title: Elastin degradation by cathepsin V requires two exosites.
abstract: Cathepsin V is a highly effective elastase and has been implicated in physiological and pathological extracellular matrix degradation. However, its mechanism of action remains elusive. Whereas human cathepsin V exhibits a potent elastolytic activity, the structurally homologous cathepsin L, which shares a 78% amino acid sequence, has only a minimal proteolytic activity toward insoluble elastin. This suggests that there are distinct structural domains that play an important role in elastinolysis. In this study, a total of 11 chimeras of cathepsins V and L were generated to identify elastin-binding domains in cathepsin V. Evaluation of these chimeras revealed two exosites contributing to the elastolytic activity of cathepsin V that are distant from the active cleft of the protease and are located in surface loop regions. Replacement of exosite 1 or 2 with analogous residues from cathepsin L led to a 75 and 43% loss in the elastolytic activity, respectively. Replacement of both exosites yielded a non-elastase variant similar to that of cathepsin L. Identification of these exosites may contribute to the design of inhibitors that will only affect the elastolytic activity of cysteine cathepsins without interfering with other physiological protease functions.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Faculty of Medicine, Center for Blood Research, and.
authors.forename: Xin
authors.initials: X
authors.lastname: Du
authors.forename: Nelson L H
authors.initials: NL
authors.lastname: Chen
authors.forename: Andre
authors.initials: A
authors.lastname: Wong
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: Dieter
authors.initials: D
authors.lastname: Brömme
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D056668:Cathepsin L / Q000737:chemistry* / Q000378:metabolism; D002403:Cathepsins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004549:Elastin / Q000737:chemistry / Q000378:metabolism*; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D059748:Proteolysis; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D004549:Elastin; D002403:Cathepsins; D010196:Pancreatic Elastase; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C117627:CTSV protein, human
keywords: Cathepsin V; Cysteine Protease; Elastase; Elastin; Exosite; Extracellular Matrix Proteins; Protein Degradation; Site-directed Mutagenesis; Substrate Specificity