The authors have declared that no competing interests exist.
Conceived and designed the experiments: JYK BDC. Performed the experiments: JYK EDP JSC BDC. Analyzed the data: JYK JSC EDP BDC. Contributed reagents/materials/analysis tools: BDC BFC. Wrote the paper: JYK BDC.
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
Embryonic morphogenesis, wound healing, and many pathological processes such as tumor metastasis involve cellular processes that are integrated and exquisitely regulated at multiple levels, many of which remain poorly understood. Among these, the dynamics of extracellular matrix (ECM) remodeling has been the focus of intense investigation for many years. The matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases best known for their ability to hydrolyze ECM components
Over the past four decades, MMP research has employed an enormous diversity of biochemical, molecular, cell-biological and high-throughput techniques. However, it has become increasingly apparent that, because of the complexity of the post-translational regulation of MMPs, assays that elucidate the activity of these enzymes, especially
Activity-based protein profiling (ABPP) uses a chemical probe that targets active MMPs and becomes covalently bound to the protease. This technique provides quantitative information about the abundance of active proteases, as well as qualitative information about their location and, potentially, their molecular identity. We have used a hydroxamate-benzophenone probe (HxBP) that has previously been demonstrated to specifically target active MMPs
The MMP labeling probes used were versions of this structure (compound 4 in
We show that this probe can be used to label active MMPs in complex mixtures both
This study was carried out under the auspices of the University of New Brunswick Animal Care committee, and all animal used was governed by protocols in strict accordance with the recommendations of the Canadian Council for Animal Care. Zebrafish and
Sexually mature
Zebrafish embryos were obtained by natural spawning of wild type fish maintained on a 14-hour light/10-hour dark cycle
HxBP probes were synthesized as described in Saghatelian
Human recombinant MMP-2 was dissolved in activation buffer (50 mM NaCl, 50 mM Tris, 0.005% Triton X-100, pH 7.5) to a final concentration of 1 µg/ml in the presence or absence of 1 mM
20 µl of each sample was resolved on a 12% polyacrylamide gel for 2 hours at 120 V then horizontally transferred onto an Immobilon-P (Millipore) transfer membrane at 70 V for 2 hours. The blot was incubated in blocking buffer (5% BSA in phosphate-buffered saline with 0.01% Tween-20 (PBSTw)) overnight at 4°C to prevent nonspecific binding, and then incubated in Streptavidin-HRP conjugate (Invitrogen) (diluted 1∶10000 in blocking buffer) overnight at 4°C. The blot was then washed three times for 15 minutes in PBSTw to remove unbound streptavidin-HRP and detection of biotinylated proteins was performed using an enhanced chemiluminescence (ECL) kit (Pierce) according to the manufacturer’s directions.
Hatched zebrafish embryos and larvae between 96 hpf to 168 hpf were terminally anesthetized in 0.4 mg/ml carbonate buffered tricaine and homogenized in 10 µl lysis buffer (150 mM NaCl, 10 mM HEPES pH 7.5, 10 mM CaCl2, 0.1% Triton X-100, 2× Protease Inhibitor (Sigma-Aldrich) (a cocktail of 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatin A, E-64, bestatin, leupeptin and aprotinin protease inhibitors, but which notably lacks EDTA and other MMP inhibitors)) per embryo. To verify the probe’s MMP-specificity, some samples were pre-incubated with 2.5 µM of GM6001 (Chemicon) inhibitor. HxBP was added to a final concentration of 5 nM and incubated overnight at 4°C. Tubes were then cross-linked with 120.0 mJ/cm2 of UV irradiation (Stratalinker 1800) (some control tubes were not cross-linked).
Three volumes of acetone were added to precipitate proteins, followed by a 20 minute incubation on ice. Samples were spun at 21,000 g at room temperature for 10 minutes and the pellets washed in 300 mM guanidine hydrochloride in 95% ethanol with 2.5% glycerol thrice for 10 minutes to remove acetone. Pellets were then washed twice in 95% ethanol with 3.5% glycerol for another 10 minutes each to remove residual salts. Finally pellets were dissolved in 2× SDS-PAGE reducing sample buffer with 3 M urea (at a ratio of 7.5 µL per embryo) and centrifuged to remove insoluble debris before being resolved on a 10% polyacrylamide gel at 120 V for 2 hours and transferred to PVDF (Immobilon-P) at 60 V for 3 hours. After blocking in 10% Bovine Serum Albumin (BSA) overnight, the membrane was incubated in 1∶10000 Streptavidin-HRP for 2 hours, washed thrice in PBSTw and biotinylated proteins detected by ECL (Pierce).
Embryos were anesthetized in 0.04 mg/mL buffered tricaine solution and mounted in 3% methylcellulose dissolved in embryo rearing media (ERM). Using a pressure-based microinjection apparatus (ASI), these embryos were injected with a 5 µM solution of rhodamine-tagged HxBP dissolved in distilled water. Embryos were injected in the head between the developing eyes, or in the trunk dorsal to the yolk extension, allowed to recover for 5 minutes in fresh ERM, and then cross-linked with either 48.0 or 72.0 mJ/cm2 of UV irradiation (Stratalinker 1800). Control embryos were either pre-injected with 0.01 mg/ml GM6001 (diluted from a or not cross-linked as indicated. Embryos were fixed in 4% paraformaldehyde in PBS at 4°C overnight. After three washes in phosphate-buffered saline with 0.1% Triton X-100 (PBSTx), embryos were mounted for viewing using a Leica SP-2 confocal microscope.
Samples were spun to remove insoluble organic matter and the supernatant was collected. 5 mM Rhodamine-biotin azide was covalently linked to the alkyne moiety of the HxBP probe using click chemistry
For samples that were to be resolved by SDS-PAGE, beads were boiled for 15 minutes in 5× reducing SDS-PAGE sample buffer, run on a 10% polyacrylamide gel for 2 hours at 120 V, and either silver stained and photographed, or blotted to a PVDF membrane. Proteins that were transferred to a PVDF membrane were blocked overnight in blocking buffer (PBSTw with 5% BSA) at 4°C, then incubated overnight at 4°C in a 1∶10000 dilution of Streptavidin-HRP. After three 10 minute washes in PBSTw, the membrane was detected using ECL (Pierce).
Frozen tadpole homogenates were thawed, spun and diluted 1∶4 with 5X non-reducing sample buffer (225 mM Tris pH 6.8, 50% glycerol, 5% SDS, 0.05% bromophenol blue) and left at room temperature for 5 minutes. Samples were run on a 10% polyacrylamide gel containing 0.1% bovine gelatin for 3 hours at 110 V. The gel was then washed twice for 20 minutes in renaturing buffer (1.25% Triton X-100 dissolved in 25 mM Tris pH 7.5) and twice for 20 minutes in developing buffer (50 mM Tris pH 7.5, 5 mM CaCl2, 0.1 mM ZnSO4, 0.025% Brij35) at room temperature with gentle agitation, then placed in fresh developing buffer and incubated at 28°C for 48 hours. The gel was stained with Coomassie R-250, and then photographed once lytic bands were clearly visible.
In order to verify that the HxBP probe is able to label MMP-2 in our hands, we incubated full-length human recombinant MMP-2 (hrMMP-2), either with or without APMA activation, with biotinylated HxBP, UV-cross-linked, then blotted and probed with streptavidin-HRP. Consistently with the results of Saghatelian
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 (h
It is well established that T3-treated
A single ∼70 kD biotinylated protein is detected in the clicked proteomes of T3-induced tail homogenates (
Live
We sought to determine whether, in addition to facilitating their biochemical characterization, this activity-based probe could be used to visualize the distribution of active MMPs in living tissues. Because the optical properties of the
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.
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.
To verify that the probe is interacting specifically with MMPs, we attempted to attenuate the HxBP labeling by competition with the higher affinity MMP inhibitor GM6001 (for GM6001 Ki = 1.1 nM, vs. 13.0 nM for HxBP against hrMMP-2
In the 72 hpf embryos not pre-injected with GM6001, labeling is most notable surrounding the developing retina, in the myotomes, developing myotome boundaries and the horizontal myoseptum, in migrating meschencyme, the developing circulatory system (especially in the dorsal aorta, posterior cardinal vein and intersomitic vessels), and in the surface epithelium. High magnification images illustrating details of these and other staining patterns are shown in
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.
In the head of 24 hpf embryos, labeling is strong in the mesenchymal tissues, especially surrounding the retina and lens of the developing eye and surrounding the otic vesicle (
In 96 hpf embryos, we observe labeling in the horizontal myoseptum, myotome boundaries, cloaca, mesenchymal cells, and developing circulatory system (
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.
We were unable to extract sufficient quantities of HxBP-tagged metalloproteinases from zebrafish embryos characterize them biochemically, so we cannot unequivocally identify the proteins labeled in these samples. However, based on the results using
Because of their importance in development, normal physiology and disease processes, MMPs have been the focus of intense scrutiny for many years. However, because of the complexity, and largely post-translational nature of their regulation, the assays of gene expression that have proven so valuable in developing our understanding of the regulation of so many other biological processes are not as easily applied to the understanding of MMP regulation. Recent advances in the development of fluorogenic MMP substrates have been developed into methods that allow the detection of MMP activity
Tagging molecules with HxBP-Rh
The patterns of
It is interesting to note that both cell-specific and interstitial labeling can be observed in these preparations, given that both membrane-bound and secreted forms of MMPs are know to be expressed in zebrafish at this time
Although the axial resolution of our optical sections is not sufficient to eliminate the possibility that the HxBP labeling we observe associated with myofibrils is genuinely intracellular, there is an increasing body of evidence indicating that MMPs, and MMP-2 in particular, are both present and playing important intracellular roles
Combining immunostaining with
The authors would like to thank Robyn Shortt and Robyn O’Keefe for their excellent animal care, and two anonymous reviewers for insightful and constructive comments on the manuscript.