Limits...
Incorporation of pentraxin 3 into hyaluronan matrices is tightly regulated and promotes matrix cross-linking.

Baranova NS, Inforzato A, Briggs DC, Tilakaratna V, Enghild JJ, Thakar D, Milner CM, Day AJ, Richter RP - J. Biol. Chem. (2014)

Bottom Line: We found that PTX3 binds neither to HA alone nor to HA films containing TSG-6.Interestingly, prior encounter with IαI was required for effective incorporation of PTX3 into TSG-6-loaded HA films.We propose that this mechanism is essential for correct assembly of the COC matrix and may also have general implications in other inflammatory processes that are associated with HA cross-linking.

View Article: PubMed Central - PubMed

Affiliation: From the CIC biomaGUNE, 20009 Donostia-San Sebastian, Spain.

Show MeSH

Related in: MedlinePlus

PTX3 differentially modulates the interactions of Link_TSG6 and full length TSG-6 with HA.A, PTX3 modulates Link_TSG6 interaction with HA. Binding isotherms obtained from titration of Link_TSG-6 into an HA film, monitored by SE, in the presence or absence of 0.3 μm PTX3 in the solution phase. A fit (solid line) of the titration data for Link_TSG6 (filled triangles) with the Hill equation gave a Hill exponent close to 1.0, confirming a simple non-cooperative interaction between HA and Link_TSG6. In the presence of PTX3, the interaction is more complex. The sigmoidal shape of the data (filled circles) indicates cooperative binding. A fit with the Hill equation (dashed line) indeed provides a Hill exponent of 3.6 (i.e. much larger than 1.0). B, competition of PTX3 and HA for TSG-6 binding. Binding isotherms obtained from titration of rhTSG-6 into an HA film, alone (filled circles) and in the presence of 0.3 μm (filled triangles) or 1 μm (empty triangles) PTX3 in the solution phase. At a bulk rhTSG-6 concentration of 0.45 μm, an ∼3-fold decrease in the surface density of adsorbed rhTSG-6 can be detected in the presence of 0.3 μm PTX3. At 1 μm PTX3, the decrease in binding becomes even more pronounced.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215230&req=5

Figure 3: PTX3 differentially modulates the interactions of Link_TSG6 and full length TSG-6 with HA.A, PTX3 modulates Link_TSG6 interaction with HA. Binding isotherms obtained from titration of Link_TSG-6 into an HA film, monitored by SE, in the presence or absence of 0.3 μm PTX3 in the solution phase. A fit (solid line) of the titration data for Link_TSG6 (filled triangles) with the Hill equation gave a Hill exponent close to 1.0, confirming a simple non-cooperative interaction between HA and Link_TSG6. In the presence of PTX3, the interaction is more complex. The sigmoidal shape of the data (filled circles) indicates cooperative binding. A fit with the Hill equation (dashed line) indeed provides a Hill exponent of 3.6 (i.e. much larger than 1.0). B, competition of PTX3 and HA for TSG-6 binding. Binding isotherms obtained from titration of rhTSG-6 into an HA film, alone (filled circles) and in the presence of 0.3 μm (filled triangles) or 1 μm (empty triangles) PTX3 in the solution phase. At a bulk rhTSG-6 concentration of 0.45 μm, an ∼3-fold decrease in the surface density of adsorbed rhTSG-6 can be detected in the presence of 0.3 μm PTX3. At 1 μm PTX3, the decrease in binding becomes even more pronounced.

Mentions: Surprisingly, PTX3 did not show any measurable binding if the HA film was preloaded with full-length TSG-6 instead of Link_TSG6. Consistent with this, subsequent incubation with the MNB4 antibody, which recognizes the N-terminal domain of PTX3, did not lead to any increase in binding signal (i.e. it did not appreciably affect the rhTSG-6 unbinding curve) (Fig. 2B). However, from Fig. 1, D and E, as well as previous reports (12, 30, 60), we know that rhTSG6 can interact with PTX3 in the absence of HA. Thus, it would appear that the interaction of rhTSG-6 with PTX3 is perturbed by HA. To shed light on this, we performed binding assays in which the HA film was exposed to a constant amount of PTX3, along with increasing concentrations of either Link_TSG6 (Fig. 3A) or rhTSG-6 (Fig. 3B). For comparison, both TSG-6 constructs were also titrated into the HA film in the absence of PTX3 and were found to exhibit distinct binding (Fig. 3), as has been noted previously (40); binding of rhTSG-6 to HA is characterized by a pronounced positive cooperativity, and the K0.5 is about 5-fold lower than for Link_TSG6.


Incorporation of pentraxin 3 into hyaluronan matrices is tightly regulated and promotes matrix cross-linking.

Baranova NS, Inforzato A, Briggs DC, Tilakaratna V, Enghild JJ, Thakar D, Milner CM, Day AJ, Richter RP - J. Biol. Chem. (2014)

PTX3 differentially modulates the interactions of Link_TSG6 and full length TSG-6 with HA.A, PTX3 modulates Link_TSG6 interaction with HA. Binding isotherms obtained from titration of Link_TSG-6 into an HA film, monitored by SE, in the presence or absence of 0.3 μm PTX3 in the solution phase. A fit (solid line) of the titration data for Link_TSG6 (filled triangles) with the Hill equation gave a Hill exponent close to 1.0, confirming a simple non-cooperative interaction between HA and Link_TSG6. In the presence of PTX3, the interaction is more complex. The sigmoidal shape of the data (filled circles) indicates cooperative binding. A fit with the Hill equation (dashed line) indeed provides a Hill exponent of 3.6 (i.e. much larger than 1.0). B, competition of PTX3 and HA for TSG-6 binding. Binding isotherms obtained from titration of rhTSG-6 into an HA film, alone (filled circles) and in the presence of 0.3 μm (filled triangles) or 1 μm (empty triangles) PTX3 in the solution phase. At a bulk rhTSG-6 concentration of 0.45 μm, an ∼3-fold decrease in the surface density of adsorbed rhTSG-6 can be detected in the presence of 0.3 μm PTX3. At 1 μm PTX3, the decrease in binding becomes even more pronounced.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215230&req=5

Figure 3: PTX3 differentially modulates the interactions of Link_TSG6 and full length TSG-6 with HA.A, PTX3 modulates Link_TSG6 interaction with HA. Binding isotherms obtained from titration of Link_TSG-6 into an HA film, monitored by SE, in the presence or absence of 0.3 μm PTX3 in the solution phase. A fit (solid line) of the titration data for Link_TSG6 (filled triangles) with the Hill equation gave a Hill exponent close to 1.0, confirming a simple non-cooperative interaction between HA and Link_TSG6. In the presence of PTX3, the interaction is more complex. The sigmoidal shape of the data (filled circles) indicates cooperative binding. A fit with the Hill equation (dashed line) indeed provides a Hill exponent of 3.6 (i.e. much larger than 1.0). B, competition of PTX3 and HA for TSG-6 binding. Binding isotherms obtained from titration of rhTSG-6 into an HA film, alone (filled circles) and in the presence of 0.3 μm (filled triangles) or 1 μm (empty triangles) PTX3 in the solution phase. At a bulk rhTSG-6 concentration of 0.45 μm, an ∼3-fold decrease in the surface density of adsorbed rhTSG-6 can be detected in the presence of 0.3 μm PTX3. At 1 μm PTX3, the decrease in binding becomes even more pronounced.
Mentions: Surprisingly, PTX3 did not show any measurable binding if the HA film was preloaded with full-length TSG-6 instead of Link_TSG6. Consistent with this, subsequent incubation with the MNB4 antibody, which recognizes the N-terminal domain of PTX3, did not lead to any increase in binding signal (i.e. it did not appreciably affect the rhTSG-6 unbinding curve) (Fig. 2B). However, from Fig. 1, D and E, as well as previous reports (12, 30, 60), we know that rhTSG6 can interact with PTX3 in the absence of HA. Thus, it would appear that the interaction of rhTSG-6 with PTX3 is perturbed by HA. To shed light on this, we performed binding assays in which the HA film was exposed to a constant amount of PTX3, along with increasing concentrations of either Link_TSG6 (Fig. 3A) or rhTSG-6 (Fig. 3B). For comparison, both TSG-6 constructs were also titrated into the HA film in the absence of PTX3 and were found to exhibit distinct binding (Fig. 3), as has been noted previously (40); binding of rhTSG-6 to HA is characterized by a pronounced positive cooperativity, and the K0.5 is about 5-fold lower than for Link_TSG6.

Bottom Line: We found that PTX3 binds neither to HA alone nor to HA films containing TSG-6.Interestingly, prior encounter with IαI was required for effective incorporation of PTX3 into TSG-6-loaded HA films.We propose that this mechanism is essential for correct assembly of the COC matrix and may also have general implications in other inflammatory processes that are associated with HA cross-linking.

View Article: PubMed Central - PubMed

Affiliation: From the CIC biomaGUNE, 20009 Donostia-San Sebastian, Spain.

Show MeSH
Related in: MedlinePlus