Abstract
The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover. Within the matrix metalloproteinase (MMP) family distinct preferences for collagen types are seen. The substrate determinants that may guide these specificities are unknown. In this study, we have utilized 12 triple-helical substrates in combination with 10 MMPs to better define the contributions of substrate sequence and thermal stability toward triple helicase activity and collagen specificity. In general, MMP-13 was found to be distinct from MMP-8 and MT1-MMP(Delta279-523), in that enhanced substrate thermal stability has only a modest effect on activity, regardless of sequence. This result correlates to the unique collagen specificity of MMP-13 compared with MMP-8 and MT1-MMP, in that MMP-13 hydrolyzes type II collagen efficiently, whereas MMP-8 and MT1-MMP are similar in their preference for type I collagen. In turn, MMP-1 was the least efficient of the collagenolytic MMPs at processing increasingly thermal stable triple helices and thus favors type III collagen, which has a relatively flexible cleavage site. Gelatinases (MMP-2 and MMP-9(Delta444-707)) appear incapable of processing more stable helices and are thus mechanistically distinct from collagenolytic MMPs. The collagen specificity of MMPs appears to be based on a combination of substrate sequence and thermal stability. Analysis of the hydrolysis of triple-helical peptides by an MMP mutant indicated that Tyr(210) functions in triple helix binding and hydrolysis, but not in processing triple helices of increasing thermal stabilities. Further exploration of MMP active sites and exosites, in combination with substrate conformation, may prove valuable for additional dissection of collagenolysis and yield information useful in the design of more selective MMP inhibitors.
Highlights
Current studies identify at least 25 different collagen types, each with a specific role in the extracellular matrix [1, 2]
Collagenolysis-related exosites have been identified within the matrix metalloproteinase (MMP)-1 catalytic domain [19] and the active site cleft [20] and hypothesized in analogous regions in MMP-2, MMP-8, MMP-9, and MMP-13 [21]
Unique interactions between the catalytic and hemopexin-like domains are found in pro-MMP-1 compared with MMP-1 [25], suggesting that binding of substrate may result in considerable conformational changes within MMPs
Summary
Current studies identify at least 25 different collagen types, each with a specific role in the extracellular matrix [1, 2]. Whatever the collagen specificity determinants are, they must reflect differences in MMP collagen-binding and active sites. Unique interactions between the catalytic and hemopexin-like domains are found in pro-MMP-1 compared with MMP-1 [25], suggesting that binding of substrate may result in considerable conformational changes within MMPs. dissecting out collagen specificity elements requires identification of specific MMP-binding sites and their roles in the steps of collagenolysis. Substitution of Leu by Cys(Mob) in the P1Ј subsite of a consensus MMP substrate led to increased specificity toward MT1-MMP These observations lead us to believe that by modifying substrate sequence and thermal stability, we can better rationalize the collagen specificities of MMPs. In this study we explore the effects of substitutions in the P2 and P1Ј substrate subsites on MMP triple-helical specificity. MMP-8 mutant triple-helical peptidase activities have been quantified to evaluate the role of Tyr210 on substrate selectivity, as this residue in MMP-1 and MMP-8 had been shown previously to be an important participant in collagenolysis [19, 28]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
