Expression, mutagenèse dirigée et mécanisme d'action de la collagénase interstitielle (MMP-13) de souris
(1998)
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- Authors
- Supervisors
- Eeckhout, Yves
- Abstract
- Collagenases are the only vertebrate proteinases able to cleave the triple helical core of the major fibrillar collagens. In the present study, techniques derived from molecular genetics were applied to investigate properties, catalytic mechanisms and physiopathological roles of mouse interstitial collagenase (collagenase-3 or MMP-13), whose human homologue has been implicated in osteogenesis, tumor invasion and in the breakdown of articular cartilage. <BR> Mouse collagenase-3 has first been expressed in E. coli. About 40% of the renatured enzyme has been recovered as latent proenzyme whose spontaneous activation and subsequent degradation have limited its further use. The use of the E. coli-expressed protein has, however, contributed to the finding, in collaboration with Dr. S. Krane, of a novel property of collagenase-3, that is not shared by collagenases-1 and -2: the ability to cleave type I collagen at a second site located in the non-helical N-terminal telopeptides, downstream of the intra- and inter-molecular crosslinks. This aminotelopeptidase activity depolymerises crosslinked collagen chains and appears sufficient for normal remodelling of type I collagen until mouse adulthood, but not later (Krane et al., 1996). <BR> In order to improve the latency and stability of recombinant collagenase-3, the insect cell/Baculovirus system has been used for its expression. In this system, collagenase-3 is secreted as a glycosylated, latent and stable zymogen. Using this recombinant proenzyme, we have contributed to a collaborative study with Cr. P. Carmeliet on aortic aneurysm formation in transgenic mice with a deficiency of apolipoprotein E, singly or combined with tPA or uPA deficiency. We have shown that physiological concentrations of mouse of human plasmin activate procollagenase-3 in vitro and that wild-type or tPA -/- macrophages, but not uPA-/- ones, activate procollagenase-3 in the presence of plasminogen. These results point to an in vivo role of uPA and plasmin in the activation of protocollagenase-3 and of other related proMMPs (Carmeliet et al., 1997). <BR> The catalytic domain of mouse collagenase-3 has been expressed in E. coli, in order to study its enzymatic properties and, in collaboration with Dr. E. Meyer, its crystal structure. This approach enabled us to observe that the catalytic domain of collagenase-3 is devoid of collagen-helicase activity but retains its aminotelopeptidase activity. Site-directed mutagenesis of the catalytic domain did not, however, reveal residues critical for the aminotelopeptidase activity. <BR> Full length chimeric mouse collagenase-3/stromelysin-1 enzymes were expressed in the Baculovirus system to identify sequences of the hemopexin domain that are critical for the cleavage of the triple helix of type I collagen. Our observations indicate that the surface region of the first hemopexin blade contains determinants of the collagen-helicase activity. They also suggest the involvement of other C-terminal determinants and confirm that the helicase activity results more from the entire three-dimensional structure than from a few sequences conserved amongst all collagenases
- Affiliations
Citations
Lemaitre, V. (1998). Expression, mutagenèse dirigée et mécanisme d’action de la collagénase interstitielle (MMP-13) de souris. https://hdl.handle.net/2078.5/111493