title: RGD-dependent binding of procathepsin X to integrin alphavbeta3 mediates cell-adhesive properties.
abstract: Secreted lysosomal cysteine proteases (cathepsins) are involved in degradation and remodeling of the extracellular matrix, thus contributing to cell adhesion and migration. Among the eleven human lysosomal cysteine proteases, only procathepsin X contains an RGD motif located in a highly exposed region of the propeptide, which may allow binding of the proenzyme to RGD-recognizing integrins. Here, we have tested procathepsin X for cell-adhesive properties and found that it supports integrin alpha(v)beta(3)-dependent attachment and spreading of human umbilical vein endothelial cells. Using site-directed mutants of procathepsin X, we proved that this effect is mediated by the RGD sequence within the proregion of the protease. Endogenous procathepsin X is transported to the plasma membrane, accumulates in vesicles at lamellipodia of the human umbilical vein endothelial cell, and is partly associated with the cell surface, as shown by immunofluorescence. In addition, procathepsin X is partly co-localized with integrin beta(3), as detected by immunogold electron microscopy. A direct interaction between endogenous procathepsin X and alpha(v)beta(3) was demonstrated by co-immunoprecipitation. Moreover, surface plasmon resonance analysis revealed significant and RGD-dependent binding of procathepsin X to integrin alpha(v)beta(3). Our results provide for the first time evidence that the extracellular function of cathepsin X may include binding to integrins thereby modulating the attachment of migrating cells to ECM components.
authors.affiliation: Division of Clinical Chemistry and Clinical Biochemistry in the Department of Surgery, Ludwig-Maximilians-University and Adolf-Butenandt Institute, Ludwig-Maximilians-University, 80336 Munich, Germany.
authors.forename: Annette M
authors.initials: AM
authors.lastname: Lechner
authors.forename: Irmgard
authors.initials: I
authors.lastname: Assfalg-Machleidt
authors.forename: Stefan
authors.initials: S
authors.lastname: Zahler
authors.forename: Mechthild
authors.initials: M
authors.lastname: Stoeckelhuber
authors.forename: Werner
authors.initials: W
authors.lastname: Machleidt
authors.forename: Marianne
authors.initials: M
authors.lastname: Jochum
authors.forename: Dorit K
authors.initials: DK
authors.lastname: Nägler
mesh_terms: D002403:Cathepsins / Q000737:chemistry* / Q000378:metabolism; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry; D042783:Endothelial Cells / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000737:chemistry* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D011498:Protein Precursors; C047981:arginyl-glycyl-aspartic acid; D002403:Cathepsins; C516795:procathepsin X, human; D003546:Cysteine Endopeptidases