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Hydrolysis of triple-helical collagen peptide models by matrix metalloproteinases
J L Lauer-Fields1, K A Tuzinski, K i Shimokawa
1Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, Florida 33431-0991, USA. fieldsg@fau.edu
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.
Insights
Matrix metalloproteinases (MMPs) are crucial in diseases. Researchers used triple-helical peptide (THP) models to study MMP action on collagen, finding THPs effectively mimic collagen cleavage sites for MMP-1, MMP-2, and MMP-13.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Matrix metalloproteinases (MMPs) are enzymes implicated in various diseases, including arthritis, atherosclerosis, and cancer metastasis.
- Understanding MMP mechanisms, particularly their action on collagenous substrates, is vital for disease research.
Purpose of the Study:
- To construct and utilize homotrimeric triple-helical peptide (THP) models representing collagenase cleavage sites in type I and II collagen.
- To investigate the binding and proteolysis of these THP models by different MMPs.
Main Methods:
- Synthesized THPs incorporating specific sequences from alpha1(I) and alpha1(II) collagen.
- Assessed hydrolysis of THPs by various MMPs, including MMP-1, MMP-2, MMP-3, and their truncated variants.
- Performed kinetic analyses to determine catalytic efficiency (kcat/Km) and substrate binding (Km).
Main Results:
- THPs were effectively hydrolyzed by MMP-1 at specific Gly-X bonds, mimicking native collagen cleavage.
- MMP-1, MMP-2, and MMP-13 hydrolyzed the alpha1(I) THP, while MMP-3 did not.
- Kinetic analysis revealed MMP-1 hydrolysis of the alpha1(I) THP had a lower catalytic efficiency compared to native type I collagen, primarily due to Km.
- MMP-2 and MMP-13 cleaved the THP more rapidly than MMP-1, with MMP-2 exhibiting multiple cleavage sites.
Conclusions:
- Triple-helical peptide models contain sufficient information for MMP binding and proteolysis.
- The COOH-terminal domain of MMPs may be essential for orienting native collagen but not necessarily for cleaving triple-helical peptide substrates.
- Catalytic domain features significantly contribute to MMP enzyme specificity.