Hoffmann Sebastian, Berger Benedict-Tilman, Lucas Liane Rosalie, Schiele Felix, Park John Edward
Division of Cancer Immunology and Immune Modulation, Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach, Germany.
Division of High-Throughput Biology, Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach, Germany.
Cells. 2024 Dec 17;13(24):2083. doi: 10.3390/cells13242083.
Membrane proteins, especially extracellular domains, are key therapeutic targets due to their role in cell communication and associations. Yet, their functions and interactions often remain unclear. This study presents a general method to discover interactions of membrane proteins with immune cells and subsequently to deorphanize their respective receptors. We developed a comprehensive recombinant protein library of extracellular domains of human transmembrane proteins and proteins found in the ER-Golgi-lysosomal systems. Using this library, we conducted a flow-cytometric screen that identified several cell surface binding events, including an interaction between carbonic anhydrase 9 (CAH9/CA9/CAIX) and CD14 cells. Further analysis revealed this interaction was indirect and mediated via platelets bound to the monocytes. CA9, best known for its diverse roles in cancer, is a promising therapeutic target. We utilized our library to develop an AlphaLISA high-throughput screening assay, identifying CLEC2 as one robust CA9 binding partner. A five-amino-acid sequence (EDLPT) in CA9, identical to a CLEC2 binding domain in Podoplanin (PDPN), was found to be essential for this interaction. Like PDPN, CA9-induced CLEC2 signaling is mediated via Syk. A Hodgkin's lymphoma cell line (HDLM-2) endogenously expressing CA9 can activate Syk-dependent CLEC2 signaling, providing enticing evidence for a novel function of CA9 in hematological cancers. In conclusion, we identified numerous interactions with monocytes and platelets and validated one, CA9, as an endogenous CLEC2 ligand. We provide a new list of other putative CA9 interaction partners and uncovered CA9-induced CLEC2 activation, providing new insights for CA9-based therapeutic strategies.
膜蛋白，尤其是细胞外结构域，因其在细胞通讯和关联中的作用而成为关键的治疗靶点。然而，它们的功能和相互作用往往仍不清楚。本研究提出了一种通用方法，用于发现膜蛋白与免疫细胞的相互作用，并随后鉴定其各自的受体。我们构建了一个包含人类跨膜蛋白以及在内质网-高尔基体-溶酶体系统中发现的蛋白的细胞外结构域的全面重组蛋白文库。利用该文库，我们进行了流式细胞术筛选，确定了几种细胞表面结合事件，包括碳酸酐酶9（CAH9/CA9/CAIX）与CD14细胞之间的相互作用。进一步分析表明，这种相互作用是间接的，通过与单核细胞结合的血小板介导。CA9以其在癌症中的多种作用而闻名，是一个有前景的治疗靶点。我们利用我们的文库开发了一种AlphaLISA高通量筛选测定法，确定CLEC2是一种强大的CA9结合伴侣。发现CA9中的一个五氨基酸序列（EDLPT）与血小板反应蛋白（PDPN）中的一个CLEC2结合域相同，对于这种相互作用至关重要。与PDPN一样，CA9诱导的CLEC2信号传导是通过Syk介导的。内源性表达CA9的霍奇金淋巴瘤细胞系（HDLM-2）可以激活Syk依赖性CLEC2信号传导，为CA9在血液系统癌症中的新功能提供了诱人的证据。总之，我们鉴定了与单核细胞和血小板的多种相互作用，并验证了一种，即CA9，作为内源性CLEC2配体。我们提供了其他假定的CA9相互作用伴侣的新列表，并揭示了CA9诱导的CLEC2激活，为基于CA9的治疗策略提供了新的见解。
