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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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Related in: MedlinePlus

PTX3 incorporates into HA films when presented in a ternary mixture with TSG-6 and IαI.A, binding assay by SE. HA films were first exposed to 0.3 μm PTX3. After 2 min of incubation, 1 μm IαI was added, and after another 2.5 min, 0.3 μm rhTSG-6 was added. Binding ensued after the addition of rhTSG-6; the gray solid line is a linear fit revealing an initial binding rate of 6 ng/cm2/min. The protein mixtures were incubated with the HA films for 2 h. Subsequent binding of anti-PTX3 antibody, incubated at 0.08 μm, indicates successful incorporation of PTX3. The curve shown is representative of a set of measurements performed in duplicate. B, binding assay by QCM-D. Δf (blue lines) and ΔD (orange lines) are shown. Mixtures of PTX3, rhTSG-6, and IαI were exposed to HA films at final protein concentrations of 1.0, 0.6, and 0.2 μm, respectively. In one case (circles), PTX3 was first mixed with TSG-6 for 2 h and then with IαI for another 1 h (all at room temperature) before exposure to HA. In the other case (triangles), TSG-6 and IαI were mixed first (for 2 h), and then PTX3 was added (for 1 h) before exposure to HA. Clear QCM-D responses upon the subsequent addition of anti-PTX3 antibody, incubated at 0.08 μm, indicated successful incorporation of PTX3.
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Figure 5: PTX3 incorporates into HA films when presented in a ternary mixture with TSG-6 and IαI.A, binding assay by SE. HA films were first exposed to 0.3 μm PTX3. After 2 min of incubation, 1 μm IαI was added, and after another 2.5 min, 0.3 μm rhTSG-6 was added. Binding ensued after the addition of rhTSG-6; the gray solid line is a linear fit revealing an initial binding rate of 6 ng/cm2/min. The protein mixtures were incubated with the HA films for 2 h. Subsequent binding of anti-PTX3 antibody, incubated at 0.08 μm, indicates successful incorporation of PTX3. The curve shown is representative of a set of measurements performed in duplicate. B, binding assay by QCM-D. Δf (blue lines) and ΔD (orange lines) are shown. Mixtures of PTX3, rhTSG-6, and IαI were exposed to HA films at final protein concentrations of 1.0, 0.6, and 0.2 μm, respectively. In one case (circles), PTX3 was first mixed with TSG-6 for 2 h and then with IαI for another 1 h (all at room temperature) before exposure to HA. In the other case (triangles), TSG-6 and IαI were mixed first (for 2 h), and then PTX3 was added (for 1 h) before exposure to HA. Clear QCM-D responses upon the subsequent addition of anti-PTX3 antibody, incubated at 0.08 μm, indicated successful incorporation of PTX3.

Mentions: The absence of PTX3 binding in the above-described assays disproves existing hypotheses about the mechanism by which PTX3 is incorporated into HA matrices (12, 48). On the other hand, PTX3 has been shown to be an essential component in the formation of the COC matrix (12). So how is PTX3 incorporated into HA films? We hypothesized that PTX3 must encounter IαI and/or TSG-6 prior to interaction with HA in order for PTX3 to be incorporated into HA assemblies. To test this hypothesis, we first added PTX3 and IαI at bulk concentrations of 0.3 and 1 μm, respectively, to an HA film. As anticipated, this mixture did not show any HA binding activity (Fig. 1C) (45). Second, TSG-6 was added at a bulk concentration of 0.3 μm. This protein did start a binding reaction (Fig. 5A). After 2 h of incubation, all proteins in the soluble phase were removed. Following this, the anti-PTX3 antibody (MNB4) did bind to the film. In contrast, the same antibody did not bind to an HA film that contained TSG-6 (Fig. 2B). This provides strong evidence that co-incubation of PTX3 in a ternary mixture with IαI and TSG-6 promotes PTX3 incorporation into the HA film.


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 incorporates into HA films when presented in a ternary mixture with TSG-6 and IαI.A, binding assay by SE. HA films were first exposed to 0.3 μm PTX3. After 2 min of incubation, 1 μm IαI was added, and after another 2.5 min, 0.3 μm rhTSG-6 was added. Binding ensued after the addition of rhTSG-6; the gray solid line is a linear fit revealing an initial binding rate of 6 ng/cm2/min. The protein mixtures were incubated with the HA films for 2 h. Subsequent binding of anti-PTX3 antibody, incubated at 0.08 μm, indicates successful incorporation of PTX3. The curve shown is representative of a set of measurements performed in duplicate. B, binding assay by QCM-D. Δf (blue lines) and ΔD (orange lines) are shown. Mixtures of PTX3, rhTSG-6, and IαI were exposed to HA films at final protein concentrations of 1.0, 0.6, and 0.2 μm, respectively. In one case (circles), PTX3 was first mixed with TSG-6 for 2 h and then with IαI for another 1 h (all at room temperature) before exposure to HA. In the other case (triangles), TSG-6 and IαI were mixed first (for 2 h), and then PTX3 was added (for 1 h) before exposure to HA. Clear QCM-D responses upon the subsequent addition of anti-PTX3 antibody, incubated at 0.08 μm, indicated successful incorporation of PTX3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: PTX3 incorporates into HA films when presented in a ternary mixture with TSG-6 and IαI.A, binding assay by SE. HA films were first exposed to 0.3 μm PTX3. After 2 min of incubation, 1 μm IαI was added, and after another 2.5 min, 0.3 μm rhTSG-6 was added. Binding ensued after the addition of rhTSG-6; the gray solid line is a linear fit revealing an initial binding rate of 6 ng/cm2/min. The protein mixtures were incubated with the HA films for 2 h. Subsequent binding of anti-PTX3 antibody, incubated at 0.08 μm, indicates successful incorporation of PTX3. The curve shown is representative of a set of measurements performed in duplicate. B, binding assay by QCM-D. Δf (blue lines) and ΔD (orange lines) are shown. Mixtures of PTX3, rhTSG-6, and IαI were exposed to HA films at final protein concentrations of 1.0, 0.6, and 0.2 μm, respectively. In one case (circles), PTX3 was first mixed with TSG-6 for 2 h and then with IαI for another 1 h (all at room temperature) before exposure to HA. In the other case (triangles), TSG-6 and IαI were mixed first (for 2 h), and then PTX3 was added (for 1 h) before exposure to HA. Clear QCM-D responses upon the subsequent addition of anti-PTX3 antibody, incubated at 0.08 μm, indicated successful incorporation of PTX3.
Mentions: The absence of PTX3 binding in the above-described assays disproves existing hypotheses about the mechanism by which PTX3 is incorporated into HA matrices (12, 48). On the other hand, PTX3 has been shown to be an essential component in the formation of the COC matrix (12). So how is PTX3 incorporated into HA films? We hypothesized that PTX3 must encounter IαI and/or TSG-6 prior to interaction with HA in order for PTX3 to be incorporated into HA assemblies. To test this hypothesis, we first added PTX3 and IαI at bulk concentrations of 0.3 and 1 μm, respectively, to an HA film. As anticipated, this mixture did not show any HA binding activity (Fig. 1C) (45). Second, TSG-6 was added at a bulk concentration of 0.3 μm. This protein did start a binding reaction (Fig. 5A). After 2 h of incubation, all proteins in the soluble phase were removed. Following this, the anti-PTX3 antibody (MNB4) did bind to the film. In contrast, the same antibody did not bind to an HA film that contained TSG-6 (Fig. 2B). This provides strong evidence that co-incubation of PTX3 in a ternary mixture with IαI and TSG-6 promotes PTX3 incorporation into the HA film.

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