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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

Confocal projections of fibers reconstituted from hetero- (green) and homotrimeric (red) mouse tail tendon collagen before and after digestion with recombinant human MMP-1. A and B, fibers reconstituted from a 1:1 mixture of AF546-labeled homotrimers and AF647-labeled heterotrimers (0.2 mg/ml). Different sample areas were imaged before (A) and after (B) replacing the buffer with 150 nm MMP-1 in TNC and incubating for 1.5 h at 37 °C. C and D, fibers reconstituted by adding an ice-cold heterotrimer solution (0.2 mg/ml) to preformed homotrimer fibrils (0.2 mg/ml) and incubating at 32 °C for several days. Different sample areas were imaged before (C) and after (D) replacing the buffer with 150 nm MMP-1 in TNC and incubating for 3.5 h at 37 °C. E and F, fibers reconstituted by adding an ice-cold homotrimer solution (0.2 mg/ml) to preformed heterotrimer fibrils (0.2 mg/ml) and incubating at 32 °C for several days. The same area of the sample was imaged after incubating with 150 nm MMP-1 at 37 °C for 18 min (E) and 2.5 h (F).
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Figure 5: Confocal projections of fibers reconstituted from hetero- (green) and homotrimeric (red) mouse tail tendon collagen before and after digestion with recombinant human MMP-1. A and B, fibers reconstituted from a 1:1 mixture of AF546-labeled homotrimers and AF647-labeled heterotrimers (0.2 mg/ml). Different sample areas were imaged before (A) and after (B) replacing the buffer with 150 nm MMP-1 in TNC and incubating for 1.5 h at 37 °C. C and D, fibers reconstituted by adding an ice-cold heterotrimer solution (0.2 mg/ml) to preformed homotrimer fibrils (0.2 mg/ml) and incubating at 32 °C for several days. Different sample areas were imaged before (C) and after (D) replacing the buffer with 150 nm MMP-1 in TNC and incubating for 3.5 h at 37 °C. E and F, fibers reconstituted by adding an ice-cold homotrimer solution (0.2 mg/ml) to preformed heterotrimer fibrils (0.2 mg/ml) and incubating at 32 °C for several days. The same area of the sample was imaged after incubating with 150 nm MMP-1 at 37 °C for 18 min (E) and 2.5 h (F).

Mentions: To test the cleavage of such mixed fibers, we reconstituted them by in vitro fibrillogenesis from fluorescently labeled mouse homo- and heterotrimers and incubated them with MMP-1 at 37 °C. We observed complete disintegration of most fibers after several hours (Fig. 5, A and B), but we also observed small residual fibers composed almost entirely of the homotrimers (shown by arrows in Fig. 5B). The residual homotrimer fibers likely originated from segregated regions (31) composed of the homotrimers with few or no heterotrimers.


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)

Confocal projections of fibers reconstituted from hetero- (green) and homotrimeric (red) mouse tail tendon collagen before and after digestion with recombinant human MMP-1. A and B, fibers reconstituted from a 1:1 mixture of AF546-labeled homotrimers and AF647-labeled heterotrimers (0.2 mg/ml). Different sample areas were imaged before (A) and after (B) replacing the buffer with 150 nm MMP-1 in TNC and incubating for 1.5 h at 37 °C. C and D, fibers reconstituted by adding an ice-cold heterotrimer solution (0.2 mg/ml) to preformed homotrimer fibrils (0.2 mg/ml) and incubating at 32 °C for several days. Different sample areas were imaged before (C) and after (D) replacing the buffer with 150 nm MMP-1 in TNC and incubating for 3.5 h at 37 °C. E and F, fibers reconstituted by adding an ice-cold homotrimer solution (0.2 mg/ml) to preformed heterotrimer fibrils (0.2 mg/ml) and incubating at 32 °C for several days. The same area of the sample was imaged after incubating with 150 nm MMP-1 at 37 °C for 18 min (E) and 2.5 h (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Confocal projections of fibers reconstituted from hetero- (green) and homotrimeric (red) mouse tail tendon collagen before and after digestion with recombinant human MMP-1. A and B, fibers reconstituted from a 1:1 mixture of AF546-labeled homotrimers and AF647-labeled heterotrimers (0.2 mg/ml). Different sample areas were imaged before (A) and after (B) replacing the buffer with 150 nm MMP-1 in TNC and incubating for 1.5 h at 37 °C. C and D, fibers reconstituted by adding an ice-cold heterotrimer solution (0.2 mg/ml) to preformed homotrimer fibrils (0.2 mg/ml) and incubating at 32 °C for several days. Different sample areas were imaged before (C) and after (D) replacing the buffer with 150 nm MMP-1 in TNC and incubating for 3.5 h at 37 °C. E and F, fibers reconstituted by adding an ice-cold homotrimer solution (0.2 mg/ml) to preformed heterotrimer fibrils (0.2 mg/ml) and incubating at 32 °C for several days. The same area of the sample was imaged after incubating with 150 nm MMP-1 at 37 °C for 18 min (E) and 2.5 h (F).
Mentions: To test the cleavage of such mixed fibers, we reconstituted them by in vitro fibrillogenesis from fluorescently labeled mouse homo- and heterotrimers and incubated them with MMP-1 at 37 °C. We observed complete disintegration of most fibers after several hours (Fig. 5, A and B), but we also observed small residual fibers composed almost entirely of the homotrimers (shown by arrows in Fig. 5B). The residual homotrimer fibers likely originated from segregated regions (31) composed of the homotrimers with few or no heterotrimers.

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