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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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PPS increases affinity by a dual mechanism(A) TIMP-3 (0.5 nM) was incubated with either ADAMTS5-2 or ADAMTS5-5 (0.5 nM) in the presence of PPS size-exclusion chromatography fractions F1 or F5 (0.5–1000 nM, 1 h, 37°C) and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (B) TIMP-3 (100 nM) was incubated with ADAMTS5-2 or ADAMTS-5-5 (100 nM) with or without Bio-PPS (1 μM) (1 h, 37°C). Streptavidin–Sepharose beads were added and bound proteins were recovered in SDS buffer and analysed by immunoblotting with an M2 anti-FLAG antibody. The molecular mass in kDa is indicated. (C) ADAMTS5-2 (0.5 nM) was incubated with TIMP-3 (0.5 nM) in the presence of PPS (0.05 nM–50 μM) (1 h, 37°C) and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C).
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Figure 5: PPS increases affinity by a dual mechanism(A) TIMP-3 (0.5 nM) was incubated with either ADAMTS5-2 or ADAMTS5-5 (0.5 nM) in the presence of PPS size-exclusion chromatography fractions F1 or F5 (0.5–1000 nM, 1 h, 37°C) and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (B) TIMP-3 (100 nM) was incubated with ADAMTS5-2 or ADAMTS-5-5 (100 nM) with or without Bio-PPS (1 μM) (1 h, 37°C). Streptavidin–Sepharose beads were added and bound proteins were recovered in SDS buffer and analysed by immunoblotting with an M2 anti-FLAG antibody. The molecular mass in kDa is indicated. (C) ADAMTS5-2 (0.5 nM) was incubated with TIMP-3 (0.5 nM) in the presence of PPS (0.05 nM–50 μM) (1 h, 37°C) and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C).

Mentions: The ability of short (F5) and long (F1) PPS fractions to increase the ADAMTS5-2–TIMP-3 and ADAMTS5-5–TIMP-3 affinity was compared. F5 was equally active at increasing affinity between TIMP-3 and both forms of the enzyme, whereas F1 was more effective at increasing TIMP-3 affinity for ADAMTS5-2 than for ADAMTS5-5 (Figure 5A).


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 increases affinity by a dual mechanism(A) TIMP-3 (0.5 nM) was incubated with either ADAMTS5-2 or ADAMTS5-5 (0.5 nM) in the presence of PPS size-exclusion chromatography fractions F1 or F5 (0.5–1000 nM, 1 h, 37°C) and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (B) TIMP-3 (100 nM) was incubated with ADAMTS5-2 or ADAMTS-5-5 (100 nM) with or without Bio-PPS (1 μM) (1 h, 37°C). Streptavidin–Sepharose beads were added and bound proteins were recovered in SDS buffer and analysed by immunoblotting with an M2 anti-FLAG antibody. The molecular mass in kDa is indicated. (C) ADAMTS5-2 (0.5 nM) was incubated with TIMP-3 (0.5 nM) in the presence of PPS (0.05 nM–50 μM) (1 h, 37°C) 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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Show All Figures
getmorefigures.php?uid=PMC3369482&req=5

Figure 5: PPS increases affinity by a dual mechanism(A) TIMP-3 (0.5 nM) was incubated with either ADAMTS5-2 or ADAMTS5-5 (0.5 nM) in the presence of PPS size-exclusion chromatography fractions F1 or F5 (0.5–1000 nM, 1 h, 37°C) and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (B) TIMP-3 (100 nM) was incubated with ADAMTS5-2 or ADAMTS-5-5 (100 nM) with or without Bio-PPS (1 μM) (1 h, 37°C). Streptavidin–Sepharose beads were added and bound proteins were recovered in SDS buffer and analysed by immunoblotting with an M2 anti-FLAG antibody. The molecular mass in kDa is indicated. (C) ADAMTS5-2 (0.5 nM) was incubated with TIMP-3 (0.5 nM) in the presence of PPS (0.05 nM–50 μM) (1 h, 37°C) and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C).
Mentions: The ability of short (F5) and long (F1) PPS fractions to increase the ADAMTS5-2–TIMP-3 and ADAMTS5-5–TIMP-3 affinity was compared. F5 was equally active at increasing affinity between TIMP-3 and both forms of the enzyme, whereas F1 was more effective at increasing TIMP-3 affinity for ADAMTS5-2 than for ADAMTS5-5 (Figure 5A).

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