title: Activity-based labeling of matrix metalloproteinases in living vertebrate embryos.
abstract: Extracellular matrix (ECM) remodeling is a physiologically and developmentally essential process mediated by a family of zinc-dependent extracellular proteases called matrix metalloproteinases (MMPs). In addition to complex transcriptional control, MMPs are subject to extensive post-translational regulation. Because of this, classical biochemical, molecular and histological techniques that detect the expression of specific gene products provide useful but limited data regarding the biologically relevant activity of MMPs. Using benzophenone-bearing hydroxamate-based probes that interact with the catalytic zinc ion in MMPs, active proteases can be covalently 'tagged' by UV cross-linking. This approach has been successfully used to tag MMP-2 in vitro in tissue culture supernatants, and we show here that this probe tags proteins with mobilities consistent with known MMPs and detectable gelatinolytic activity in homogenates of zebrafish embryos. Furthermore, because of the transparency of the zebrafish embryo, UV-photocroslinking can be accomplished in vivo, and rhodamated benzophenone probe is detected in striking spatial patterns consistent with known distributions of active matrix remodeling in embryos. Finally, in metamorphosing Xenopus tadpoles, this probe can be used to biotinylate active MMP-2 by injecting it and cross-linking it in vivo, allowing the protein to be subsequently extracted and biochemically identified.
authors.affiliation: Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada.
authors.forename: Jonathan Y
authors.initials: JY
authors.lastname: Keow
authors.forename: Eric D
authors.initials: ED
authors.lastname: Pond
authors.forename: Justin S
authors.initials: JS
authors.lastname: Cisar
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Cravatt
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Crawford
mesh_terms: D000818:Animals; D001577:Benzophenones / Q000737:chemistry / Q000494:pharmacology; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D005109:Extracellular Matrix / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007477:Ions; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D008956:Models, Chemical; D012323:RNA Processing, Post-Transcriptional; D014466:Ultraviolet Rays; D014714:Vertebrates / Q000502:physiology*; D014982:Xenopus laevis; D015027:Zebrafish; D015032:Zinc / Q000737:chemistry
chemical_list: D001577:Benzophenones; D003432:Cross-Linking Reagents; D007477:Ions; D020782:Matrix Metalloproteinases; D020778:Matrix Metalloproteinase 2; D015032:Zinc