title: Engineered Tissue Inhibitor of Metalloproteinases-3 Variants Resistant to Endocytosis Have Prolonged Chondroprotective Activity.
abstract: Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a central inhibitor of matrix-degrading and sheddase families of metalloproteinases. Extracellular levels of the inhibitor are regulated by the balance between its retention on the extracellular matrix and its endocytic clearance by the scavenger receptor low density lipoprotein receptor-related protein 1 (LRP1). Here, we used molecular modeling to predict TIMP-3 residues potentially involved in binding to LRP1 based on the proposed LRP1 binding motif of 2 lysine residues separated by about 21 Å and mutated the candidate lysine residues to alanine individually and in pairs. Of the 22 mutants generated, 13 displayed a reduced rate of uptake by HTB94 chondrosarcoma cells. The two mutants (TIMP-3 K26A/K45A and K42A/K110A) with lowest rates of uptake were further evaluated and found to display reduced binding to LRP1 and unaltered inhibitory activity against prototypic metalloproteinases. TIMP-3 K26A/K45A retained higher affinity for sulfated glycosaminoglycans than K42A/K110A and exhibited increased affinity for ADAMTS-5 in the presence of heparin. Both mutants inhibited metalloproteinase-mediated degradation of cartilage at lower concentrations and for longer than wild-type TIMP-3, indicating that their increased half-lives improved their ability to protect cartilage. These mutants may be useful in treating connective tissue diseases associated with increased metalloproteinase activity.
authors: From the Arthritis Research UK Centre for Osteoarthritis Pathogenesis, Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Oxford OX3 7FY, United Kingdom.
authors: Christine M
authors: CM
authors: Doherty
authors: From the Arthritis Research UK Centre for Osteoarthritis Pathogenesis, Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Oxford OX3 7FY, United Kingdom.
authors: Robert
authors: R
authors: Visse
authors: the School of Computing and Engineering, University of Missouri, Kansas City, Missouri 64111, and.
authors: Deendayal
authors: D
authors: Dinakarpandian
authors: the University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors: Dudley K
authors: DK
authors: Strickland
authors: From the Arthritis Research UK Centre for Osteoarthritis Pathogenesis, Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Oxford OX3 7FY, United Kingdom.
authors: Hideaki
authors: H
authors: Nagase
authors: From the Arthritis Research UK Centre for Osteoarthritis Pathogenesis, Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Oxford OX3 7FY, United Kingdom, linda.troeberg@kennedy.ox.ac.uk.
authors: Linda
authors: L
authors: Troeberg
mesh_terms: D000071116:ADAMTS5 Protein / Q000235:genetics / Q000378:metabolism; D001859:Bone Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002356:Cartilage / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002813:Chondrosarcoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D004705:Endocytosis; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006493:Heparin / Q000378:metabolism; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics / Q000378:metabolism*
chemical_list: C475476:LRP1 protein, human; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D009363:Neoplasm Proteins; C088390:TIMP3 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3; D006493:Heparin; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human
keywords: ADAM; ADAMTS; extracellular matrix; glycosaminoglycan; low-density lipoprotein receptor-related protein 1; osteoarthritis; receptor; tissue inhibitor of metalloproteinase (TIMP)