Abstract
The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. To study the mechanisms of MMP action on collagenous substrates, we have constructed homotrimeric triple-helical peptide (THP) models of the collagenase cleavage sites in types I and II collagen. The THPs incorporate either the alpha1(I)772-786 or the alpha1(II)772-783 sequence. The alpha1(I)772-786 and alpha1(II)772-783 THPs were hydrolyzed by MMP-1 at the Gly-Ile and Gly-Leu bonds, respectively, analogous to the bonds cleaved in corresponding native collagens. Thus, the THPs contained all necessary information to direct MMP-1 binding and proteolysis. Subsequent investigations using the alpha1(I)772-786 THP showed hydrolysis by MMP-2, MMP-13, and a COOH-terminal domain-deleted MMP-1 (MMP-1(Delta(243-450))) but not by MMP-3 or a COOH-terminal domain-deleted MMP-3 (MMP-3(Delta(248-460))). Kinetic analyses showed a k(cat)/K(m) value of 1,808 s(-1) m(-1) for MMP-1 hydrolysis of alpha1(I)772-786 THP, approximately 10-fold lower than for type I collagen. The effect is caused primarily by relative K(m) values. MMP-2 and MMP-13 cleaved the THP more rapidly than MMP-1, but MMP-2 cleavage occurred at distinct multiple sites. Comparison of MMP-1 and MMP-1(Delta(243-450)) hydrolysis of alpha1(I)772-786 THP showed that both can cleave a triple-helical substrate with a slightly higher K(m) value for MMP-1(Delta(243-450)). We propose that the COOH-terminal domain of MMPs is necessary for orienting whole, native collagen molecules but may not be necessary for binding to and cleaving a THP. This proposal is consistent with the large distance between the MMP-1 catalytic and COOH-terminal domains observed by three-dimensional structural analysis and supports previous suggestions that the features of the catalytic domain contribute significantly toward enzyme specificity.
Highlights
The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis
The interstitial collagen sequences targeted by MMP-1, MMP-2, MMP-8, and MMP-13 have been identified, and a model collagenase cleavage site has been proposed based on the combination of triple-helical collagen primary, secondary, and super-secondary structures [5]
In the present study we examined the suitability of synthetic triplehelical peptide (THP) as peptide models of native collagens
Summary
Materials—All standard peptide synthesis chemicals were analytical reagent grade or better and purchased from Novabiochem (San Diego) or Fisher. Pro-MMP-2 was activated by reacting with 1 mM 4-aminophenylmercuric acetate at 37 °C for 45 min. Pro-MMP-1 was activated by reacting with 1 mM 4-aminophenylmercuric acetate and an equimolar amount of MMP-3 at 37 °C for 6 h. Pro-MMP-1(⌬243–450) and pro-MMP-3(⌬248–460) were expressed in Escherichia coli using the expression vector pET3a (Novagen), refolded from inclusion bodies, and purified as described previously [54] Both zymogens were activated as described above for the full-length pro-MMP-1 and pro-MMP-3. For the first assay method, THPs were prepared as 260 M stock solutions in phosphate-buffered saline (PBS). Initial MMP assays were carried out by incubating 40 M substrate in either PBS or TNC buffer (50 mM Tris1⁄7HCl, pH 7.5, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij-35, 0.02% NaN3) with 67 nM enzyme for 24 h at 37 °C. The slope from these plots was divided by the fluorescence change corresponding to complete hydrolysis and multiplied by the substrate concentration to obtain initial velocity in units of M/s
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