Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a key regulator of extracellular matrix turnover for its ability to inhibit matrix metalloproteinases (MMPs), adamalysin-like metalloproteinases (ADAMs) and ADAMs with thrombospondin motifs (ADAMTSs). TIMP-3 is a secreted protein whose extracellular levels are regulated by endocytosis via the low-density-lipoprotein receptor-related protein-1 (LRP-1). In this study we developed a molecule able to "trap" TIMP-3 extracellularly, thereby increasing its tissue bioavailability. LRP-1 contains four ligand-binding clusters. In order to investigate the TIMP-3 binding site on LRP-1, we generated soluble minireceptors (sLRPs) containing the four distinct binding clusters or part of each cluster. We used an array of biochemical methods to investigate the binding of TIMP-3 to different sLRPs. We found that TIMP-3 binds to the ligand-binding cluster II of the receptor with the highest affinity and a soluble minireceptor containing the N-terminal half of cluster II specifically blocked TIMP-3 internalization, without affecting the turnover of metalloproteinases. Mass spectrometry-based secretome analysis showed that this minireceptor, named T3TRAP, selectively increased TIMP-3 levels in the extracellular space and inhibited constitutive shedding of a number of cell surface proteins. In conclusion, T3TRAP represents a biological tool that can be used to modulate TIMP-3 levels in the tissue and could be potentially developed as a therapy for diseases characterized by a deficit of TIMP-3, including arthritis.
Matrix biology : journal of the International Society for Matrix Biology
69 - 79
Department of Biochemistry, Nagoya University Graduate School of Medicine, Nagoya, Japan; Department of Neuroproteomics, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich, Germany; Neuroproteomics, Klinikum rechts der Isar and Institute for Advanced Study, Technische Universität München, Munich, Germany. Electronic address: email@example.com.
Neuroglia, COS Cells, Cell Line, Tumor, Extracellular Matrix, Epithelial Cells, Animals, Cercopithecus aethiops, Humans, Recombinant Proteins, Tissue Inhibitor of Metalloproteinase-3, Transfection, Protein Interaction Mapping, Signal Transduction, Endocytosis, Gene Expression Regulation, Binding Sites, Protein Binding, Protein Transport, Kinetics, Solubility, Protein Interaction Domains and Motifs, HEK293 Cells, Molecular Sequence Annotation, Low Density Lipoprotein Receptor-Related Protein-1, Receptors, Artificial