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Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex.

Troeberg L, Mulloy B, Ghosh P, Lee MH, Murphy G, Nagase H - Biochem. J. (2012)

Bottom Line: A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase.PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites.The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl.

View Article: PubMed Central - PubMed

Affiliation: The Kennedy Institute of Rheumatology, University of Oxford, 65 Aspenlea Road, Hammersmith, London W6 8LH, UK. linda.troeberg@kennedy.ox.ac.uk

ABSTRACT
The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

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The PPS effect is electrostatic(A) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) in the absence (○) or presence (●) of 10 nM PPS and 50–500 mM NaCl (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (B) TIMP-3 (0.5 nM) was incubated with ADAMTS5-5 (0.5 nM) in the absence (○) or presence (●) of 100 nM PPS and 50–500 mM NaCl (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (C) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) and PPS (10 nM) in TNC buffer (containing 250 mM NaCl) at 37°C for times ranging between 1 and 72 h. The concentration of NaCl was then increased to 400 mM and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C).
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Figure 6: The PPS effect is electrostatic(A) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) in the absence (○) or presence (●) of 10 nM PPS and 50–500 mM NaCl (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (B) TIMP-3 (0.5 nM) was incubated with ADAMTS5-5 (0.5 nM) in the absence (○) or presence (●) of 100 nM PPS and 50–500 mM NaCl (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (C) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) and PPS (10 nM) in TNC buffer (containing 250 mM NaCl) at 37°C for times ranging between 1 and 72 h. The concentration of NaCl was then increased to 400 mM and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C).

Mentions: First, the effect of NaCl on the interaction between TIMP-3 and ADAMTS-5 was analysed. Inhibition of ADAMTS5-2 (0.5 nM) by TIMP-3 (0.5 nM) was slightly reduced at NaCl concentrations above 250 mM (Figure 6A, broken line), indicating that there is an electrostatic element to the interaction of these two proteins. In contrast, the inhibition of ADAMTS5-5 by TIMP-3 was not affected by NaCl concentrations of up to 500 mM (Figure 6B, broken line), indicating that electrostatic interactions do not play a significant role in the interaction of the TIMP-3 with the catalytic and Dis domain of ADAMTS-5.


Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex.

Troeberg L, Mulloy B, Ghosh P, Lee MH, Murphy G, Nagase H - Biochem. J. (2012)

The PPS effect is electrostatic(A) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) in the absence (○) or presence (●) of 10 nM PPS and 50–500 mM NaCl (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (B) TIMP-3 (0.5 nM) was incubated with ADAMTS5-5 (0.5 nM) in the absence (○) or presence (●) of 100 nM PPS and 50–500 mM NaCl (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (C) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) and PPS (10 nM) in TNC buffer (containing 250 mM NaCl) at 37°C for times ranging between 1 and 72 h. The concentration of NaCl was then increased to 400 mM and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: The PPS effect is electrostatic(A) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) in the absence (○) or presence (●) of 10 nM PPS and 50–500 mM NaCl (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (B) TIMP-3 (0.5 nM) was incubated with ADAMTS5-5 (0.5 nM) in the absence (○) or presence (●) of 100 nM PPS and 50–500 mM NaCl (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (C) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) and PPS (10 nM) in TNC buffer (containing 250 mM NaCl) at 37°C for times ranging between 1 and 72 h. The concentration of NaCl was then increased to 400 mM and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C).
Mentions: First, the effect of NaCl on the interaction between TIMP-3 and ADAMTS-5 was analysed. Inhibition of ADAMTS5-2 (0.5 nM) by TIMP-3 (0.5 nM) was slightly reduced at NaCl concentrations above 250 mM (Figure 6A, broken line), indicating that there is an electrostatic element to the interaction of these two proteins. In contrast, the inhibition of ADAMTS5-5 by TIMP-3 was not affected by NaCl concentrations of up to 500 mM (Figure 6B, broken line), indicating that electrostatic interactions do not play a significant role in the interaction of the TIMP-3 with the catalytic and Dis domain of ADAMTS-5.

Bottom Line: A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase.PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites.The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl.

View Article: PubMed Central - PubMed

Affiliation: The Kennedy Institute of Rheumatology, University of Oxford, 65 Aspenlea Road, Hammersmith, London W6 8LH, UK. linda.troeberg@kennedy.ox.ac.uk

ABSTRACT
The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

Show MeSH
Related in: MedlinePlus