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Inter-α-inhibitor impairs TSG-6-induced hyaluronan cross-linking.

Baranova NS, Foulcer SJ, Briggs DC, Tilakaratna V, Enghild JJ, Milner CM, Day AJ, Richter RP - J. Biol. Chem. (2013)

Bottom Line: The other type of complex is novel and binds stably but noncovalently to HA.Prolonged incubation with TSG-6 and IαI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species.These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IαI will dictate its function.

View Article: PubMed Central - PubMed

Affiliation: From the Biosurfaces Unit, CIC biomaGUNE, 20009 Donostia-San Sebastian, Spain.

ABSTRACT
Under inflammatory conditions and in the matrix of the cumulus-oocyte complex, the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-α-inhibitor (IαI). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from IαI to HA is catalyzed by TSG-6 (tumor necrosis factor-stimulated gene-6), but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of IαI and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6-mediated cross-linking of HA films is impaired in the presence of IαI and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44-positive cells. Furthermore, we find that the interaction of TSG-6 and IαI in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HC·TSG-6 complexes. The other type of complex is novel and binds stably but noncovalently to HA. Prolonged incubation with TSG-6 and IαI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IαI will dictate its function.

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Changes in the thickness of HA films upon incubation with TSG-6 and IαI. Variations in the thickness of a HA (1,083 kDa) film as a function of incubation time were quantified by colloidal probe RICM. A, incubation with a mixture of 0.3 μm TSG-6 and 1 μm IαI (no premixing, open circles; 120 min premixing, filled triangles) resulted in a minor decrease in film thickness during the first 25 min after incubation. In contrast, incubation with TSG-6 alone, at the same concentration, induced pronounced condensation of the film within the same time (open squares). Film thicknesses remained virtually unaltered throughout the subsequent 2 h of incubation. B, addition of 1 μm IαI to the TSG-6-loaded film for 30 min led to a partial recovery of the original film thickness. The data are plotted as mean values of 10 independent measurements on the same surface (± S.E.). The film thickness did not change significantly upon prolonged incubation of IαI (i.e., for a total of 120 min; data not shown).
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Figure 6: Changes in the thickness of HA films upon incubation with TSG-6 and IαI. Variations in the thickness of a HA (1,083 kDa) film as a function of incubation time were quantified by colloidal probe RICM. A, incubation with a mixture of 0.3 μm TSG-6 and 1 μm IαI (no premixing, open circles; 120 min premixing, filled triangles) resulted in a minor decrease in film thickness during the first 25 min after incubation. In contrast, incubation with TSG-6 alone, at the same concentration, induced pronounced condensation of the film within the same time (open squares). Film thicknesses remained virtually unaltered throughout the subsequent 2 h of incubation. B, addition of 1 μm IαI to the TSG-6-loaded film for 30 min led to a partial recovery of the original film thickness. The data are plotted as mean values of 10 independent measurements on the same surface (± S.E.). The film thickness did not change significantly upon prolonged incubation of IαI (i.e., for a total of 120 min; data not shown).

Mentions: Next, we studied how the covalent modification of HA films by HCs influences the morphology of HA. We had reported earlier that TSG-6, when presented alone, cross-links HA and induces HA film shrinkage (4). Indeed, addition of TSG-6 at 0.3 μm resulted in a strong decrease of the film thickness by more than 60% (Fig. 6). In contrast, the thickness of the HA film decreased only ∼20%, when TSG-6 was co-incubated with 1 μm IαI. Apparently, the cross-linking properties of TSG-6 are impaired in the presence of IαI. Beyond 30 min of incubation, the thickness remained virtually constant (Fig. 6A). The TSG-6/IαI premixing time had no significant influence on the film thickness in the co-incubation assays. However, the film only partially recovered, to 65% of its original thickness, when an already TSG-6 cross-linked HA film was exposed to IαI (Fig. 6B).


Inter-α-inhibitor impairs TSG-6-induced hyaluronan cross-linking.

Baranova NS, Foulcer SJ, Briggs DC, Tilakaratna V, Enghild JJ, Milner CM, Day AJ, Richter RP - J. Biol. Chem. (2013)

Changes in the thickness of HA films upon incubation with TSG-6 and IαI. Variations in the thickness of a HA (1,083 kDa) film as a function of incubation time were quantified by colloidal probe RICM. A, incubation with a mixture of 0.3 μm TSG-6 and 1 μm IαI (no premixing, open circles; 120 min premixing, filled triangles) resulted in a minor decrease in film thickness during the first 25 min after incubation. In contrast, incubation with TSG-6 alone, at the same concentration, induced pronounced condensation of the film within the same time (open squares). Film thicknesses remained virtually unaltered throughout the subsequent 2 h of incubation. B, addition of 1 μm IαI to the TSG-6-loaded film for 30 min led to a partial recovery of the original film thickness. The data are plotted as mean values of 10 independent measurements on the same surface (± S.E.). The film thickness did not change significantly upon prolonged incubation of IαI (i.e., for a total of 120 min; data not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3795262&req=5

Figure 6: Changes in the thickness of HA films upon incubation with TSG-6 and IαI. Variations in the thickness of a HA (1,083 kDa) film as a function of incubation time were quantified by colloidal probe RICM. A, incubation with a mixture of 0.3 μm TSG-6 and 1 μm IαI (no premixing, open circles; 120 min premixing, filled triangles) resulted in a minor decrease in film thickness during the first 25 min after incubation. In contrast, incubation with TSG-6 alone, at the same concentration, induced pronounced condensation of the film within the same time (open squares). Film thicknesses remained virtually unaltered throughout the subsequent 2 h of incubation. B, addition of 1 μm IαI to the TSG-6-loaded film for 30 min led to a partial recovery of the original film thickness. The data are plotted as mean values of 10 independent measurements on the same surface (± S.E.). The film thickness did not change significantly upon prolonged incubation of IαI (i.e., for a total of 120 min; data not shown).
Mentions: Next, we studied how the covalent modification of HA films by HCs influences the morphology of HA. We had reported earlier that TSG-6, when presented alone, cross-links HA and induces HA film shrinkage (4). Indeed, addition of TSG-6 at 0.3 μm resulted in a strong decrease of the film thickness by more than 60% (Fig. 6). In contrast, the thickness of the HA film decreased only ∼20%, when TSG-6 was co-incubated with 1 μm IαI. Apparently, the cross-linking properties of TSG-6 are impaired in the presence of IαI. Beyond 30 min of incubation, the thickness remained virtually constant (Fig. 6A). The TSG-6/IαI premixing time had no significant influence on the film thickness in the co-incubation assays. However, the film only partially recovered, to 65% of its original thickness, when an already TSG-6 cross-linked HA film was exposed to IαI (Fig. 6B).

Bottom Line: The other type of complex is novel and binds stably but noncovalently to HA.Prolonged incubation with TSG-6 and IαI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species.These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IαI will dictate its function.

View Article: PubMed Central - PubMed

Affiliation: From the Biosurfaces Unit, CIC biomaGUNE, 20009 Donostia-San Sebastian, Spain.

ABSTRACT
Under inflammatory conditions and in the matrix of the cumulus-oocyte complex, the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-α-inhibitor (IαI). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from IαI to HA is catalyzed by TSG-6 (tumor necrosis factor-stimulated gene-6), but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of IαI and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6-mediated cross-linking of HA films is impaired in the presence of IαI and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44-positive cells. Furthermore, we find that the interaction of TSG-6 and IαI in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HC·TSG-6 complexes. The other type of complex is novel and binds stably but noncovalently to HA. Prolonged incubation with TSG-6 and IαI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IαI will dictate its function.

Show MeSH
Related in: MedlinePlus