Abstract
Matrix metalloproteases (MMPs) have attracted considerable attention as critical mediators of pathological tissue remodeling processes. However it remains an unresolved challenge to detect their active forms in biological samples. To prove the efficacy of a recently developed MMP activity-based probe, we examined the content in MMP active forms of bronchoalveolar lavage fluids (BALf) from male C57BL/6 mice exposed to ultrafine carbon black nanoparticles, a model of chronic obstructive pulmonary disease. This probe was shown to label proteins, mostly expressed in BALf of mice exposed to nanoparticles. Using competition assays with a selective MMP-12 inhibitor as well as MMP-12 knock-out mice, one of these proteins was identified as the active form of the catalytic domain of MMP-12. This new probe can detect the active form of MMP-12 down to a threshold of 1 fmol. Radioactive counting showed the concentration of the active form of MMP-12 to be around 1 fmol/μl in BALf from nanoparticle-treated mice. A less sensitive probe would therefore not have detected MMP-12. As the probe can detect other MMPs in the femtomolar range, it is a potentially powerful tool for monitoring the levels of MMP active forms in various diseases.
Highlights
The detection of Matrix metalloproteases (MMPs) active forms remains a challenge
To prove the efficacy of a recently developed MMP activity-based probe, we examined the content in MMP active forms of bronchoalveolar lavage fluids (BALf) from male C57BL/6 mice exposed to ultrafine carbon black nanoparticles, a model of chronic obstructive pulmonary disease
The reactive intermediate generated by azide group in response to activation by light is known to react preferentially with nucleophilic groups, like that present on the side chain of Lys241 in MMP-12 and His241 in MMP-3. In addition to these probe-related difficulties with the labeling of different human MMPs, we reported the poor modification of murine MMP-12, because of the weak nucleophilicity at neutral pH of the arginine residue in position 241 of this MMP [26]
Summary
The detection of MMP active forms remains a challenge. Results: An endogenous active form of MMP-12 was detected in animal fluids with a new activity-based probe. This precludes the use of this probe for the detection of active forms of mMMP-12 in samples from animal models We overcame this problem, to facilitate testing for MMP in fluids or tissue extracts from animal models, by replacing the azide group in the P1Ј position of probe 1 by a trifluoromethyldiazirine (probe 2, Scheme 1), as the reactive intermediate generated by this photolabile group in response to light activation is known to react with various types of residues in close proximity [27]. Probe 2 includes radioactive tritium atoms to ensure the sensitive detection of protein covalent adducts by radioimaging and a short polyethylene linker to increase the solubility of the probe in water This novel probe displays nanomolar affinities toward a large set of human MMPs and was shown to crosslink only active forms of MMPs with very high crosslinking yields, allowing their detection in the femtomolar range in vitro [28]. In many previous studies, the detection of active forms of MMP in biological samples has been reported only after a concentration step to increase the MMP quantity, which is a major limiting factor for the detection of active forms of MMP in most samples [19, 22, 33,34,35]
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