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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

Longer PPS molecules are more effective than short PPS molecules at improving affinity(A) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) and various concentrations of PPS fractions F1–F6 (see Table 1) for 1 h at 37°C. The residual activity against Abz-TESE~SRGAIY-Dpa-KK was measured. (B) PPS fractions F1–F6 (2.5 μM) were immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with TIMP-3. Bound TIMP-3 was quantified using an M2 anti-FLAG antibody. TIMP-3 bound strongly to F1 (●, 35 dp), F2 (★, 25 dp), F3 (▲, 16 dp) and F4 (×, 11 dp), but only weakly to F5 (■, 8 dp) and F6 (▼, 8 dp). (C) PPS fractions F1–F6 (2.5 μM) were immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with ADAMTS5-2. Bound ADAMTS5-2 was quantified using an M2 anti-FLAG antibody. ADAMTS5-2 bound strongly to F1 (●), F2 (★), F3 (▲) and F4 (×), and weakly to F5 (■) and F6 (▼).
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Figure 4: Longer PPS molecules are more effective than short PPS molecules at improving affinity(A) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) and various concentrations of PPS fractions F1–F6 (see Table 1) for 1 h at 37°C. The residual activity against Abz-TESE~SRGAIY-Dpa-KK was measured. (B) PPS fractions F1–F6 (2.5 μM) were immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with TIMP-3. Bound TIMP-3 was quantified using an M2 anti-FLAG antibody. TIMP-3 bound strongly to F1 (●, 35 dp), F2 (★, 25 dp), F3 (▲, 16 dp) and F4 (×, 11 dp), but only weakly to F5 (■, 8 dp) and F6 (▼, 8 dp). (C) PPS fractions F1–F6 (2.5 μM) were immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with ADAMTS5-2. Bound ADAMTS5-2 was quantified using an M2 anti-FLAG antibody. ADAMTS5-2 bound strongly to F1 (●), F2 (★), F3 (▲) and F4 (×), and weakly to F5 (■) and F6 (▼).

Mentions: F1 (10.3 kDa, dp 35), F2 (7.3 kDa, dp 25), F3 (4.8 kDa, dp 16) and F4 (3.3 kDa, dp 11) were equally able to increase ADAMTS5-2–TIMP-3 affinity (Figure 4A), with concentrations above 10 nM enabling complete inhibition of 0.5 nM ADAMTS5-2 by 0.5 nM TIMP-3. F5 (2.4 kDa, dp 8) and F6 (2.4 kDa, dp 8) were less active, with 100-fold higher concentrations of these fractions required for a comparable increase in ADAMTS5-2–TIMP-3 affinity.


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)

Longer PPS molecules are more effective than short PPS molecules at improving affinity(A) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) and various concentrations of PPS fractions F1–F6 (see Table 1) for 1 h at 37°C. The residual activity against Abz-TESE~SRGAIY-Dpa-KK was measured. (B) PPS fractions F1–F6 (2.5 μM) were immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with TIMP-3. Bound TIMP-3 was quantified using an M2 anti-FLAG antibody. TIMP-3 bound strongly to F1 (●, 35 dp), F2 (★, 25 dp), F3 (▲, 16 dp) and F4 (×, 11 dp), but only weakly to F5 (■, 8 dp) and F6 (▼, 8 dp). (C) PPS fractions F1–F6 (2.5 μM) were immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with ADAMTS5-2. Bound ADAMTS5-2 was quantified using an M2 anti-FLAG antibody. ADAMTS5-2 bound strongly to F1 (●), F2 (★), F3 (▲) and F4 (×), and weakly to F5 (■) and F6 (▼).
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Figure 4: Longer PPS molecules are more effective than short PPS molecules at improving affinity(A) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) and various concentrations of PPS fractions F1–F6 (see Table 1) for 1 h at 37°C. The residual activity against Abz-TESE~SRGAIY-Dpa-KK was measured. (B) PPS fractions F1–F6 (2.5 μM) were immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with TIMP-3. Bound TIMP-3 was quantified using an M2 anti-FLAG antibody. TIMP-3 bound strongly to F1 (●, 35 dp), F2 (★, 25 dp), F3 (▲, 16 dp) and F4 (×, 11 dp), but only weakly to F5 (■, 8 dp) and F6 (▼, 8 dp). (C) PPS fractions F1–F6 (2.5 μM) were immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with ADAMTS5-2. Bound ADAMTS5-2 was quantified using an M2 anti-FLAG antibody. ADAMTS5-2 bound strongly to F1 (●), F2 (★), F3 (▲) and F4 (×), and weakly to F5 (■) and F6 (▼).
Mentions: F1 (10.3 kDa, dp 35), F2 (7.3 kDa, dp 25), F3 (4.8 kDa, dp 16) and F4 (3.3 kDa, dp 11) were equally able to increase ADAMTS5-2–TIMP-3 affinity (Figure 4A), with concentrations above 10 nM enabling complete inhibition of 0.5 nM ADAMTS5-2 by 0.5 nM TIMP-3. F5 (2.4 kDa, dp 8) and F6 (2.4 kDa, dp 8) were less active, with 100-fold higher concentrations of these fractions required for a comparable increase in ADAMTS5-2–TIMP-3 affinity.

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