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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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Related in: MedlinePlus

PPS improves the affinity between full-length ADAMTS-5 and TIMP-3Enzymes were incubated with TIMP-3 in the absence (○) or presence (●) of 25 nM PPS (for 1 h at 37°C) and residual activity was determined. (A) TIMP-3 (0.5–5 nM) inhibition of full-length ADAMTS-5 (0.5 nM). (B) N-TIMP-3 (0.5–5 nM) inhibition of full-length ADAMTS-5 (0.5 nM). (C) TIMP-1 (50–1000 nM) inhibition of ADAMTS-5 (0.5 nM). (D) TIMP-3 (0.4–25 nM) inhibition of MMP-1 catalytic domain (1 nM). (E) TIMP-3 (0.1–1 nM) inhibition of MMP-2 (0.02 nM). (F) TIMP-3 (0.4–25 nM) inhibition of MMP-3 catalytic domain (1 nM). Continuous lines represent the best fit obtained by non-linear regression analysis of the data using eqn (1).
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Figure 1: PPS improves the affinity between full-length ADAMTS-5 and TIMP-3Enzymes were incubated with TIMP-3 in the absence (○) or presence (●) of 25 nM PPS (for 1 h at 37°C) and residual activity was determined. (A) TIMP-3 (0.5–5 nM) inhibition of full-length ADAMTS-5 (0.5 nM). (B) N-TIMP-3 (0.5–5 nM) inhibition of full-length ADAMTS-5 (0.5 nM). (C) TIMP-1 (50–1000 nM) inhibition of ADAMTS-5 (0.5 nM). (D) TIMP-3 (0.4–25 nM) inhibition of MMP-1 catalytic domain (1 nM). (E) TIMP-3 (0.1–1 nM) inhibition of MMP-2 (0.02 nM). (F) TIMP-3 (0.4–25 nM) inhibition of MMP-3 catalytic domain (1 nM). Continuous lines represent the best fit obtained by non-linear regression analysis of the data using eqn (1).

Mentions: In the absence of PPS, a Ki value of 0.56±0.03 nM was determined, in agreement with previous findings [9]. In the presence of 25 nM (0.1 μg/ml) PPS, Ki was too low to be determined, with 0.5 nM ADAMTS-5 completely inhibited by an equimolar amount of TIMP-3 (Figure 1A). The affinity of N-TIMP-3 for full-length ADAMTS-5 was similarly increased by PPS (Figure 1B). Accurate determination of the improved Ki values was not possible, as ADAMTS-5 concentrations below 0.5 nM cannot be reliably assayed with the currently available substrates. Heparin also increased the affinity between TIMP-3 and full-length ADAMTS-5, although 10-fold higher heparin concentrations were required. Other glycosaminoglycans, such as dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and hyaluronic acid had no effect on affinity at concentrations up to 1 μM.


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)

PPS improves the affinity between full-length ADAMTS-5 and TIMP-3Enzymes were incubated with TIMP-3 in the absence (○) or presence (●) of 25 nM PPS (for 1 h at 37°C) and residual activity was determined. (A) TIMP-3 (0.5–5 nM) inhibition of full-length ADAMTS-5 (0.5 nM). (B) N-TIMP-3 (0.5–5 nM) inhibition of full-length ADAMTS-5 (0.5 nM). (C) TIMP-1 (50–1000 nM) inhibition of ADAMTS-5 (0.5 nM). (D) TIMP-3 (0.4–25 nM) inhibition of MMP-1 catalytic domain (1 nM). (E) TIMP-3 (0.1–1 nM) inhibition of MMP-2 (0.02 nM). (F) TIMP-3 (0.4–25 nM) inhibition of MMP-3 catalytic domain (1 nM). Continuous lines represent the best fit obtained by non-linear regression analysis of the data using eqn (1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369482&req=5

Figure 1: PPS improves the affinity between full-length ADAMTS-5 and TIMP-3Enzymes were incubated with TIMP-3 in the absence (○) or presence (●) of 25 nM PPS (for 1 h at 37°C) and residual activity was determined. (A) TIMP-3 (0.5–5 nM) inhibition of full-length ADAMTS-5 (0.5 nM). (B) N-TIMP-3 (0.5–5 nM) inhibition of full-length ADAMTS-5 (0.5 nM). (C) TIMP-1 (50–1000 nM) inhibition of ADAMTS-5 (0.5 nM). (D) TIMP-3 (0.4–25 nM) inhibition of MMP-1 catalytic domain (1 nM). (E) TIMP-3 (0.1–1 nM) inhibition of MMP-2 (0.02 nM). (F) TIMP-3 (0.4–25 nM) inhibition of MMP-3 catalytic domain (1 nM). Continuous lines represent the best fit obtained by non-linear regression analysis of the data using eqn (1).
Mentions: In the absence of PPS, a Ki value of 0.56±0.03 nM was determined, in agreement with previous findings [9]. In the presence of 25 nM (0.1 μg/ml) PPS, Ki was too low to be determined, with 0.5 nM ADAMTS-5 completely inhibited by an equimolar amount of TIMP-3 (Figure 1A). The affinity of N-TIMP-3 for full-length ADAMTS-5 was similarly increased by PPS (Figure 1B). Accurate determination of the improved Ki values was not possible, as ADAMTS-5 concentrations below 0.5 nM cannot be reliably assayed with the currently available substrates. Heparin also increased the affinity between TIMP-3 and full-length ADAMTS-5, although 10-fold higher heparin concentrations were required. Other glycosaminoglycans, such as dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and hyaluronic acid had no effect on affinity at concentrations up to 1 μM.

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