Keow JY et al. (2012), Activity-based labeling of matrix metalloprotei...

XB-ART-45871

PLoS One 2012 Jan 01;78:e43434. doi: 10.1371/journal.pone.0043434.

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Activity-based labeling of matrix metalloproteinases in living vertebrate embryos.

Keow JY , Pond ED , Cisar JS , Cravatt BF , Crawford BD .

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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.

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Species referenced: Xenopus laevis
Genes referenced: avd mmp2

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	Figure 2. Hx-BP labels active MMPs in vitro.135 ng of human recombinant MMP-2 (hrMMP-2) was incubated with 0.5 nmol of biotinylated HxBP, with or without activation by APMA and with or without UV crosslinking, then resolved by SDS-PAGE and blotted to PVDF membrane. Streptavidin-HRP detection of biotinylated hrMMP-2 is dependent on both the activation status of the protease, and on exposure to UV light (hv).
	Figure 3. HxBP labels its target proteins in vivo.Live Xenopus tadpoles were injected with HxBP-alkyne, allowed to recover, exposed to UV light, and then sacrificed and protein extracts of the tails were subjected to click chemistry to biotinylate HxBP-tagged proteins. In panel A, biotinylated proteins were affinity purified by avidin chromatography, resolved by SDS-PAGE and detected by silver staining. A single protein with a mobility consistent with xMMP2 is detectable in the lane containing the HxBP-labeled proteome of the larvae that were induced to express xMMP2 by treatment with T3, and then biotinylated by click chemistry. In panel B, tail homogenates were split and run in parallel on a gelatin zymography gel (left) and for blotting to PVDF (right). Blots were probed with streptavidin-HRP, and revealed a single labeled protein at the same mobility as the gelatinolytic xMMP2 band on the zymograph.
	Figure 4. HxBP can be UV-photocrosslinked in vivo, and detected in situ by confocal microscopy.Composite confocal micrographs of 24 hpf zebrafish embryos injected anteriorly with 50 µM trifunctional HxBP probe, recovered, and exposed to increasing levels of UV irradiation reveals increasingly spatially structured patterns of fluorescence up to 72 mJ/cm2. Structures exhibiting strong HxBP labeling in 24 hpf embryos include the retina (r) and lens (l), head mesenchyme (hm), hatching gland (hg), perichordal sheath (ps), isolated notochord cells in the elongating region of the notochord (n), maturing myotome boundaries (mb), mesenchymal tissues of the trunk and tail (m), and the basement membrane underlying the epithelium dorsal to the elongating tail (bm). Asterisks mark the point of injection.
	Figure 5. HxBP is labeling metalloproteinases in vivo.Composite confocal micrographs of 72 hpf zebrafish embryos injected anteriorly (*) with 50 µM of trifunctional HxBP probe either with (B) or without (A) competition from unlabeled GM6001. Embryos pre-injected with GM6001 show dramatically attenuated HxBP labeling, requiring the data shown in panel B to be collected using 42% increased gain in order to be detectable. Structures showing strong labeling in HxBP labeled 72 hpf embryos include proliferative myofibrils along the anterior lateral midline (m), maturing myotome boundaries (mb), the horizontal myoseptum (hm), individual migratory mesenchyme cells (mc), and the developing vasculature including the dorsal aorta (da), posterior cardinal vein (pcv) and intersomitic vessels (isv). Asterisk marks the injection site. Scale bar is 500 µM.
	Figure 6. Details of HxBP labeling patterns reveal dynamics of active matrix remodeling.Z-projections of high magnification confocal micrographs illustrating patterns of HxBP labeling in both 24 hpf (A, B) and 72 hpf (C–H) zebrafish embryos. A) The developing zebrafish eye at 24 hpf, with HxBP labeling migratory mesenchyme (arrowhead), retinal epithelium (r), the choroid fissure and the hatching gland (hg). The lens shows no HxBP labeling, suggesting that matrix remodeling is absent in the lens. B) HxBP labeling the epithelial (e) and mesenchymal tissue (m) around the otic vesicle (ov), but no labeling within the otic vesicle. Primes are single focal planes of confocal stacks used to generate the non-prime panels. C) A ventrolateral view of the head of a 72 hpf embryo showing strong labeling in the developing scleral ossicles. Strong labeling is also evident in individual migratory mesenchyme cells and developing vasculature. D) lateral view of the anterior trunk showing strong labeling in the maturing myotome boundaries (mb), horizontal myoseptum (hm) and in individual myofibrils (mf) situated in the proliferative zone. E) Epithelia of the lateral head and dorsal aspect of the eye showing strong labeling in individual cells and patches of contiguous epithelial cells. F) lateral view of the tail showing strong labeling in migratory mesenchyme cells (mc), as well as labeling in the dorsal aorta (da), posterior cardinal vein (pcv) and intersomitic vessels (isv). G) lateral view of the surface epithelia covering the anterior trunk illustrating the ‘chicken wire’ patterning of HxBP labeling surrounding the periphery of the cells in this tissue. H) A dorsolateral view of the eye, illustrating strong HxBP labeling of mesenchymal cells invading across the surface of the retina and surrounding the lens (l). Scale bars are 50 µm in all panels.
	Figure 7. HxBP labeling in a 96 hpf swimming larva.Composite of confocal projections taken of a 96 hpf larva injected (at asterisk) with 50 µM trifunctional HxBP. Strong labeling is evident throughout the developing circulatory system, most notably in the looping vessels of the gill arches (g), the dorsal aorta (da), posterior cardinal vein (pcv), intersomitic vessels (isv), and hyaloid artery (ha). Labeling is also notable in individual migratory mesenchyme cells (mc), the protease-rich stomach (s), horizontal myoseptum (hm) and maturing craniofacial cartilages. Scale bar is 500 µm.
	Figure 1. Structure of the Hx-PByne probe.The MMP labeling probes used were versions of this structure (compound 4 in [1]). The hydroxamic acid moiety (pink) chelates the catalytic zinc ion, and the benzophenone moiety (green) functions as a UV-photoactivateable crosslinking group. The terminal alkyne (red) allows the addition of groups such as biotin or rhodamine by click chemistry [1].

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