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Role of the netrin-like domain of procollagen C-proteinase enhancer-1 in the control of metalloproteinase activity.

Bekhouche M, Kronenberg D, Vadon-Le Goff S, Bijakowski C, Lim NH, Font B, Kessler E, Colige A, Nagase H, Murphy G, Hulmes DJ, Moali C - J. Biol. Chem. (2010)

Bottom Line: The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity.Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family.In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate.

View Article: PubMed Central - PubMed

Affiliation: From the Institut de Biologie et Chimie des Protéines, CNRS/Université de Lyon UMR 5086, IFR128, 69367 Lyon, France.

ABSTRACT
The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments.

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Binding of PCPE-1 to BMP-1 studied by surface plasmon resonance (Biacore). A, concentration dependence of binding of full-length PCPE-1 (12.5, 25, 50, 100, 200, 400, 800, 1600, 3200, and 6400 nm) to immobilized BMP-1 (500 RU). Inset, fit of the response at infinite time (RU eq) as a function of PCPE-1 concentration using the equation given under “Experimental Procedures.” B, interactions of PCPE-1 fragments CUB1CUB2, NTRt, and CUB2-NTR (all at 500 nm) with immobilized BMP-1 (1463 RU).
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Figure 4: Binding of PCPE-1 to BMP-1 studied by surface plasmon resonance (Biacore). A, concentration dependence of binding of full-length PCPE-1 (12.5, 25, 50, 100, 200, 400, 800, 1600, 3200, and 6400 nm) to immobilized BMP-1 (500 RU). Inset, fit of the response at infinite time (RU eq) as a function of PCPE-1 concentration using the equation given under “Experimental Procedures.” B, interactions of PCPE-1 fragments CUB1CUB2, NTRt, and CUB2-NTR (all at 500 nm) with immobilized BMP-1 (1463 RU).

Mentions: For the analysis of kinetic experiments, double referencing (subtraction of the control sensorgram and then subtraction of a sensorgram obtained with buffer alone) were used. Because it was not possible to fit the association and dissociation curves shown in Fig. 4A using the usual binding models, responses during the association phase were plotted against 1/t, and a linear fit was used to calculate steady state values at infinite time (36). These values were then plotted against PCPE-1 concentrations, and the curve was fitted using the equation RUeq = (Rmax1 × [PCPE-1]/(KD1 + [PCPE-1])) + (Rmax2 × [PCPE-1]/(KD2 + [PCPE-1])) with Kaleidagraph software.


Role of the netrin-like domain of procollagen C-proteinase enhancer-1 in the control of metalloproteinase activity.

Bekhouche M, Kronenberg D, Vadon-Le Goff S, Bijakowski C, Lim NH, Font B, Kessler E, Colige A, Nagase H, Murphy G, Hulmes DJ, Moali C - J. Biol. Chem. (2010)

Binding of PCPE-1 to BMP-1 studied by surface plasmon resonance (Biacore). A, concentration dependence of binding of full-length PCPE-1 (12.5, 25, 50, 100, 200, 400, 800, 1600, 3200, and 6400 nm) to immobilized BMP-1 (500 RU). Inset, fit of the response at infinite time (RU eq) as a function of PCPE-1 concentration using the equation given under “Experimental Procedures.” B, interactions of PCPE-1 fragments CUB1CUB2, NTRt, and CUB2-NTR (all at 500 nm) with immobilized BMP-1 (1463 RU).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2871463&req=5

Figure 4: Binding of PCPE-1 to BMP-1 studied by surface plasmon resonance (Biacore). A, concentration dependence of binding of full-length PCPE-1 (12.5, 25, 50, 100, 200, 400, 800, 1600, 3200, and 6400 nm) to immobilized BMP-1 (500 RU). Inset, fit of the response at infinite time (RU eq) as a function of PCPE-1 concentration using the equation given under “Experimental Procedures.” B, interactions of PCPE-1 fragments CUB1CUB2, NTRt, and CUB2-NTR (all at 500 nm) with immobilized BMP-1 (1463 RU).
Mentions: For the analysis of kinetic experiments, double referencing (subtraction of the control sensorgram and then subtraction of a sensorgram obtained with buffer alone) were used. Because it was not possible to fit the association and dissociation curves shown in Fig. 4A using the usual binding models, responses during the association phase were plotted against 1/t, and a linear fit was used to calculate steady state values at infinite time (36). These values were then plotted against PCPE-1 concentrations, and the curve was fitted using the equation RUeq = (Rmax1 × [PCPE-1]/(KD1 + [PCPE-1])) + (Rmax2 × [PCPE-1]/(KD2 + [PCPE-1])) with Kaleidagraph software.

Bottom Line: The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity.Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family.In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate.

View Article: PubMed Central - PubMed

Affiliation: From the Institut de Biologie et Chimie des Protéines, CNRS/Université de Lyon UMR 5086, IFR128, 69367 Lyon, France.

ABSTRACT
The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments.

Show MeSH
Related in: MedlinePlus