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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 inhibits the catalytic activity of TSG-6 in HC transfer. In a solution-phase assay, 0.27 μm TSG-6, 1.8 μm IαI, and 20 μm b-HA10 were co-incubated with or without 1.8 μm PTX3 for various times and subsequently analyzed by Western blots with streptavidin-conjugated Alexa 488, which recognizes biotin in b-HA10·HC complexes. A, Western blot with co-incubation times indicated. B, densitometric analysis of Western blots. Error bars, S.E. from three blots. Data are representative of two independent experiments.
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Figure 7: PTX3 inhibits the catalytic activity of TSG-6 in HC transfer. In a solution-phase assay, 0.27 μm TSG-6, 1.8 μm IαI, and 20 μm b-HA10 were co-incubated with or without 1.8 μm PTX3 for various times and subsequently analyzed by Western blots with streptavidin-conjugated Alexa 488, which recognizes biotin in b-HA10·HC complexes. A, Western blot with co-incubation times indicated. B, densitometric analysis of Western blots. Error bars, S.E. from three blots. Data are representative of two independent experiments.

Mentions: To test this hypothesis, we performed complementary HC transfer assays in solution, in which catalytic amounts of TSG-6 (28) were co-incubated with IαI and a biotinylated HA oligosaccharide (b-HA10) in the absence/presence of PTX3 (Fig. 7). In the absence of PTX3, transfer rates remained virtually unchanged throughout the total 4-h incubation time (based on the visualization of b-HA10·HC species; see Fig. 7A), consistent with the linear response in Fig. 6B. In contrast, in the presence of PTX3, transfer proceeded throughout the first hour but then essentially halted. These data provide independent confirmation that PTX3 reduces the ability of TSG-6 to transfer heavy chains and are fully consistent with our proposal that PTX3 inhibits recycling of TSG-6. It should be noted that the experiments in Figs. 6 and 7 were conducted at different temperatures (24 and 37 °C, respectively) and that the rate of encounter between HA and the proteins is higher with HA in the solution phase, which probably explains the different time scales over which this inhibition was observed.


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 inhibits the catalytic activity of TSG-6 in HC transfer. In a solution-phase assay, 0.27 μm TSG-6, 1.8 μm IαI, and 20 μm b-HA10 were co-incubated with or without 1.8 μm PTX3 for various times and subsequently analyzed by Western blots with streptavidin-conjugated Alexa 488, which recognizes biotin in b-HA10·HC complexes. A, Western blot with co-incubation times indicated. B, densitometric analysis of Western blots. Error bars, S.E. from three blots. Data are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: PTX3 inhibits the catalytic activity of TSG-6 in HC transfer. In a solution-phase assay, 0.27 μm TSG-6, 1.8 μm IαI, and 20 μm b-HA10 were co-incubated with or without 1.8 μm PTX3 for various times and subsequently analyzed by Western blots with streptavidin-conjugated Alexa 488, which recognizes biotin in b-HA10·HC complexes. A, Western blot with co-incubation times indicated. B, densitometric analysis of Western blots. Error bars, S.E. from three blots. Data are representative of two independent experiments.
Mentions: To test this hypothesis, we performed complementary HC transfer assays in solution, in which catalytic amounts of TSG-6 (28) were co-incubated with IαI and a biotinylated HA oligosaccharide (b-HA10) in the absence/presence of PTX3 (Fig. 7). In the absence of PTX3, transfer rates remained virtually unchanged throughout the total 4-h incubation time (based on the visualization of b-HA10·HC species; see Fig. 7A), consistent with the linear response in Fig. 6B. In contrast, in the presence of PTX3, transfer proceeded throughout the first hour but then essentially halted. These data provide independent confirmation that PTX3 reduces the ability of TSG-6 to transfer heavy chains and are fully consistent with our proposal that PTX3 inhibits recycling of TSG-6. It should be noted that the experiments in Figs. 6 and 7 were conducted at different temperatures (24 and 37 °C, respectively) and that the rate of encounter between HA and the proteins is higher with HA in the solution phase, which probably explains the different time scales over which this inhibition was observed.

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