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Region Hovedstaden - en del af Københavns Universitetshospital
Udgivet

A Survey of Novel Ligands of the Members of the Mannose Receptor Protein Family

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

  1. Tumor cell MT1-MMP is dispensable for osteosarcoma tumor growth, bone degradation and lung metastasis

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

  2. The collagen receptor uPARAP/Endo180 regulates collectins through unique structural elements in its FNII domain

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

  3. Cellular uptake of collagens and implications for immune cell regulation in disease

    Publikation: Bidrag til tidsskriftReviewForskningpeer review

  4. Immune regulation by fibroblasts in tissue injury depends on uPARAP-mediated uptake of collectins

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

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Temporal and spatial regulation of proteins in the extracellular environment is essential during health and disease. Regulatory mechanisms exist at the levels of transcription, translation, and secretion, as well as targeting secreted proteins for degradation. One example of the latter is clearance of extracellular proteins by receptor-mediated endocytosis for intracellular degradation.
Two endocytic receptors with this role are the mannose receptor (MR) protein family members, MR and urokinase plasminogen-activator receptor-associated protein (uPARAP). The two receptors share the ability to internalize large cleavage products from structural collagens through their fibronectin type II (FNII) domain. uPARAP mediated collagen internalization for intracellular degradation plays a role in bone development, reducing aberrant collagen accumulation during fibrosis, and extracellular matrix degradation during cancer invasion. In addition to collagens, MR binds glycoprotein hormones and glycosylated lysosomal enzymes, and acts as a pattern recognition receptor in the innate immune defense, whereas uPARAP has been shown more recently to bind collectins, a group of immune defense molecules. At the start of this thesis work, it appeared likely that additional ligands for these receptors might exist.
The work presented in this thesis is a survey of the molecular determinants involved in ligand binding by uPARAP, along with an unbiased, discovery-based search for novel ligands for uPARAP and MR, applying mass spectrometry (MS)-based proteomics.
The first manuscript presents the results of an investigation of the recent and rather puzzling observation that uPARAP’s ability to bind collectins of the innate immune defense, is not shared by the structurally related collagen receptor, MR. The study entails an examination of the molecular elements in uPARAP responsible for binding to the collectins, mannose binding lectin (MBL) and surfactant protein D (SP-D), using an in vitro internalization assay. The study revealed that a protruding loop of ten amino acids in the FNII domain, essential for collagen binding, is also required for uPARAP´s binding of the collectins. Furthermore, we identified single amino acid residues within the FNII domain uniquely involved in collectin binding. This provided important clues to the lack of collectin binding by MR, as these residues are absent in MR’s FNII domain. In addition, the study found significant differences in binding of MBL and SP-D by uPARAP. The active lectin domain of uPARAP is important for binding of SP-D, but not MBL, and uPARAP can bind MBL, but not SP-D, immobilized on the surface of pathogen particles. We suggest that the observed differences can be explained by the distinct structural properties of SP-D and MBL oligomers, and that they potentially contribute to a differential regulation of these innate immune components.
In the second manuscript, the matricellular protein thrombospondin-1 (TSP-1) was discovered as a novel ligand of uPARAP. TSP-1 is considered highly important for the regulation of angiogenesis and most likely is also involved in other important physiological and pathological processes, such as bone development, pulmonary homeostasis, fibrosis, and cancer. Therefore, the finding of a novel potential regulator of TSP-1 may be of central importance. This finding was the result of an unbiased search for uPARAP ligands. Novel ligands were searched through a comparison of the abundance levels of proteins, identified by MS, in endosomal-enriched subcellular fractions from wild type and uPARAP depleted human osteosarcoma cells in culture, leading to the identification of TSP-1 as a candidate ligand. Subsequent investigations, using an in vitro internalization assay, confirmed uPARAP as a novel endocytic receptor of TSP-1 and revealed that uPARAP-mediated endocytosis leads to intracellular 4
degradation of TSP-1 in the lysosomes. Importantly, this study is the first to show receptor-mediated endocytosis as an important mechanism in regulating extracellular TSP-1 levels. By assaying the levels of TSP-1 in the culture medium from osteosarcoma cells and uPARAP-transfected CHO-K1 cells, uPARAP mediated endocytosis was found to be a clearance mechanism for both endogenous and exogenous TSP-1. The binding site for TSP-1 was mapped to the FNII domain, the domain also responsible for collagen and collectin binding. However, we found that within this domain, TSP-1 binding involves elements that are not critical for either collagen or collectin binding. Remarkably, as also found for the interaction with collectins, uPARAP´s interaction with TSP-1 was unique in the sense that the binding capability was not shared with MR. Heparin was found to inhibit TSP-1 endocytosis by uPARAP.
The third manuscript presents the results of an MS-based proteomics analysis of the role of MR-mediated endocytosis in vivo, comparing bronchoalveolar lavage fluid (BALF) from wild type and MR knock out mice. This led to the discovery of the matricellular protein, TSP-4, as a novel candidate ligand for MR, and MR was confirmed as an endocytic receptor for TSP-4 in vitro. This study is the first to identify a receptor responsible for internalization of TSP-4, and importantly provides evidence for receptor-mediated endocytosis of TSP-4 as a clearance mechanism of endogenous TSP-4 in vivo. The analysis revealed additional proteins in the BALF that were significantly more abundant in the absence of MR. These included previously described MR ligands; collagen type IV-VI and several lysosomal enzymes, and these findings substantiate the usefulness of MS-based proteomics for discovery and validation of endocytic receptor ligands.
The results presented in this thesis reveal important novel information about the molecular elements involved in ligand binding by uPARAP, the discovery of uPARAP and MR as endocytic receptors for two members of the TSP family, and their role in the regulation of the extracellular levels of these matricellular proteins with diverse and important biological roles.
OriginalsprogEngelsk
ForlagEget Forlag
Antal sider118
StatusUdgivet - apr. 2020

ID: 60798703