf-2 酶的生理活性及其在肿瘤微环境中的活性。
