Receptor-mediated cellular uptake of specific ligands constitutes an important step in the dynamic regulation of individual protein levels in extracellular fluids. With a focus on the inflammatory lung, we here performed a proteomics-based search for novel ligands regulated by the mannose receptor (MR), a macrophage-expressed endocytic receptor. WT and MR-deficient mice were exposed to lipopolysaccharide, after which the protein content in their lung epithelial lining fluid was compared by tandem mass tag-based mass spectrometry. More than 1200 proteins were identified in the epithelial lining fluid using this unbiased approach, but only six showed a statistically different abundance. Among these, an unexpected potential new ligand, thrombospondin-4 (TSP-4), displayed a striking 17-fold increased abundance in the MR-deficient mice. Experiments using exogenous addition of TSP-4 to MR-transfected CHO cells or MR-positive alveolar macrophages confirmed that TSP-4 is a ligand for MR-dependent endocytosis. Similar studies revealed that the molecular interaction with TSP-4 depends on both the lectin activity and the fibronectin type-II domain of MR and that a closely related member of the TSP family, TSP-5, is also efficiently internalized by the receptor. This was unlike the other members of this protein family, including TSPs −1 and −2, which are ligands for a close MR homologue known as urokinase plasminogen activator receptor-associated protein. Our study shows that MR takes part in the regulation of TSP-4, an important inflammatory component in the injured lung, and that two closely related endocytic receptors, expressed on different cell types, undertake the selective endocytosis of distinct members of the TSP family.

中文翻译：

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一项基于蛋白质组学的调查揭示了血小板反应蛋白 4 作为受损伤肺中甘露糖受体调节的配体


受体介导的细胞对特定配体的摄取构成了细胞外液中个体蛋白质水平动态调节的重要步骤。以炎症性肺部为重点，我们进行了基于蛋白质组学的研究，寻找受甘露糖受体（MR）（一种巨噬细胞表达的内吞受体）调节的新型配体。将 WT 和 MR 缺陷小鼠暴露于脂多糖，然后通过基于串联质量标签的质谱法比较其肺上皮衬里液中的蛋白质含量。使用这种公正的方法在上皮衬里液中鉴定出了 1200 多种蛋白质，但只有 6 种蛋白质的丰度存在统计学差异。其中，一种意想不到的潜在新配体，血小板反应蛋白-4 (TSP-4)，在 MR 缺陷小鼠中表现出惊人的 17 倍丰度增加。使用外源添加 TSP-4 至 MR 转染的 CHO 细胞或 MR 阳性肺泡巨噬细胞的实验证实，TSP-4 是 MR 依赖性内吞作用的配体。类似的研究表明，与 TSP-4 的分子相互作用取决于凝集素活性和 MR 的纤连蛋白 II 型结构域，并且 TSP 家族的密切相关成员 TSP-5 也能被受体有效内化。这与该蛋白家族的其他成员不同，包括 TSP -1 和 -2，它们是称为尿激酶纤溶酶原激活剂受体相关蛋白的紧密 MR 同源物的配体。我们的研究表明，MR 参与 TSP-4 的调节，TSP-4 是受损肺部的重要炎症成分，并且在不同细胞类型上表达的两种密切相关的内吞受体承担 TSP 家族不同成员的选择性内吞作用。