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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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Modes of PPS binding to ADAMTS-5–TIMP-3 complexesShort PPS chains increase the affinity between TIMP-3 and ADAMTS5-5 or ADAMTS5-2 by altering their interaction with the charged environment [mode (i)]. Longer PPS chains further increase affinity through an increase in the Gibbs free energy of binding brought about by linking the low-affinity binding sites on TIMP-3 and ADAMTS5-5 [mode (ii)]. Such longer PPS chains are even more effective with ADAMTS5-2, as their length enables them to interact with multiple positively charged regions of the enzyme, increasing the affinity of the trimolecular complex [mode (iii)].
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Figure 7: Modes of PPS binding to ADAMTS-5–TIMP-3 complexesShort PPS chains increase the affinity between TIMP-3 and ADAMTS5-5 or ADAMTS5-2 by altering their interaction with the charged environment [mode (i)]. Longer PPS chains further increase affinity through an increase in the Gibbs free energy of binding brought about by linking the low-affinity binding sites on TIMP-3 and ADAMTS5-5 [mode (ii)]. Such longer PPS chains are even more effective with ADAMTS5-2, as their length enables them to interact with multiple positively charged regions of the enzyme, increasing the affinity of the trimolecular complex [mode (iii)].

Mentions: Similarly, truncated forms of ADAMTS-5 with reduced PPS binding ability showed decreased sensitivity to the PPS-mediated affinity increase. Deletion of the C-terminal TS-2 domain has no detectable effect on matrix binding or aggrecanolytic activity of ADAMTS-5 [10], and had little or no effect on PPS susceptibility (Figure 2B). The Sp domain has previously been suggested to bind to glycosaminoglycans, as it promotes aggrecan hydrolysis [10] and binding to the extracellular matrix [10]. We found that deletion of the Sp domain (ADAMTS5-3) markedly reduced both direct binding to PPS and susceptibility to the PPS-mediated affinity increase (Figures 2B and 2C). The Sp domain thus contains a major glycosaminoglycan-binding site of ADAMTS-5 (site B in Figure 7). The CysR domain has been shown to promote aggrecan hydrolysis, extracellular matrix binding and susceptibility to exosite inhibition by PPS [6,10], but we found that deletion of this domain (ADAMTS5-4) had little effect on binding to PPS or susceptibility to the PPS-mediated affinity increase. The TS-1 domain is reported to contribute to aggrecan binding and hydrolysis by ADAMTS-4 [20], and deletion of this domain in ADAMTS-5 (ADAMTS5-5) reduced direct binding to PPS, but had little effect on the affinity increase (Figures 2B and 2C). Although ADAMTS5-5 bound weakly to PPS, some binding was still evident at higher concentrations, in agreement with previous findings that this form of the enzyme retains some heparin-binding ability [21]. These data thus indicate that, in addition to the major glycosaminoglycan-binding site on the Sp domain of ADAMTS-5, the TS-1 and catalytic/Dis domains contain additional binding sites (site A in Figure 7). As ADAMTS5-1 and ADAMTS5-2 contain all of these binding sites, they bind to PPS with the highest affinity, and thus require low PPS concentrations for trimolecular complex formation. Truncated forms of ADAMTS-5 that lack the Sp domain bind to PPS with lower affinity and require 100-fold higher PPS concentrations for trimolecular complex formation.


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)

Modes of PPS binding to ADAMTS-5–TIMP-3 complexesShort PPS chains increase the affinity between TIMP-3 and ADAMTS5-5 or ADAMTS5-2 by altering their interaction with the charged environment [mode (i)]. Longer PPS chains further increase affinity through an increase in the Gibbs free energy of binding brought about by linking the low-affinity binding sites on TIMP-3 and ADAMTS5-5 [mode (ii)]. Such longer PPS chains are even more effective with ADAMTS5-2, as their length enables them to interact with multiple positively charged regions of the enzyme, increasing the affinity of the trimolecular complex [mode (iii)].
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Modes of PPS binding to ADAMTS-5–TIMP-3 complexesShort PPS chains increase the affinity between TIMP-3 and ADAMTS5-5 or ADAMTS5-2 by altering their interaction with the charged environment [mode (i)]. Longer PPS chains further increase affinity through an increase in the Gibbs free energy of binding brought about by linking the low-affinity binding sites on TIMP-3 and ADAMTS5-5 [mode (ii)]. Such longer PPS chains are even more effective with ADAMTS5-2, as their length enables them to interact with multiple positively charged regions of the enzyme, increasing the affinity of the trimolecular complex [mode (iii)].
Mentions: Similarly, truncated forms of ADAMTS-5 with reduced PPS binding ability showed decreased sensitivity to the PPS-mediated affinity increase. Deletion of the C-terminal TS-2 domain has no detectable effect on matrix binding or aggrecanolytic activity of ADAMTS-5 [10], and had little or no effect on PPS susceptibility (Figure 2B). The Sp domain has previously been suggested to bind to glycosaminoglycans, as it promotes aggrecan hydrolysis [10] and binding to the extracellular matrix [10]. We found that deletion of the Sp domain (ADAMTS5-3) markedly reduced both direct binding to PPS and susceptibility to the PPS-mediated affinity increase (Figures 2B and 2C). The Sp domain thus contains a major glycosaminoglycan-binding site of ADAMTS-5 (site B in Figure 7). The CysR domain has been shown to promote aggrecan hydrolysis, extracellular matrix binding and susceptibility to exosite inhibition by PPS [6,10], but we found that deletion of this domain (ADAMTS5-4) had little effect on binding to PPS or susceptibility to the PPS-mediated affinity increase. The TS-1 domain is reported to contribute to aggrecan binding and hydrolysis by ADAMTS-4 [20], and deletion of this domain in ADAMTS-5 (ADAMTS5-5) reduced direct binding to PPS, but had little effect on the affinity increase (Figures 2B and 2C). Although ADAMTS5-5 bound weakly to PPS, some binding was still evident at higher concentrations, in agreement with previous findings that this form of the enzyme retains some heparin-binding ability [21]. These data thus indicate that, in addition to the major glycosaminoglycan-binding site on the Sp domain of ADAMTS-5, the TS-1 and catalytic/Dis domains contain additional binding sites (site A in Figure 7). As ADAMTS5-1 and ADAMTS5-2 contain all of these binding sites, they bind to PPS with the highest affinity, and thus require low PPS concentrations for trimolecular complex formation. Truncated forms of ADAMTS-5 that lack the Sp domain bind to PPS with lower affinity and require 100-fold higher PPS concentrations for trimolecular complex formation.

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