title: Cellular fibronectin binds to lysyl oxidase with high affinity and is critical for its proteolytic activation.
abstract: Lysyl oxidase (LOX) is a copper-containing amine oxidase known to catalyze the covalent cross-linking of fibrillar collagens and elastin at peptidyl lysine residues. In addition, its involvement in cancer, wound healing, cell motility, chemotaxis, and differentiation reflect a remarkable functional diversity of LOX. To investigate novel mechanisms of LOX regulation and function, we performed a yeast two-hybrid screen to identify LOX-interacting proteins. Three overlapping positive clones were identified as C-terminal fragments of fibronectin (FN). Glutathione S-transferase pull-downs and solid phase binding assays confirmed this interaction. LOX binds to the cellular form of FN (cFN) with a dissociation constant (K(d)) of 2.5 nm. This was comparable with our measured K(d) of LOX binding to tropoelastin (1.9 nm) and type I collagen (5.2 nm), but LOX demonstrated a much lower binding affinity for the plasma form of FN (pFN). Immunofluorescent microscopy revealed co-localization of FN and LOX in normal human tissues, where these proteins may interact in vivo. LOX enzymatic activity assays showed that cFN does not seem to be a substrate of LOX. However, cFN can act as a scaffold for enzymatically active 30-kDa LOX. Furthermore, in FN-null mouse embryonic fibroblasts, we observed dramatically decreased proteolytic processing of the 45-kDa LOX proenzyme to the 30-kDa active form, with a corresponding decrease in LOX enzyme activity. Our results suggest that the FN matrix may provide specific microenvironments to regulate LOX catalytic activity.
authors.affiliation: Cardiovascular Research Center, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822, USA.
authors.forename: Ben
authors.initials: B
authors.lastname: Fogelgren
authors.forename: Noémi
authors.initials: N
authors.lastname: Polgár
authors.forename: Kornélia Molnárné
authors.initials: KM
authors.lastname: Szauter
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Ujfaludi
authors.forename: Rozália
authors.initials: R
authors.lastname: Laczkó
authors.forename: Keith S K
authors.initials: KS
authors.lastname: Fong
authors.forename: Katalin
authors.initials: K
authors.lastname: Csiszar
mesh_terms: D000818:Animals; D002384:Catalysis; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D008249:Protein-Lysine 6-Oxidase / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014333:Tropoelastin / Q000737:chemistry; D020798:Two-Hybrid System Techniques
chemical_list: D018076:DNA, Complementary; D005353:Fibronectins; D011993:Recombinant Fusion Proteins; D014333:Tropoelastin; D008249:Protein-Lysine 6-Oxidase; D005982:Glutathione Transferase; D008239:Lysine