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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 does not bind to HA films that had previously been exposed to a mixture of IαI/TSG-6.A, binding assays by SE. 1 μm IαI and 0.3 μm TSG-6 were sequentially added to the HA film without premixing. In this case, the film contains an additional fraction of non-covalently but stably bound protein (45). The gray solid line is a linear fit revealing an initial binding rate of 13 ng/cm2/min. Incubation with 0.3 μm PTX3 does not affect the surface density of the film. The lack of a significant response upon incubation with 0.08 μm anti-PTX3 antibody (MNB4) confirms the absence of PTX3 binding. The curve shown is representative of a set of measurements performed in duplicate. B, HA films are permeable to PTX3. b-PTX3 was added to SAv-covered surfaces without any further functionalization (solid line) or in the presence of HA (837 kDa) films with a surface density of 35 ± 5 ng/cm2. HA films were presented pure (dashed line) or following exposure to 0.3 μm rhTSG-6 (solid line with open circles) or to a mixture of 1 μm IαI and 0.3 μm TSG-6 (premixed for 1 min before the addition to the HA film; solid line with filled squares). Only initial binding is shown. Binding with similar rates is observed for all surfaces. Because PTX3 alone did not show binding on any of these surfaces, the binding of b-PTX3 must occur via the biotin moiety to SAv, indicating that all HA films are permeable to PTX3.
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Figure 4: PTX3 does not bind to HA films that had previously been exposed to a mixture of IαI/TSG-6.A, binding assays by SE. 1 μm IαI and 0.3 μm TSG-6 were sequentially added to the HA film without premixing. In this case, the film contains an additional fraction of non-covalently but stably bound protein (45). The gray solid line is a linear fit revealing an initial binding rate of 13 ng/cm2/min. Incubation with 0.3 μm PTX3 does not affect the surface density of the film. The lack of a significant response upon incubation with 0.08 μm anti-PTX3 antibody (MNB4) confirms the absence of PTX3 binding. The curve shown is representative of a set of measurements performed in duplicate. B, HA films are permeable to PTX3. b-PTX3 was added to SAv-covered surfaces without any further functionalization (solid line) or in the presence of HA (837 kDa) films with a surface density of 35 ± 5 ng/cm2. HA films were presented pure (dashed line) or following exposure to 0.3 μm rhTSG-6 (solid line with open circles) or to a mixture of 1 μm IαI and 0.3 μm TSG-6 (premixed for 1 min before the addition to the HA film; solid line with filled squares). Only initial binding is shown. Binding with similar rates is observed for all surfaces. Because PTX3 alone did not show binding on any of these surfaces, the binding of b-PTX3 must occur via the biotin moiety to SAv, indicating that all HA films are permeable to PTX3.

Mentions: A, proteins involved in HA matrix stabilization. Ternary and quaternary structures are schematically shown, with the sizes of all proteins and their subunits approximately to scale and known interactions indicated by arrows. Subunits that were used separately in addition to the complete protein are enclosed within dashed boxes. B, schematic illustration of the platform for solid-phase binding assays. A gold support was modified with a protein-repellent oligoethylene glycol monolayer functionalized with biotin, followed by the formation of a dense monolayer of well oriented SAv. HA, Link_TSG6, or PTX3 was grafted to the SAv layer via biotin tags. HA chains were site-specifically functionalized with biotin at their reducing end and could therefore be immobilized at controlled orientation. Proteins were functionalized through primary amines and therefore might be immobilized in a variety of orientations. The thickness of the oligoethylene glycol monolayer and the dimensions of SAv, Link_TSG6, and the PTX3 octamer are drawn approximately to scale; the thickness of the HA brush and the mean distance between HA anchor points are reduced by 10–20-fold for illustrative purposes. C, interaction of PTX3, IαI, and a mixture of IαI/PTX3 with HA film. A control shows that 1 μm IαI, 0.3 μm PTX3, and a mixture of these proteins do not bind to HA in the absence of TSG-6. The start and duration of the incubation with different samples are indicated (arrows). After each incubation step, the solution phase was replaced by buffer. QCM-D did not show any significant interaction between the HA film (58 kDa) and IαI, PTX3, or a PTX3/IαI mixture. The employed HA films are easily permeated by the proteins (cf.Fig. 4B). The control measurements therefore also confirm that our streptavidin-coated surfaces are resistant to nonspecific binding of IαI or PTX3, alone or in a mixture. D, interaction of surface-bound Link_TSG6 with octamer-forming wild type PTX3 and dimer-forming N_PTX3_MUT. Interactions were measured by QCM-D. Biotinylated Link_TSG6 (b-Link_TSG6) but not Link_TSG6 without biotin was immobilized to a streptavidin monolayer, and binding of PTX3 constructs was monitored. The bulk PTX3 concentration refers to the molar concentration of PTX3 monomers. E, interaction of surface-bound PTX3 with Link_TSG6 and rhTSG-6. Interactions were measured by SE. b-PTX3 was immobilized, and binding of Link_TSG6 and rhTSG-6 was monitored. F, interaction of surface-bound PTX3 with IαI. Interactions were measured by QCM-D. b-PTX3 was immobilized, and binding of IαI was monitored. The curves shown in C–F are representative of sets of measurements performed at least in duplicate.


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 does not bind to HA films that had previously been exposed to a mixture of IαI/TSG-6.A, binding assays by SE. 1 μm IαI and 0.3 μm TSG-6 were sequentially added to the HA film without premixing. In this case, the film contains an additional fraction of non-covalently but stably bound protein (45). The gray solid line is a linear fit revealing an initial binding rate of 13 ng/cm2/min. Incubation with 0.3 μm PTX3 does not affect the surface density of the film. The lack of a significant response upon incubation with 0.08 μm anti-PTX3 antibody (MNB4) confirms the absence of PTX3 binding. The curve shown is representative of a set of measurements performed in duplicate. B, HA films are permeable to PTX3. b-PTX3 was added to SAv-covered surfaces without any further functionalization (solid line) or in the presence of HA (837 kDa) films with a surface density of 35 ± 5 ng/cm2. HA films were presented pure (dashed line) or following exposure to 0.3 μm rhTSG-6 (solid line with open circles) or to a mixture of 1 μm IαI and 0.3 μm TSG-6 (premixed for 1 min before the addition to the HA film; solid line with filled squares). Only initial binding is shown. Binding with similar rates is observed for all surfaces. Because PTX3 alone did not show binding on any of these surfaces, the binding of b-PTX3 must occur via the biotin moiety to SAv, indicating that all HA films are permeable to PTX3.
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Figure 4: PTX3 does not bind to HA films that had previously been exposed to a mixture of IαI/TSG-6.A, binding assays by SE. 1 μm IαI and 0.3 μm TSG-6 were sequentially added to the HA film without premixing. In this case, the film contains an additional fraction of non-covalently but stably bound protein (45). The gray solid line is a linear fit revealing an initial binding rate of 13 ng/cm2/min. Incubation with 0.3 μm PTX3 does not affect the surface density of the film. The lack of a significant response upon incubation with 0.08 μm anti-PTX3 antibody (MNB4) confirms the absence of PTX3 binding. The curve shown is representative of a set of measurements performed in duplicate. B, HA films are permeable to PTX3. b-PTX3 was added to SAv-covered surfaces without any further functionalization (solid line) or in the presence of HA (837 kDa) films with a surface density of 35 ± 5 ng/cm2. HA films were presented pure (dashed line) or following exposure to 0.3 μm rhTSG-6 (solid line with open circles) or to a mixture of 1 μm IαI and 0.3 μm TSG-6 (premixed for 1 min before the addition to the HA film; solid line with filled squares). Only initial binding is shown. Binding with similar rates is observed for all surfaces. Because PTX3 alone did not show binding on any of these surfaces, the binding of b-PTX3 must occur via the biotin moiety to SAv, indicating that all HA films are permeable to PTX3.
Mentions: A, proteins involved in HA matrix stabilization. Ternary and quaternary structures are schematically shown, with the sizes of all proteins and their subunits approximately to scale and known interactions indicated by arrows. Subunits that were used separately in addition to the complete protein are enclosed within dashed boxes. B, schematic illustration of the platform for solid-phase binding assays. A gold support was modified with a protein-repellent oligoethylene glycol monolayer functionalized with biotin, followed by the formation of a dense monolayer of well oriented SAv. HA, Link_TSG6, or PTX3 was grafted to the SAv layer via biotin tags. HA chains were site-specifically functionalized with biotin at their reducing end and could therefore be immobilized at controlled orientation. Proteins were functionalized through primary amines and therefore might be immobilized in a variety of orientations. The thickness of the oligoethylene glycol monolayer and the dimensions of SAv, Link_TSG6, and the PTX3 octamer are drawn approximately to scale; the thickness of the HA brush and the mean distance between HA anchor points are reduced by 10–20-fold for illustrative purposes. C, interaction of PTX3, IαI, and a mixture of IαI/PTX3 with HA film. A control shows that 1 μm IαI, 0.3 μm PTX3, and a mixture of these proteins do not bind to HA in the absence of TSG-6. The start and duration of the incubation with different samples are indicated (arrows). After each incubation step, the solution phase was replaced by buffer. QCM-D did not show any significant interaction between the HA film (58 kDa) and IαI, PTX3, or a PTX3/IαI mixture. The employed HA films are easily permeated by the proteins (cf.Fig. 4B). The control measurements therefore also confirm that our streptavidin-coated surfaces are resistant to nonspecific binding of IαI or PTX3, alone or in a mixture. D, interaction of surface-bound Link_TSG6 with octamer-forming wild type PTX3 and dimer-forming N_PTX3_MUT. Interactions were measured by QCM-D. Biotinylated Link_TSG6 (b-Link_TSG6) but not Link_TSG6 without biotin was immobilized to a streptavidin monolayer, and binding of PTX3 constructs was monitored. The bulk PTX3 concentration refers to the molar concentration of PTX3 monomers. E, interaction of surface-bound PTX3 with Link_TSG6 and rhTSG-6. Interactions were measured by SE. b-PTX3 was immobilized, and binding of Link_TSG6 and rhTSG-6 was monitored. F, interaction of surface-bound PTX3 with IαI. Interactions were measured by QCM-D. b-PTX3 was immobilized, and binding of IαI was monitored. The curves shown in C–F are representative of sets of measurements performed at least in duplicate.

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