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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

Binding of TIMP-3 to PPS is a prerequisite for the affinity increase(A) Immunoblot showing the comparable reactivity of recombinant TIMP-3 (lane 1, 10 ng) and TIMP-3(K26/27/30/76E+R163/K165Q) (lane 2, 10 ng) with a polyclonal rabbit anti-TIMP-3 antibody. The molecular mass in kDa is indicated. (B) TIMP-3(K26/27/30/76E+R163/K165Q) (0.5–10 nM) was incubated with ADAMTS5-2 (0.5 nM) in the absence (○) or in the presence (●) of 25 nM PPS (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). Continuous and broken lines represent the best fit obtained by non-linear regression analysis of the data using eqn (1). (C) PPS (2.5 μM) was immobilized on glycosaminoglyan-binding multi-well plates and subsequent binding of TIMP-3 (●) or TIMP-3(K26/27/30/76E+R163/K165Q) (○) (0.3–40 nM) was detected using a polyclonal rabbit anti-TIMP-3 antibody.
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Figure 3: Binding of TIMP-3 to PPS is a prerequisite for the affinity increase(A) Immunoblot showing the comparable reactivity of recombinant TIMP-3 (lane 1, 10 ng) and TIMP-3(K26/27/30/76E+R163/K165Q) (lane 2, 10 ng) with a polyclonal rabbit anti-TIMP-3 antibody. The molecular mass in kDa is indicated. (B) TIMP-3(K26/27/30/76E+R163/K165Q) (0.5–10 nM) was incubated with ADAMTS5-2 (0.5 nM) in the absence (○) or in the presence (●) of 25 nM PPS (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). Continuous and broken lines represent the best fit obtained by non-linear regression analysis of the data using eqn (1). (C) PPS (2.5 μM) was immobilized on glycosaminoglyan-binding multi-well plates and subsequent binding of TIMP-3 (●) or TIMP-3(K26/27/30/76E+R163/K165Q) (○) (0.3–40 nM) was detected using a polyclonal rabbit anti-TIMP-3 antibody.

Mentions: A TIMP-3 mutant with low PPS-binding ability was used to assess whether the affinity increase requires TIMP-3 binding to PPS. Lee et al. [13] mutated several positively charged residues of TIMP-3 to generate TIMP-3(K26/27/30/76E+R163/K165Q), which was unable to bind to the extracellular matrix. We postulated that this mutant would also exhibit reduced binding to PPS. We recombinantly expressed and purified the mutant with a C-terminal His6 tag (Figure 3A), and confirmed it to be an effective ADAMTS inhibitor, with a Ki value of 1.34±0.04 nM for ADAMTS5-2, comparable with that of wild-type TIMP-3.


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)

Binding of TIMP-3 to PPS is a prerequisite for the affinity increase(A) Immunoblot showing the comparable reactivity of recombinant TIMP-3 (lane 1, 10 ng) and TIMP-3(K26/27/30/76E+R163/K165Q) (lane 2, 10 ng) with a polyclonal rabbit anti-TIMP-3 antibody. The molecular mass in kDa is indicated. (B) TIMP-3(K26/27/30/76E+R163/K165Q) (0.5–10 nM) was incubated with ADAMTS5-2 (0.5 nM) in the absence (○) or in the presence (●) of 25 nM PPS (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). Continuous and broken lines represent the best fit obtained by non-linear regression analysis of the data using eqn (1). (C) PPS (2.5 μM) was immobilized on glycosaminoglyan-binding multi-well plates and subsequent binding of TIMP-3 (●) or TIMP-3(K26/27/30/76E+R163/K165Q) (○) (0.3–40 nM) was detected using a polyclonal rabbit anti-TIMP-3 antibody.
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Related In: Results  -  Collection

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Figure 3: Binding of TIMP-3 to PPS is a prerequisite for the affinity increase(A) Immunoblot showing the comparable reactivity of recombinant TIMP-3 (lane 1, 10 ng) and TIMP-3(K26/27/30/76E+R163/K165Q) (lane 2, 10 ng) with a polyclonal rabbit anti-TIMP-3 antibody. The molecular mass in kDa is indicated. (B) TIMP-3(K26/27/30/76E+R163/K165Q) (0.5–10 nM) was incubated with ADAMTS5-2 (0.5 nM) in the absence (○) or in the presence (●) of 25 nM PPS (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). Continuous and broken lines represent the best fit obtained by non-linear regression analysis of the data using eqn (1). (C) PPS (2.5 μM) was immobilized on glycosaminoglyan-binding multi-well plates and subsequent binding of TIMP-3 (●) or TIMP-3(K26/27/30/76E+R163/K165Q) (○) (0.3–40 nM) was detected using a polyclonal rabbit anti-TIMP-3 antibody.
Mentions: A TIMP-3 mutant with low PPS-binding ability was used to assess whether the affinity increase requires TIMP-3 binding to PPS. Lee et al. [13] mutated several positively charged residues of TIMP-3 to generate TIMP-3(K26/27/30/76E+R163/K165Q), which was unable to bind to the extracellular matrix. We postulated that this mutant would also exhibit reduced binding to PPS. We recombinantly expressed and purified the mutant with a C-terminal His6 tag (Figure 3A), and confirmed it to be an effective ADAMTS inhibitor, with a Ki value of 1.34±0.04 nM for ADAMTS5-2, comparable with that of wild-type TIMP-3.

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