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Molecular mechanism of type I collagen homotrimer resistance to mammalian collagenases.

Han S, Makareeva E, Kuznetsova NV, DeRidder AM, Sutter MB, Losert W, Phillips CL, Visse R, Nagase H, Leikin S - J. Biol. Chem. (2010)

Bottom Line: Similar MMP-1 binding to the two isoforms and similar cleavage efficiency of unwound alpha1(I) and alpha2(I) chains suggested increased stability and less efficient unwinding of the homotrimer triple helix at the collagenase cleavage site.The unwinding, necessary for placing individual chains inside the catalytic cleft of the enzyme, was the rate-limiting cleavage step for both collagen isoforms.Comparative analysis of the homo- and heterotrimer cleavage kinetics revealed that MMP-1 binding promotes stochastic helix unwinding, resolving the controversy between different models of collagenase action.

View Article: PubMed Central - PubMed

Affiliation: Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Type I collagen cleavage is crucial for tissue remodeling, but its homotrimeric isoform is resistant to all collagenases. The homotrimers occur in fetal tissues, fibrosis, and cancer, where their collagenase resistance may play an important physiological role. To understand the mechanism of this resistance, we studied interactions of alpha1(I)(3) homotrimers and normal alpha1(I)(2)alpha2(I) heterotrimers with fibroblast collagenase (MMP-1). Similar MMP-1 binding to the two isoforms and similar cleavage efficiency of unwound alpha1(I) and alpha2(I) chains suggested increased stability and less efficient unwinding of the homotrimer triple helix at the collagenase cleavage site. The unwinding, necessary for placing individual chains inside the catalytic cleft of the enzyme, was the rate-limiting cleavage step for both collagen isoforms. Comparative analysis of the homo- and heterotrimer cleavage kinetics revealed that MMP-1 binding promotes stochastic helix unwinding, resolving the controversy between different models of collagenase action.

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Related in: MedlinePlus

Equilibrium binding of MMP-1(E200A) to homo- and heterotrimeric mouse-tail-tendon type I collagen at room temperature. A, number (N) of bound MMP-1(E200A) at 10–65 μm concentration of free molecules was well fitted by a straight line with the intercept at N = 1. B, after subtracting this fitted low affinity binding, the number of MMP-1(E200A) bound below 5 μm was consistent with high affinity binding (Kd = 1.3 ± 0.3 μm) at a single site. Hom, homotrimer (dashed lines in A and B); Het, heterotrimer (solid lines in A and B).
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Figure 3: Equilibrium binding of MMP-1(E200A) to homo- and heterotrimeric mouse-tail-tendon type I collagen at room temperature. A, number (N) of bound MMP-1(E200A) at 10–65 μm concentration of free molecules was well fitted by a straight line with the intercept at N = 1. B, after subtracting this fitted low affinity binding, the number of MMP-1(E200A) bound below 5 μm was consistent with high affinity binding (Kd = 1.3 ± 0.3 μm) at a single site. Hom, homotrimer (dashed lines in A and B); Het, heterotrimer (solid lines in A and B).

Mentions: Equilibrium microdialysis experiments revealed low affinity binding (Kd > 50 μm) of at least five MMP-1(E200A) per collagen triple helix (Fig. 3A) and high affinity binding (Kd = 1.3 ± 0.3 μm) of a single MMP-1(E200A) at the cleavage site (Fig. 3B). The binding to the homo- and heterotrimers was identical. The high affinity Kd value was consistent with that reported in Ref. 24 for guinea pig heterotrimers and with the Km value in our kinetic experiments. Thus, the homotrimer resistance to MMP-1 could not be attributed to weaker enzyme binding and had to be related to triple helix unwinding and/or cleavage rate of individual chains.


Molecular mechanism of type I collagen homotrimer resistance to mammalian collagenases.

Han S, Makareeva E, Kuznetsova NV, DeRidder AM, Sutter MB, Losert W, Phillips CL, Visse R, Nagase H, Leikin S - J. Biol. Chem. (2010)

Equilibrium binding of MMP-1(E200A) to homo- and heterotrimeric mouse-tail-tendon type I collagen at room temperature. A, number (N) of bound MMP-1(E200A) at 10–65 μm concentration of free molecules was well fitted by a straight line with the intercept at N = 1. B, after subtracting this fitted low affinity binding, the number of MMP-1(E200A) bound below 5 μm was consistent with high affinity binding (Kd = 1.3 ± 0.3 μm) at a single site. Hom, homotrimer (dashed lines in A and B); Het, heterotrimer (solid lines in A and B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903388&req=5

Figure 3: Equilibrium binding of MMP-1(E200A) to homo- and heterotrimeric mouse-tail-tendon type I collagen at room temperature. A, number (N) of bound MMP-1(E200A) at 10–65 μm concentration of free molecules was well fitted by a straight line with the intercept at N = 1. B, after subtracting this fitted low affinity binding, the number of MMP-1(E200A) bound below 5 μm was consistent with high affinity binding (Kd = 1.3 ± 0.3 μm) at a single site. Hom, homotrimer (dashed lines in A and B); Het, heterotrimer (solid lines in A and B).
Mentions: Equilibrium microdialysis experiments revealed low affinity binding (Kd > 50 μm) of at least five MMP-1(E200A) per collagen triple helix (Fig. 3A) and high affinity binding (Kd = 1.3 ± 0.3 μm) of a single MMP-1(E200A) at the cleavage site (Fig. 3B). The binding to the homo- and heterotrimers was identical. The high affinity Kd value was consistent with that reported in Ref. 24 for guinea pig heterotrimers and with the Km value in our kinetic experiments. Thus, the homotrimer resistance to MMP-1 could not be attributed to weaker enzyme binding and had to be related to triple helix unwinding and/or cleavage rate of individual chains.

Bottom Line: Similar MMP-1 binding to the two isoforms and similar cleavage efficiency of unwound alpha1(I) and alpha2(I) chains suggested increased stability and less efficient unwinding of the homotrimer triple helix at the collagenase cleavage site.The unwinding, necessary for placing individual chains inside the catalytic cleft of the enzyme, was the rate-limiting cleavage step for both collagen isoforms.Comparative analysis of the homo- and heterotrimer cleavage kinetics revealed that MMP-1 binding promotes stochastic helix unwinding, resolving the controversy between different models of collagenase action.

View Article: PubMed Central - PubMed

Affiliation: Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Type I collagen cleavage is crucial for tissue remodeling, but its homotrimeric isoform is resistant to all collagenases. The homotrimers occur in fetal tissues, fibrosis, and cancer, where their collagenase resistance may play an important physiological role. To understand the mechanism of this resistance, we studied interactions of alpha1(I)(3) homotrimers and normal alpha1(I)(2)alpha2(I) heterotrimers with fibroblast collagenase (MMP-1). Similar MMP-1 binding to the two isoforms and similar cleavage efficiency of unwound alpha1(I) and alpha2(I) chains suggested increased stability and less efficient unwinding of the homotrimer triple helix at the collagenase cleavage site. The unwinding, necessary for placing individual chains inside the catalytic cleft of the enzyme, was the rate-limiting cleavage step for both collagen isoforms. Comparative analysis of the homo- and heterotrimer cleavage kinetics revealed that MMP-1 binding promotes stochastic helix unwinding, resolving the controversy between different models of collagenase action.

Show MeSH
Related in: MedlinePlus