Incorporation of pentraxin 3 into hyaluronan matrices is tightly regulated and promotes matrix cross-linking.
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.
Affiliation: From the CIC biomaGUNE, 20009 Donostia-San Sebastian, Spain.
- C-Reactive Protein/chemistry*
- Extracellular Matrix/chemistry/physiology*
- Hyaluronic Acid/chemistry*
- Serum Amyloid P-Component/chemistry*
- Cell Adhesion Molecules/chemistry
- Cell Line
- Drosophila melanogaster
- Ovarian Follicle/metabolism
- Protein Binding
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Figure 8: Western blot analysis of protein material incorporated into HA films.A–C, HA films were incubated with proteins in binary (IαI/TSG-6) and ternary (IαI/TSG-6/PTX3) mixtures, as shown in Figs. 4A and 5, respectively. Western blots were made from fractions obtained by stepwise elution with 2 and 8 m GdnHCl and by digestion with hyaluronidase (HA-ase). Collected material was analyzed by Western blots with anti-PTX3 (A), anti-IαI (B), and anti-TSG-6 (C) antibodies. The control reaction mix of TSG-6, HA14, and IαI is expected to contain a total amount of 100 ng of TSG-6 and 25 ng of IαI, the control lane for PTX3 is expected to contain 500 pg of PTX3, and the detection limits are estimated to be around 5 ng for TSG-6, 0.5 ng for IαI, and 50 pg for PTX3. D, direct comparison of IαI and TSG-6 proteins running in 4–12% NuPAGE BisTris gels (used in B and C) and 10% Tris/Tricine gels (used by Rugg et al. (28)), stained with Coomassie Blue. The same standards were used for both gel types (as indicated), and their apparent molecular masses were assigned following the manufacturer's indications for NuPAGE BisTris 4–12 with MES (as in B and C) and Tris-glycine gels (as in Ref. 28), respectively. IαI and TSG-6 were mixed (1.8 and 2.7 μm, respectively) and co-incubated at 4 °C using the standard conditions (as described in Ref. 28). Immediately after mixing (0 h), bands for intact IαI and TSG-6 are dominant; after 2 h of co-incubation, additional bands for HC·bikunin/HC·TSG-6 appear. In the 10% Tris/Tricine gel, IαI, HC·bikunin, the HC·TSG-6 doublet, and TSG-6 run at apparent molecular masses of ∼220, ∼130, ∼120, and ∼38 kDa, respectively, consistent with Rugg et al. (28), where these had been identified by Edman degradation and mass spectrometry. In the 4–12% NuPAGE BisTris gels, the apparent molecular masses for IαI and TSG-6 are ∼170 and ∼34 kDa, respectively; the HC·bikunin and HC·TSG-6 species run together with an apparent molecular mass of ∼98 kDa. HMW IαI, a high molecular weight form of IαI with three or four HCs attached, which is a minor species within the IαI preparation purified from serum that also forms as a by-product of HC·TSG-6 complex formation (see Ref. 29).
To analyze the composition of the HA film after protein incubation, we performed Western blot analyses (with anti-PTX3, anti-TSG-6, and anti-IαI antibodies) of material collected from the incubation assays in Figs. 4A and 5 after exposure sequentially to 2 m GdnHCl, 8 m GdnHCl, and hyaluronidase. The anti-PTX3 antibody MNB4 (Fig. 8A) revealed the two strongest bands to be in the 8 m GdnHCl eluate from the ternary protein mixture; the apparent molecular masses of these bands (about 45 and 90 kDa) were identical to that of the PTX3 monomer and dimer that are present in a control sample. No bands were found at these positions in the 2 m GdnHCl eluates and the hyaluronidase digests (or the 8 m GdnHCl eluate from the IαI/TSG-6 binary mixture). This indicates that most, if not all, PTX3 is very tightly yet non-covalently bound to the HA matrix. Some immunoreactive bands were observed at 55 and 28 kDa in samples from 2 m GdnHCl washes (Fig. 8A, labeled as a and b, respectively). These might correspond to the heavy and light chains, respectively, of antibody leftovers in the measurement chamber from the injections performed in Figs. 4A and 5. Also a band at about 65 kDa (Fig. 8A, labeled as c) is present, probably due to sample contamination with BSA, that is nonspecifically recognized by the applied antibody.