Galectin-3结合蛋白抑制细胞外肝素6-O-内硫酸化酶Sulf-2。

IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Molecular & Cellular Proteomics Pub Date : 2024-07-01 Epub Date: 2024-06-01 DOI:10.1016/j.mcpro.2024.100793
Aswini Panigrahi, Julius Benicky, Reem Aljuhani, Pritha Mukherjee, Zora Nováková, Cyril Bařinka, Radoslav Goldman
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引用次数: 0

摘要

人类细胞外 6-O-内切硫酸化酶 Sulf-1 和 Sulf-2 是唯一能在合成后改变硫酸肝素蛋白多糖(HSPG)的 6-O 硫酸化的酶,它们能调节 HSPG 与许多蛋白质的相互作用。Sulf-2在不同癌症中的致癌作用已被证实,我们已经证明Sulf-2与头颈部鳞状细胞癌(HNSCC)的不良生存结果有关。尽管 Sulf-2 蛋白非常重要,但有关其在肿瘤微环境中的直接蛋白-蛋白相互作用的信息却非常有限。在这项研究中,我们利用单克隆抗体(mAb)亲和纯化和质谱分析技术,在 HNSCC 细胞系的条件培养基中鉴定出 galectin-3 结合蛋白(LG3BP)是 Sulf-2 的高度特异性结合伙伴。我们利用重组蛋白在体外验证了它们之间的直接相互作用,并证明与 Sulf-2 共价结合的硫酸软骨素(CS)会影响与 LG3BP 的结合。我们通过生成缺乏 CS 的突变 Sulf-2 蛋白,证实了 CS 链对相互作用的重要性。重要的是,我们发现 LG3BP 在体外以浓度依赖的方式抑制 Sulf-2 的活性。因此,在球形细胞培养中加入 LG3BP 可抑制 HNSCC 细胞侵入 Matrigel。因此,Sulf-2与LG3BP的相互作用可能会调节Sulf-2酶的生理活性及其在肿瘤微环境中的活性。
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Galectin-3-Binding Protein Inhibits Extracellular Heparan 6-O-Endosulfatase Sulf-2.

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.

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来源期刊
Molecular & Cellular Proteomics
Molecular & Cellular Proteomics 生物-生化研究方法
CiteScore
11.50
自引率
4.30%
发文量
131
审稿时长
84 days
期刊介绍: The mission of MCP is to foster the development and applications of proteomics in both basic and translational research. MCP will publish manuscripts that report significant new biological or clinical discoveries underpinned by proteomic observations across all kingdoms of life. Manuscripts must define the biological roles played by the proteins investigated or their mechanisms of action. The journal also emphasizes articles that describe innovative new computational methods and technological advancements that will enable future discoveries. Manuscripts describing such approaches do not have to include a solution to a biological problem, but must demonstrate that the technology works as described, is reproducible and is appropriate to uncover yet unknown protein/proteome function or properties using relevant model systems or publicly available data. Scope: -Fundamental studies in biology, including integrative "omics" studies, that provide mechanistic insights -Novel experimental and computational technologies -Proteogenomic data integration and analysis that enable greater understanding of physiology and disease processes -Pathway and network analyses of signaling that focus on the roles of post-translational modifications -Studies of proteome dynamics and quality controls, and their roles in disease -Studies of evolutionary processes effecting proteome dynamics, quality and regulation -Chemical proteomics, including mechanisms of drug action -Proteomics of the immune system and antigen presentation/recognition -Microbiome proteomics, host-microbe and host-pathogen interactions, and their roles in health and disease -Clinical and translational studies of human diseases -Metabolomics to understand functional connections between genes, proteins and phenotypes
期刊最新文献
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