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Abstract
The cleavage of bovine collagen I by neutrophil collagenase MMP-8 has been followed at pH 7.4, 37 degrees C. The behavior of the whole enzyme molecule (whMMP-8), displaying both the catalytic domain and the hemopexin-like domain, has been compared under the same experimental conditions with that of the catalytic domain only. The main observation is that whMMP-8 cleaves bovine collagen I only at a single specific site, as already reported by many others (Mallya, S. K., Mookhtiar, K. A., Gao, Y., Brew, K., Dioszegi, M., Birkedal-Hansen, H., and van Wart, H. E. (1990) Biochemistry 29, 10628-10634; Knäuper, V., Osthues, A., DeClerk, Y. A., Langley, K. A., Bläser, J., and Tschesche, H. (1993) Biochem. J. 291, 847-854; Marini, S., Fasciglione, G. F., De Sanctis, G., D'Alessio, S., Politi, V., and Coletta, M. (2000) J. Biol. Chem. 275, 18657-18663), whereas the catalytic domain lacks this specificity and cleaves the collagen molecule at multiple sites. Furthermore, a meaningful difference is observed for the cleavage features displayed by two forms of the catalytic domain, which differ for the N terminus resulting from the activation process (i.e. the former Met(80) of the proenzyme (MetMMP-8) and the former Phe(79) of the proenzyme (PheMMP-8)). Thus, the PheMMP-8 species is characterized by a much faster k(cat)/K(m), fully attributable to a lower K(m), suggesting that the conformation of the catalytic domain, induced by the insertion of this N-terminal residue in a specific pocket (Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Piper, M., Tschesche, H., and Bode, W. (1994) FEBS Lett. 338, 227-233), brings about a better, although less discriminatory, recognition process of cleavage site(s) on bovine collagen I.
Highlights
Collagen I is one of the major components of the extracellular matrix for most tissues, such as skin, tendon, blood vessels, cartilage, bones, and basal laminae [20]
Several studies have shown that, the actual enzymatic action of collagenases occurs in the catalytic domain, the hemopexin-like domain plays an important role, because its neutrophil collagenase; whole neutrophil collagenase MMP-8 (whMMP-8), whole MMP-8 molecule; MetMMP-8, catalytic domain of MMP-8, where the N terminus is Met80 of the former proenzyme; PheMMP-8, catalytic domain of MMP-8, where the N terminus is Phe79 of the former proenzyme
It appears immediately obvious that whereas whMMP-8 cleaves the bovine collagen only at one site, showing only two fragments after 3 and 6 h, as already reported by us [22], both MetMMP-8 and PheMMP-8 show multiple cleavage sites, with several bands of differing molecular weight. This observation clearly indicates that the removal of the hemopexin-like domain in MMP-8 brings about a loss of specificity in recognizing the cleavage site on bovine collagen between Gly775 and the residue in position 776, which is instead peculiar to the enzymatic action of whMMP-8 [4]
Summary
Isolation of whMMP-8 and expression, purification, and refolding of recombinant MetMMP-8 and PheMMP-8 were carried out as previously reported [6]. Isolated purified whMMP-8 proenzyme was activated by incubating 0.1 ml of a 0.1 g/ml procollagenase solution with p-aminophenyl mercuric acetate (Sigma) at 37 °C for 2 h. The purity of whMMP-8, MetMMP-8 and PheMMP-8 was checked by SDS-polyacrylamide gel electrophoresis according to Laemmli [30]. Zymography was performed as follows: 2 l of purified MMP-8 was mixed with a 5-fold excess of sample buffer (0.25 M Tris, 0.8% SDS, 10% glycerol, and 0.05% bromphenol blue) and run on 12% SDS-polyacrylamide gels containing either 1 mg/ml gelatin or bovine collagen I as described previously [30]. Lane 1, intact bovine collagen I; lane M, molecular weight markers; lanes 2–7, SDS-polyacrylamide gel of samples after 1.5 (lanes 2– 4) and 6 h (lanes 5–7) of digestion at 37 °C. Electrophoretic spots, corresponding to different fragmentation intermediates of bovine collagen I, were analyzed as a function of time by laser densitometry (LKB 2202 UltraScan), and their intensity was calibrated (in order to obtain concentration values) using standard collagen solutions
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