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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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TSG-6 interacts with high affinity with HC1, HC2, and HC3.A–F, representative figures of TSG-6 (analyte) interacting with immobilized rHC1 (A and B), rHC2 (C and D), and rHC3 (E and F) shown by surface plasmon resonance in HBS-T with 1 mm MgCl2 and 1 mm CaCl2 (A, C, and E) or in HBS-T with 10 mm EDTA (B, D, and F). Raw data and curve fits (with a 1:1 Langmuir model) are shown for TSG-6 concentrations of 6.25, 12.5, 25, 50, and 100 nm. KD values were determined from the mean of three experiments ± standard deviation. Stepwise changes in response at the end of sample injection are due to RI changes upon solution exchange, modeled by BiaEval 1:1 Langmuir model fitting. Other binding models tested did not improve the fit significantly. G, SDS-PAGE analysis of purified recombinant rHC1, rHC2, and rHC3 (each with an N-terminal His-tag (MAHHHHHHVGTGSNDDDDKSPDP)). 3 μg of rHC1, rHC2, and rHC3, respectively, were loaded per lane and run under reducing conditions, indicating that these preparations are >95% pure.
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Figure 2: TSG-6 interacts with high affinity with HC1, HC2, and HC3.A–F, representative figures of TSG-6 (analyte) interacting with immobilized rHC1 (A and B), rHC2 (C and D), and rHC3 (E and F) shown by surface plasmon resonance in HBS-T with 1 mm MgCl2 and 1 mm CaCl2 (A, C, and E) or in HBS-T with 10 mm EDTA (B, D, and F). Raw data and curve fits (with a 1:1 Langmuir model) are shown for TSG-6 concentrations of 6.25, 12.5, 25, 50, and 100 nm. KD values were determined from the mean of three experiments ± standard deviation. Stepwise changes in response at the end of sample injection are due to RI changes upon solution exchange, modeled by BiaEval 1:1 Langmuir model fitting. Other binding models tested did not improve the fit significantly. G, SDS-PAGE analysis of purified recombinant rHC1, rHC2, and rHC3 (each with an N-terminal His-tag (MAHHHHHHVGTGSNDDDDKSPDP)). 3 μg of rHC1, rHC2, and rHC3, respectively, were loaded per lane and run under reducing conditions, indicating that these preparations are >95% pure.

Mentions: To better understand the possible interactions between the involved proteins, we analyzed binding of recombinantly produced HC1, HC2, and HC3 (rHC1, rHC2, and rHC3), individually immobilized on a C1 chip, to TSG-6 (the analyte) by surface plasmon resonance (Fig. 2, A–F); SDS-PAGE of purified rHC proteins is shown in Fig. 2G. Analysis of the binding data revealed that all three HCs interact rapidly and strongly with TSG-6, in a divalent cation-independent manner (i.e., in the presence of either Ca2+/Mg2+ or EDTA), each with a dissociation constant (KD) of 10 nm. On rates and off rates for the binding of the three HCs to TSG-6 were all very similar (kon = 1 × 105m−1 s−1, koff = 7 × 10−4 s−1), suggesting that they all bind (via conserved residues) to a common interaction site on TSG-6.


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)

TSG-6 interacts with high affinity with HC1, HC2, and HC3.A–F, representative figures of TSG-6 (analyte) interacting with immobilized rHC1 (A and B), rHC2 (C and D), and rHC3 (E and F) shown by surface plasmon resonance in HBS-T with 1 mm MgCl2 and 1 mm CaCl2 (A, C, and E) or in HBS-T with 10 mm EDTA (B, D, and F). Raw data and curve fits (with a 1:1 Langmuir model) are shown for TSG-6 concentrations of 6.25, 12.5, 25, 50, and 100 nm. KD values were determined from the mean of three experiments ± standard deviation. Stepwise changes in response at the end of sample injection are due to RI changes upon solution exchange, modeled by BiaEval 1:1 Langmuir model fitting. Other binding models tested did not improve the fit significantly. G, SDS-PAGE analysis of purified recombinant rHC1, rHC2, and rHC3 (each with an N-terminal His-tag (MAHHHHHHVGTGSNDDDDKSPDP)). 3 μg of rHC1, rHC2, and rHC3, respectively, were loaded per lane and run under reducing conditions, indicating that these preparations are >95% pure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: TSG-6 interacts with high affinity with HC1, HC2, and HC3.A–F, representative figures of TSG-6 (analyte) interacting with immobilized rHC1 (A and B), rHC2 (C and D), and rHC3 (E and F) shown by surface plasmon resonance in HBS-T with 1 mm MgCl2 and 1 mm CaCl2 (A, C, and E) or in HBS-T with 10 mm EDTA (B, D, and F). Raw data and curve fits (with a 1:1 Langmuir model) are shown for TSG-6 concentrations of 6.25, 12.5, 25, 50, and 100 nm. KD values were determined from the mean of three experiments ± standard deviation. Stepwise changes in response at the end of sample injection are due to RI changes upon solution exchange, modeled by BiaEval 1:1 Langmuir model fitting. Other binding models tested did not improve the fit significantly. G, SDS-PAGE analysis of purified recombinant rHC1, rHC2, and rHC3 (each with an N-terminal His-tag (MAHHHHHHVGTGSNDDDDKSPDP)). 3 μg of rHC1, rHC2, and rHC3, respectively, were loaded per lane and run under reducing conditions, indicating that these preparations are >95% pure.
Mentions: To better understand the possible interactions between the involved proteins, we analyzed binding of recombinantly produced HC1, HC2, and HC3 (rHC1, rHC2, and rHC3), individually immobilized on a C1 chip, to TSG-6 (the analyte) by surface plasmon resonance (Fig. 2, A–F); SDS-PAGE of purified rHC proteins is shown in Fig. 2G. Analysis of the binding data revealed that all three HCs interact rapidly and strongly with TSG-6, in a divalent cation-independent manner (i.e., in the presence of either Ca2+/Mg2+ or EDTA), each with a dissociation constant (KD) of 10 nm. On rates and off rates for the binding of the three HCs to TSG-6 were all very similar (kon = 1 × 105m−1 s−1, koff = 7 × 10−4 s−1), suggesting that they all bind (via conserved residues) to a common interaction site on TSG-6.

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