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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.

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PTX3 in a mixture with IαI incorporates into TSG-6-loaded HA films.A, the HA film was first loaded with 0.3 μm TSG-6, excess protein was removed from the solution phase, and a mixture of 0.3 μm PTX3, 1 μm IαI was added (premixed for 1 min). The addition of the PTX3/IαI mixture first enhanced desorption until a mass fraction of 35% remained, and thereafter incorporation of material into the HA film started. The addition of anti-PTX3, after the removal of excess protein in the solution phase, confirmed PTX3 incorporation into the film. Only a small fraction of about 12% could be eluted with 2 m GdnHCl, whereas most material was eluted with 8 m GdnHCl. A fraction of 10% remained bound in 8 m GdnHCl but could be largely digested by hyaluronidase. The increase in the desorption rate upon the addition of IαI/PTX3 can be appreciated from the linear fits to the data shortly before (green dashed line; 3.7 ng/cm2/min) and after (red dashed line; 27 ng/cm2/min) protein addition. The adsorption process setting in 10 min after incubation with IαI/PTX3 was fitted by an exponential (gray dashed line). The fit revealed a maximal surface density of 660 ng/cm2 and a half-time of about 90 min. The curve shown is representative of a set of measurements performed in duplicate. B, equivalent measurement with IαI instead of a mixture of IαI and PTX3, with linear fits to the data shortly before (green dashed line; 2.9 ng/cm2/min) and after (orange dashed line; 31 ng/cm2/min) IαI addition, reproduced from Fig. 1B in Ref. 45. In this case, the adsorption process, commencing 10 min after incubation with IαI, could be fitted with a straight line (gray dashed line).
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Figure 6: PTX3 in a mixture with IαI incorporates into TSG-6-loaded HA films.A, the HA film was first loaded with 0.3 μm TSG-6, excess protein was removed from the solution phase, and a mixture of 0.3 μm PTX3, 1 μm IαI was added (premixed for 1 min). The addition of the PTX3/IαI mixture first enhanced desorption until a mass fraction of 35% remained, and thereafter incorporation of material into the HA film started. The addition of anti-PTX3, after the removal of excess protein in the solution phase, confirmed PTX3 incorporation into the film. Only a small fraction of about 12% could be eluted with 2 m GdnHCl, whereas most material was eluted with 8 m GdnHCl. A fraction of 10% remained bound in 8 m GdnHCl but could be largely digested by hyaluronidase. The increase in the desorption rate upon the addition of IαI/PTX3 can be appreciated from the linear fits to the data shortly before (green dashed line; 3.7 ng/cm2/min) and after (red dashed line; 27 ng/cm2/min) protein addition. The adsorption process setting in 10 min after incubation with IαI/PTX3 was fitted by an exponential (gray dashed line). The fit revealed a maximal surface density of 660 ng/cm2 and a half-time of about 90 min. The curve shown is representative of a set of measurements performed in duplicate. B, equivalent measurement with IαI instead of a mixture of IαI and PTX3, with linear fits to the data shortly before (green dashed line; 2.9 ng/cm2/min) and after (orange dashed line; 31 ng/cm2/min) IαI addition, reproduced from Fig. 1B in Ref. 45. In this case, the adsorption process, commencing 10 min after incubation with IαI, could be fitted with a straight line (gray dashed line).

Mentions: To test whether PTX3 influences the competition between IαI and HA for TSG-6, we performed another sequential incubation assay (Fig. 6A); here IαI and PTX3 were premixed and then exposed to a TSG-6-loaded HA film (where the TSG-6 had been removed from the bulk solution and the HA-associated protein was beginning to dissociate). Linear fits allowed us to approximate the unbinding rates just before (3.7 ng/cm2/min; green dashed line) and after the addition of IαI/PTX3 (27 ng/cm2/min; red dashed line). Comparison with the displacement induced by IαI alone (Fig. 6B; reproduced from Ref. 45) did not reveal any significant difference. Also, the fraction of stably but non-covalently bound material that persisted after incubation with IαI and PTX3 was comparable with the fraction displaced by IαI alone (30 and 35%, respectively). Hence, PTX3 does not affect the propensity of IαI to displace TSG-6 from HA. More generally, this finding would indicate that PTX3 does not influence the initial interaction between TSG-6 and IαI.


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 in a mixture with IαI incorporates into TSG-6-loaded HA films.A, the HA film was first loaded with 0.3 μm TSG-6, excess protein was removed from the solution phase, and a mixture of 0.3 μm PTX3, 1 μm IαI was added (premixed for 1 min). The addition of the PTX3/IαI mixture first enhanced desorption until a mass fraction of 35% remained, and thereafter incorporation of material into the HA film started. The addition of anti-PTX3, after the removal of excess protein in the solution phase, confirmed PTX3 incorporation into the film. Only a small fraction of about 12% could be eluted with 2 m GdnHCl, whereas most material was eluted with 8 m GdnHCl. A fraction of 10% remained bound in 8 m GdnHCl but could be largely digested by hyaluronidase. The increase in the desorption rate upon the addition of IαI/PTX3 can be appreciated from the linear fits to the data shortly before (green dashed line; 3.7 ng/cm2/min) and after (red dashed line; 27 ng/cm2/min) protein addition. The adsorption process setting in 10 min after incubation with IαI/PTX3 was fitted by an exponential (gray dashed line). The fit revealed a maximal surface density of 660 ng/cm2 and a half-time of about 90 min. The curve shown is representative of a set of measurements performed in duplicate. B, equivalent measurement with IαI instead of a mixture of IαI and PTX3, with linear fits to the data shortly before (green dashed line; 2.9 ng/cm2/min) and after (orange dashed line; 31 ng/cm2/min) IαI addition, reproduced from Fig. 1B in Ref. 45. In this case, the adsorption process, commencing 10 min after incubation with IαI, could be fitted with a straight line (gray dashed line).
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Figure 6: PTX3 in a mixture with IαI incorporates into TSG-6-loaded HA films.A, the HA film was first loaded with 0.3 μm TSG-6, excess protein was removed from the solution phase, and a mixture of 0.3 μm PTX3, 1 μm IαI was added (premixed for 1 min). The addition of the PTX3/IαI mixture first enhanced desorption until a mass fraction of 35% remained, and thereafter incorporation of material into the HA film started. The addition of anti-PTX3, after the removal of excess protein in the solution phase, confirmed PTX3 incorporation into the film. Only a small fraction of about 12% could be eluted with 2 m GdnHCl, whereas most material was eluted with 8 m GdnHCl. A fraction of 10% remained bound in 8 m GdnHCl but could be largely digested by hyaluronidase. The increase in the desorption rate upon the addition of IαI/PTX3 can be appreciated from the linear fits to the data shortly before (green dashed line; 3.7 ng/cm2/min) and after (red dashed line; 27 ng/cm2/min) protein addition. The adsorption process setting in 10 min after incubation with IαI/PTX3 was fitted by an exponential (gray dashed line). The fit revealed a maximal surface density of 660 ng/cm2 and a half-time of about 90 min. The curve shown is representative of a set of measurements performed in duplicate. B, equivalent measurement with IαI instead of a mixture of IαI and PTX3, with linear fits to the data shortly before (green dashed line; 2.9 ng/cm2/min) and after (orange dashed line; 31 ng/cm2/min) IαI addition, reproduced from Fig. 1B in Ref. 45. In this case, the adsorption process, commencing 10 min after incubation with IαI, could be fitted with a straight line (gray dashed line).
Mentions: To test whether PTX3 influences the competition between IαI and HA for TSG-6, we performed another sequential incubation assay (Fig. 6A); here IαI and PTX3 were premixed and then exposed to a TSG-6-loaded HA film (where the TSG-6 had been removed from the bulk solution and the HA-associated protein was beginning to dissociate). Linear fits allowed us to approximate the unbinding rates just before (3.7 ng/cm2/min; green dashed line) and after the addition of IαI/PTX3 (27 ng/cm2/min; red dashed line). Comparison with the displacement induced by IαI alone (Fig. 6B; reproduced from Ref. 45) did not reveal any significant difference. Also, the fraction of stably but non-covalently bound material that persisted after incubation with IαI and PTX3 was comparable with the fraction displaced by IαI alone (30 and 35%, respectively). Hence, PTX3 does not affect the propensity of IαI to displace TSG-6 from HA. More generally, this finding would indicate that PTX3 does not influence the initial interaction between TSG-6 and IαI.

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