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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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Ternary interaction of HA, TSG-6, and IαI as a function of TSG-6/IαI premixing time. TSG-6 at 0.3 μm was exposed to HA (837 kDa) films either alone or in a mixture with 1 μm IαI for 2 h. Proteins were premixed ex situ for different periods of time (0–120 min). A, initial rate of binding to the HA film. B, protein fractions that were resistant to elution in 2 m GuHCl, as percentages of total bound protein. The data plotted correspond to one measurement or to the mean of two or three independent measurements (with error bars indicating maximal/minimal measured values).
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Figure 4: Ternary interaction of HA, TSG-6, and IαI as a function of TSG-6/IαI premixing time. TSG-6 at 0.3 μm was exposed to HA (837 kDa) films either alone or in a mixture with 1 μm IαI for 2 h. Proteins were premixed ex situ for different periods of time (0–120 min). A, initial rate of binding to the HA film. B, protein fractions that were resistant to elution in 2 m GuHCl, as percentages of total bound protein. The data plotted correspond to one measurement or to the mean of two or three independent measurements (with error bars indicating maximal/minimal measured values).

Mentions: Intrigued by the large differences observed between the co-incubation assays with 120 min and those without premixing (Fig. 3A), we tested how the premixing time influences the initial binding rate of protein into the HA film (Fig. 4). With increasing premixing time, the initial binding rate of the TSG-6/IαI mixture decreased rapidly (Fig. 4A). Already after 1 min of premixing, the initial binding rate had reached levels comparable to those observed for 120 min. The fraction of very tightly bound material (i.e., stable to 2 m GuHCl), on the other hand, was only influenced to a small extent; i.e., with ∼70% bound at 0 and 1 min premixing time and ∼85% at 15 and 120 min (Fig. 4B). Apparently, there is a short time window for premixing, not longer than a few minutes, that influences the amount of incorporation of material into HA films.


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)

Ternary interaction of HA, TSG-6, and IαI as a function of TSG-6/IαI premixing time. TSG-6 at 0.3 μm was exposed to HA (837 kDa) films either alone or in a mixture with 1 μm IαI for 2 h. Proteins were premixed ex situ for different periods of time (0–120 min). A, initial rate of binding to the HA film. B, protein fractions that were resistant to elution in 2 m GuHCl, as percentages of total bound protein. The data plotted correspond to one measurement or to the mean of two or three independent measurements (with error bars indicating maximal/minimal measured values).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Ternary interaction of HA, TSG-6, and IαI as a function of TSG-6/IαI premixing time. TSG-6 at 0.3 μm was exposed to HA (837 kDa) films either alone or in a mixture with 1 μm IαI for 2 h. Proteins were premixed ex situ for different periods of time (0–120 min). A, initial rate of binding to the HA film. B, protein fractions that were resistant to elution in 2 m GuHCl, as percentages of total bound protein. The data plotted correspond to one measurement or to the mean of two or three independent measurements (with error bars indicating maximal/minimal measured values).
Mentions: Intrigued by the large differences observed between the co-incubation assays with 120 min and those without premixing (Fig. 3A), we tested how the premixing time influences the initial binding rate of protein into the HA film (Fig. 4). With increasing premixing time, the initial binding rate of the TSG-6/IαI mixture decreased rapidly (Fig. 4A). Already after 1 min of premixing, the initial binding rate had reached levels comparable to those observed for 120 min. The fraction of very tightly bound material (i.e., stable to 2 m GuHCl), on the other hand, was only influenced to a small extent; i.e., with ∼70% bound at 0 and 1 min premixing time and ∼85% at 15 and 120 min (Fig. 4B). Apparently, there is a short time window for premixing, not longer than a few minutes, that influences the amount of incorporation of material into HA films.

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