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Insights into Collagen Uptake by C-type Mannose Receptors from the Crystal Structure of Endo180 Domains 1-4.

ABSTRACT: The C-type mannose receptor and its homolog Endo180 (or uPARAP, for urokinase plasminogen activator receptor-associated protein) mediate the endocytic uptake of collagen by macrophages and fibroblasts. This process is required for normal tissue remodeling, but also facilitates the growth and dissemination of tumors. We have determined the crystal structure at 2.5 Å resolution of the N-terminal region of Endo180, consisting of a ricin-like domain, a fibronectin type II (FN2) domain, and two C-type lectin (CTL) domains. The L-shaped arrangement of these domains creates a shallow trench spanning the FN2 and CTL1 domains, which was shown by mutagenesis to bind triple-helical and denatured collagen. Small-angle X-ray scattering showed that the L-shaped structure is maintained in solution at neutral and acidic pH, irrespective of calcium ion loading. Collagen binding was equally unaffected by acidic pH, suggesting that collagen release in endosomes is not regulated by changes within the Endo180 N-terminal region.

SUBMITTER: Paracuellos P

PROVIDER: S-EPMC4635314 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Insights into Collagen Uptake by C-type Mannose Receptors from the Crystal Structure of Endo180 Domains 1-4.

Paracuellos Patricia P Briggs David C DC Carafoli Federico F Lončar Tan T Hohenester Erhard E

Structure (London, England : 1993) 20151015 11

The C-type mannose receptor and its homolog Endo180 (or uPARAP, for urokinase plasminogen activator receptor-associated protein) mediate the endocytic uptake of collagen by macrophages and fibroblasts. This process is required for normal tissue remodeling, but also facilitates the growth and dissemination of tumors. We have determined the crystal structure at 2.5 Å resolution of the N-terminal region of Endo180, consisting of a ricin-like domain, a fibronectin type II (FN2) domain, and two C-typ ...[more]

PMID: 26481812

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Project description:Tissue remodelling is dependent on the integration of signals that control turnover of ECM (extracellular matrix). Breakdown and endocytosis of collagen, a major component of the ECM, is central to this process. Whereas controlled secretion of matrix-degrading enzymes (such as matrix metalloproteinases) has long been known to mediate ECM breakdown, it is becoming clear that uPARAP/Endo180 (where uPARAP stands for urokinase plasminogen activator receptor-associated protein) serves as a receptor that mediates endocytosis of collagen by several types of cells. In the liver, the stellate cells play a major role in turnover of ECM including collagens. These cells synthesize various collagens and also produce matrix metalloproteinases. In the present study, we investigated the capacity of rat hepatic stellate cells to endocytose and degrade 125I-labelled heat-denatured collagen I. It was found that the collagen is efficiently taken up and degraded by these cells. Degradation was inhibited by inhibitors of lysosomal proteases (leupeptin and E-64d) and the vacuolar proton pump (concanamycin A), indicating that it takes place in lysosomes. Furthermore, endocytosed FITC-labelled collagen was shown to reach late endocytic compartments in which it colocalized with LysoTracker (a marker of late endocytic compartments). Competition experiments showed that uPA and unlabelled collagen are capable of inhibiting binding and uptake of [125I]collagen in a dose-dependent manner. Moreover, Western-blot analysis of cell lysate (using a polyclonal rabbit human-Endo180 antiserum) revealed a single band at 180 kDa. In addition, the antiserum was capable of reducing [125I]collagen binding to the cell surface. Finally, using two primers designed from the human uPARAP/Endo180 mRNA sequence, the expression of uPARAP/Endo180 mRNA was detected by reverse transcriptase-PCR. These results together suggest that uPARAP/Endo180 mediates endocytosis of collagen in rat liver stellate cells.

Project description:BackgroundGlioblastoma multiforme (GBM, WHO grade IV) is the most common and most malignant of astrocytic brain tumors, and is associated with rapid invasion into neighboring tissue. In other tumor types it is well established that such invasion involves a complex interaction between tumor cells and locally produced extracellular matrix. In GBMs, surprisingly little is known about the associated matrix components, in particular the fibrillar proteins such as collagens that are known to play a key role in the invasion of other tumor types.Methodology/principal findingsIn this study we have used both the Masson's trichrome staining and a high resolution multiple immunofluorescence labeling method to demonstrate that intratumoral fibrillar collagens are an integral part of the extracellular matrix in a subset of GBMs. Correlated with this collagen deposition we observed high level expression of the collagen-binding receptor Endo180 (CD280) in the tumor cells. Further, interrogation of multiple expression array datasets identified Endo180 as one of the most highly upregulated transcripts in grade IV GBMs compared to grade III gliomas. Using promoter analysis studies we show that this increased expression is, in part, mediated via TGF-beta signaling. Functionally, we demonstrate that Endo180 serves as the major collagen internalization receptor in GBM cell lines and provide the first evidence that this activity is critical for the invasion of GBM cells through fibrillar collagen matrices.Conclusions/significanceThis study demonstrates, for the first time, that fibrillar collagens are extensively deposited in GBMs and that the collagen internalization receptor Endo180 is both highly expressed in these tumors and that it serves to mediate the invasion of tumor cells through collagen-containing matrices. Together these data provide important insights into the mechanism of GBM invasion and identify Endo180 as a potential target to limit matrix turnover by glioma cells and thereby restrict tumor progression.

Dataset Information

Insights into Collagen Uptake by C-type Mannose Receptors from the Crystal Structure of Endo180 Domains 1-4.

Publications

Insights into Collagen Uptake by C-type Mannose Receptors from the Crystal Structure of Endo180 Domains 1-4.

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