A proteomics-based survey reveals thrombospondin-4 as a ligand regulated by the mannose receptor in the injured lung

• Published in: The Journal of biological chemistry Vol. 300; no. 5; p. 107284
• Main Authors: Nørregaard, Kirstine S., Jürgensen, Henrik J., Heltberg, Signe S., Gårdsvoll, Henrik, Bugge, Thomas H., Schoof, Erwin M., Engelholm, Lars H., Behrendt, Niels
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2024; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; CHO Cells; Cricetulus; Endocytosis; Lectins, C-Type - genetics; Lectins, C-Type - metabolism; Ligands; Lipopolysaccharides - toxicity; Lung - metabolism; Lung - pathology; Lung Injury - metabolism; Lung Injury - pathology; macrophage; Macrophages, Alveolar - metabolism; Macrophages, Alveolar - pathology; Mannose Receptor; Mannose-Binding Lectins - genetics; Mannose-Binding Lectins - metabolism; mass spectrometry (MS); Mice; Mice, Knockout; protein turnover; proteomics; Proteomics - methods; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Thrombospondins - genetics; Thrombospondins - metabolism; urokinase plasminogen activator-associated protein (uPARAP/Endo180); AF647; RT; proteomics; IHC; MR; MS; BALF; urokinase plasminogen activator-associated protein (uPARAP/Endo180); Fn-II; macrophage; SCX; SPS MS3; BAL; LPS; PLA2R; TSP; BSA; protein turnover; uPARAP; endocytosis; TMT; AGC; ELF; mass spectrometry (MS)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2024.107284

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.