The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.
Introduction
In the majority of mammals, ovulation is immediately preceded by the formation of a viscoelastic extracellular matrix by the cumulus cells that surround the oocyte (
1.
, 2.
). The production of this matrix drives the expansion of the cumulus cell-oocyte complex (COC), 3
protecting the COC during its expulsion from the follicle, allowing its pickup and transport by the oviduct and providing a large surface area that facilitates sperm capture in vivo (The abbreviations used are:
COC
cumulus-oocyte complex
CSchondroitin sulfate
CUBcomplement C1r/C1s, Uegf, and BMP1
CUB_CCUB module and C-terminal peptide from human TSG-6
HAhyaluronan
bHA10HA decasaccharide biotinylated at the reducing end
HCheavy chain
rHC1 and rHC2recombinant HC1 and HC2, respectively
rhTSG-6recombinant human TSG-6
HC·HAhyaluronan with heavy chains covalently attached
HC·TSG-6covalent complex of TSG-6 and a heavy chain
IαIinter-α-inhibitor
Link_TSG6recombinant Link module from human TSG-6
SPRsurface plasmon resonance
TricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
MIDASmetal ion-dependent adhesion site.
3.
, 4.
5.
). The high molecular weight polysaccharide hyaluronan (HA) is a key structural component of the cumulus matrix; this non-sulfated glycosaminoglycan, composed entirely of repeating disaccharides of glucuronic acid and N-acetyl glucosamine, is organized into a cross-linked network during cumulus expansion, providing stability and the required mechanical properties of the COC (6.
, 7.
8.
). There is compelling evidence that this HA-rich matrix is stabilized through the cooperative action of inter-α-inhibitor (IαI), pentraxin-3, and TSG-6 (TNF-stimulated gene-6) (9.
10.
, 11.
, 12.
, 13.
, 14.
15.
); all three of these glycoproteins have been implicated as being essential, in the mouse at least (- Salustri A.
- Garlanda C.
- Hirsch E.
- De Acetis M.
- Maccagno A.
- Bottazzi B.
- Doni A.
- Bastone A.
- Mantovani G.
- Beck Peccoz P.
- Salvatori G.
- Mahoney D.J.
- Day A.J.
- Siracusa G.
- Romani L.
- Mantovani A.
PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization.
Development. 2004; 131: 1577-1586
2.
), because functional depletion/inhibition of any one of them greatly impairs COC expansion, leading to female infertility. Recent biophysical analysis has also demonstrated that human IαI, pentraxin-3, and TSG-6 are sufficient for the formation of a cross-linked matrix in model HA films (16.
). Most of these components (i.e. HA, pentraxin-3, and TSG-6) are produced by the cumulus cells in response to the gonadotropin surge (11.
, 15.
, - Salustri A.
- Garlanda C.
- Hirsch E.
- De Acetis M.
- Maccagno A.
- Bottazzi B.
- Doni A.
- Bastone A.
- Mantovani G.
- Beck Peccoz P.
- Salvatori G.
- Mahoney D.J.
- Day A.J.
- Siracusa G.
- Romani L.
- Mantovani A.
PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization.
Development. 2004; 131: 1577-1586
17.
, 18.
), which also leads to the altered permeability of the blood-follicle barrier, allowing IαI to enter from the blood (1.
).IαI is composed of three protein chains (bikinun, heavy chain 1 (HC1), and HC2) that are held together covalently by a chondroitin sulfate (CS) chain (
19.
, 20.
21.
); the CS, which contains both sulfated and non-sulfated regions (22.
, 23.
24.
), is attached to bikunin via a standard glycosaminoglycan linkage, and the HCs are attached to this proteoglycan via ester bonds between their C-terminal aspartic acid residues and C6-hydroxylates of N-acetyl galactosamine sugars of the CS chain (25.
). Importantly, both HC1 and HC2 of IαI can be covalently transferred onto the C6-hydroxyls of the N-acetyl glucosamine sugars in HA to form HC·HA, which are sometimes referred to as SHAP-HA (26.
27.
, 28.
29.
). The formation of these complexes, in which HA is probably decorated with multiple HCs (30.
), is essential for ovulation/fertilization; deletion of the bikunin gene, which abolishes the biosynthesis of IαI and consequently the production of HC·HA, leads to a lack of cumulus expansion and greatly impaired fertility in mice (9.
, 10.
).Deletion of pentraxin-3 also impairs the incorporation of HA into the COC matrix but does not affect the formation of HC·HA (
15.
). It is likely that pentraxin-3, which has no inherent HA-binding activity (- Salustri A.
- Garlanda C.
- Hirsch E.
- De Acetis M.
- Maccagno A.
- Bottazzi B.
- Doni A.
- Bastone A.
- Mantovani G.
- Beck Peccoz P.
- Salvatori G.
- Mahoney D.J.
- Day A.J.
- Siracusa G.
- Romani L.
- Mantovani A.
PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization.
Development. 2004; 131: 1577-1586
15.
, - Salustri A.
- Garlanda C.
- Hirsch E.
- De Acetis M.
- Maccagno A.
- Bottazzi B.
- Doni A.
- Bastone A.
- Mantovani G.
- Beck Peccoz P.
- Salvatori G.
- Mahoney D.J.
- Day A.J.
- Siracusa G.
- Romani L.
- Mantovani A.
PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization.
Development. 2004; 131: 1577-1586
16.
), contributes to cross-linking of HC·HA complexes via interactions with the attached HC (31.
); pentraxin-3 is an octameric protein composed of eight identical subunits (connected by disulfide bonds) through which it may bind simultaneously to multiple HCs linking HC·HA complexes together (16.
, 32.
, - Inforzato A.
- Rivieccio V.
- Morreale A.P.
- Bastone A.
- Salustri A.
- Scarchilli L.
- Verdoliva A.
- Vincenti S.
- Gallo G.
- Chiapparino C.
- Pacello L.
- Nucera E.
- Serlupi-Crescenzi O.
- Day A.J.
- Bottazzi B.
- Mantovani A.
- De Santis R.
- Salvatori G.
Structural characterization of PTX3 disulphide bond network and its multimeric status in cumulus matrix organization.
J. Biol. Chem. 2008; 283: 10147-10161
33.
- Inforzato A.
- Baldock C.
- Jowitt T.A.
- Holmes D.F.
- Lindstedt R.
- Marcellini M.
- Rivieccio V.
- Briggs D.C.
- Kadler K.E.
- Verdoliva A.
- Bottazzi B.
- Mantovani A.
- Salvatori G.
- Day A.J.
The angiogenic inhibitor long pentraxin PTX3 forms an asymmetric octamer with two binding sites for FGF2.
J. Biol. Chem. 2010; 285: 17681-17692
34.
).TSG-6 is a 35-kDa (single chain) protein composed mainly of contiguous Link and CUB modules that are flanked by N- and C-terminal sequences of 18 and 29 amino acids, respectively (
35.
, 36.
). It has been found to be crucial in the formation of HC·HA, and the COCs from TSG-6−/− mice (that failed to expand) contained no detectable HC·HA complexes (12.
). TSG-6 was shown to play a direct role in the transfer of HCs from IαI onto HA via the formation of covalent intermediates (HC1·TSG-6 and HC2·TSG-6) and act as a catalyst in this process (37.
). These HC·TSG-6 complexes (37.
, 38.
) are linked through ester bonds between Ser-28 of TSG-6 (in its N-terminal region) and the C-terminal aspartates of the HCs mentioned above (39.
); free HCs are unable to form these complexes with TSG-6 (or its individual domains) because this requires the presence of ester bonds connecting HCs to the CS chain of bikunin that are made during the biosynthesis of IαI (see Ref. 37.
).Thus, it is clear that the formation of HC·HA involves two sequential transesterification reactions (
37.
, 39.
, 40.
). However, beyond this, the molecular bases of HC·TSG-6 complex formation and HC transfer onto HA are not particularly well understood. It is known that these processes are divalent cation-dependent, but there is a lack of consensus on the identity of metal ions required and their locations within the IαI and TSG-6 proteins (37.
, 40.
, 41.
42.
); the TSG-6 CUB module has been predicted to contain an Mg2+-binding site based on homology with other CUB domains (37.
). Furthermore, it is far from clear how IαI and TSG-6 interact leading up to the formation of the HC·TSG-6 intermediates (40.
, 43.
, 44.
) or indeed how HA is recognized by these complexes during HC transfer (45.
). For example, full-length TSG-6 has been shown to interact (non-covalently) with bikunin·CS as well as HC1 and HC2 (- Higman V.A.
- Briggs D.C.
- Mahoney D.J.
- Blundell C.D.
- Sattelle B.M.
- Dyer D.P.
- Green D.E.
- DeAngelis P.L.
- Almond A.
- Milner C.M.
- Day A.J.
A refined model for the TSG-6 Link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.
J. Biol. Chem. 2014; 289: 5619-5634
40.
, 44.
, 46.
). The former is probably mediated (at least in part) through the binding of the Link module to the CS moiety (40.
), consistent with its ability to bind to CS and the non-sulfated glycosaminoglycan chondroitin (45.
, - Higman V.A.
- Briggs D.C.
- Mahoney D.J.
- Blundell C.D.
- Sattelle B.M.
- Dyer D.P.
- Green D.E.
- DeAngelis P.L.
- Almond A.
- Milner C.M.
- Day A.J.
A refined model for the TSG-6 Link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.
J. Biol. Chem. 2014; 289: 5619-5634
47.
); however, the region of TSG-6 that interacts with the HCs is not known. The TSG-6 Link module, for which NMR and x-ray structures are determined (48.
, 49.
50.
), also mediates the interaction of TSG-6 with HA (see Ref. 51.
). However, the well characterized HA-binding groove in TSG-6 (45.
, - Higman V.A.
- Briggs D.C.
- Mahoney D.J.
- Blundell C.D.
- Sattelle B.M.
- Dyer D.P.
- Green D.E.
- DeAngelis P.L.
- Almond A.
- Milner C.M.
- Day A.J.
A refined model for the TSG-6 Link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.
J. Biol. Chem. 2014; 289: 5619-5634
49.
, 52.
) may not be used for HA recognition by the HC·TSG-6 complexes during HC transfer (45.
).- Higman V.A.
- Briggs D.C.
- Mahoney D.J.
- Blundell C.D.
- Sattelle B.M.
- Dyer D.P.
- Green D.E.
- DeAngelis P.L.
- Almond A.
- Milner C.M.
- Day A.J.
A refined model for the TSG-6 Link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.
J. Biol. Chem. 2014; 289: 5619-5634
TSG-6 also binds directly to pentraxin-3 using a site on the Link module that does not overlap with its HA-binding surface, leading to the hypothesis that pentraxin-3-TSG-6 complexes could cross-link HA chains (
15.
). Although this is apparently not the case for full-length TSG-6 (- Salustri A.
- Garlanda C.
- Hirsch E.
- De Acetis M.
- Maccagno A.
- Bottazzi B.
- Doni A.
- Bastone A.
- Mantovani G.
- Beck Peccoz P.
- Salvatori G.
- Mahoney D.J.
- Day A.J.
- Siracusa G.
- Romani L.
- Mantovani A.
PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization.
Development. 2004; 131: 1577-1586
16.
), it has not been ruled out that TSG-6 can play a direct structural role in the organization of the cumulus matrix via its HA-binding properties (see Refs. 12.
, 13.
, 51.
, 53.
, and 54.
).It is worthy of mention that HC·HA complexes are also formed in contexts other than ovulation and that, at present, TSG-6 is the only known transferase that can mediate their production. Most likely this is an ancient process in vertebrates, which predates cumulus expansion (
55.
). HC·HA complexes form wherever IαI, TSG-6, and HA come into contact (see Refs. 37.
and 55.
). Given that in most tissues, TSG-6 is only expressed during inflammation (36.
, 56.
), it is not surprising that HC·HA complexes are most often associated with inflammatory processes and disease (30.
, 57.
58.
, 59.
60.
). Current evidence suggests that decoration of HA with HCs has an important role in modulating cell adhesion and cell phenotype (44.
, 61.
, 62.
) and that certain HC·HA complexes can mediate protective effects (62.
63.
, 64.
, 65.
66.
). The precise activity of HC·HA complexes (and whether they are protective or pathological) will probably depend on their exact composition (e.g. number and type of HCs, size of HA, etc.) and the identity of other associated structural/signaling molecules (44.
).Here we report the tertiary structure of the CUB module from human TSG-6 and the determination of its role in HC transfer. We have also clarified which metal ions are required for HC·TSG-6 complex formation, identifying divalent cation-binding sites in both IαI and TSG-6 that mediate an initial non-covalent interaction. Furthermore, we have demonstrated that it is the HC transferase activity of TSG-6, rather than HA binding, that is crucial for murine COC expansion.
Discussion
Here, through combined structural and biophysical approaches, we have determined the role of metal ions in the formation of covalent HC·TSG-6 complexes that act as intermediates in HC transfer onto HA. We have also found that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the HA-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.
As illustrated in Fig. 10, HC·TSG-6 complex formation is a divalent cation-dependent reaction (
37.
, 40.
), which our data reveal is mediated by Ca2+, bound to a site within the TSG-6 CUB module. The Glu-183 residue, which is involved in chelating the Ca2+ ion, makes a major contribution to the non-covalent interaction with HCs of IαI (e.g. via their Mg2+-containing MIDAS motifs), which precedes formation of the covalent bond between TSG-6 and HC. Tyr-47 in the Link module of TSG-6 then contributes to HA recognition by the HC·TSG-6 complex during “HC transfer,” but other residues implicated previously in HA binding in free TSG-6 (Tyr-94 and Tyr-113) are not involved in this process.
FIGURE 10Schematic model of the metal ion-dependent interaction of TSG-6 with IαI, leading to the formation of HC·HA complexes via an HC·TSG-6 intermediate. i, the CUB_C domain of TSG-6 interacts via its Glu-183 residue (red space filling) with the MIDAS site (*) of the HCs (illustrated for HC1) in a metal ion-dependent manner, leading to the formation of an initial non-covalent HC·TSG-6 complex. The Glu-183 amino acid conformation (and the surrounding structure) is stabilized by the presence of bound Ca2+, which is essential for the interaction with HCs. ii, the non-covalent HC·TSG-6 complex then converts to a covalent complex via the formation of an ester bond (red) between Ser-28 of TSG-6 (green space filling) and the C-terminal aspartic acid residue of an HC (). HA is recognized (yellow arrow) by a composite surface involving Tyr-47 of the TSG-6 Link module (pink) and residues of the HC, leading to the covalent transfer of the HC from TSG-6 onto HA. Previous studies have demonstrated that HC transfer is a divalent cation-dependent process (), and therefore it is likely to involve the metal ion-dependent interaction in i to stabilize the enzyme complex. iii, formation of HC·HA leads to release of TSG-6 (not shown), which can then interact with a new IαI molecule and catalyze the formation of further HC·HA.
39.
37.
From the results presented here, it is apparent that Ca2+ ion binding is not necessary for the CUB module to fold, but rather it plays a role in providing local structural organization (FIGURE 1, FIGURE 3) (e.g. of surrounding loops). In particular, it probably orients the functionally important Glu-183 amino acid residue, allowing it to interact with the HC. In this regard, our recent crystal studies on human HC1 have revealed that its von Willebrand factor A domain contains a Mg2+-containing MIDAS motif, where mutation of the chelating Asp-298 residue abolishes metal ion binding without having any effect on the overall structure.4 As shown here, the D298A mutant of rHC1 also abolishes binding to the TSG-6 CUB_C domain (Table 2), providing compelling evidence that this Mg2+ ion has a critical function in HC·TSG-6 formation. These findings suggest the intriguing possibility that the metal ion-dependent interaction of TSG-6 with IαI may be mediated by the Glu-183 side chain carboxylate (of the CUB module) co-chelating the magnesium ion within the MIDAS of the HC (i.e. reminiscent of interactions between von Willebrand factor A domains of integrins and their RGD-containing ligands (
90.
)).The discovery that TSG-6 has a Ca2+ ion-binding site explains why previously we found it unnecessary to add any calcium to form HC·TSG-6 and HC·HA complexes in vitro (
37.
, 45.
). This is because the rhTSG-6 used in these assays already contained calcium (i.e. based on “as purified” CUB_C being fully calcium ion-bound) (Fig. 3). Other studies did, however, indicate a requirement of Ca2+ for complex formation and HC transfer (- Higman V.A.
- Briggs D.C.
- Mahoney D.J.
- Blundell C.D.
- Sattelle B.M.
- Dyer D.P.
- Green D.E.
- DeAngelis P.L.
- Almond A.
- Milner C.M.
- Day A.J.
A refined model for the TSG-6 Link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.
J. Biol. Chem. 2014; 289: 5619-5634
41.
, 42.
). In our assay system, the addition of Mg2+ ions is required when we are using preparations of IαI where metal ions have been removed during purification (83.
). Importantly, differences in the sources of protein reagents and assay conditions probably explain the lack of consistency in the conclusions reached previously on the role of metal ions in the formation of HC·TSG-6 and HC·HA (37.
, 40.
, 41.
42.
).Our interaction analyses described here indicate that the full-length TSG-6 can interact with HC1 at two distinct sites: a metal ion-independent interaction with the Link domain and a metal ion-dependent interaction with the CUB module (Fig. 5). Interestingly, both interactions have Kd values of ∼2 nm (Table 2), which is very similar to the affinity (∼5 nm) for the interaction of rhTSG-6 with rHC1 and rHC2 (
44.
). Therefore, it seems unlikely that simultaneous binding of the CUB and Link modules to HCs can occur (because the affinity for rhTSG-6 would then be considerably higher). In this regard, it is reasonable to suggest that of these it is the CUB-mediated (Ca2+ ion- and Glu-183-dependent) interaction that is critical for HC·TSG-6 complex formation, given the evidence supporting the role of divalent cations (37.
, 40.
, 41.
) and Glu-183 (FIGURE 1, FIGURE 4A) in this process. On the other hand, the interaction with HC via the TSG-6 Link module might provide a mechanism whereby HC·TSG-6 complexes can remain bound to HC·HA and play a role in further catalysis of HC transfer, as suggested previously (44.
).TSG-6 can also bind weakly (180 nm) and metal ion-independently to the bikunin·CS component of IαI (
40.
, 46.
); this is probably mediated (at least in part) through the recognition of the CS chain by the Link module (47.
). Interestingly, this CS moiety has been clearly implicated as being necessary for HC·TSG-6 formation (40.
), requiring a particular sulfation pattern in the glycosaminoglycan linkage region in order for IαI to act as a substrate (24.
); chondroitin (91.
, 92.
) and the CS chain of bikunin·CS (93.
), which has non-sulfated “chondroitin-like” regions (23.
), can act as weak substrates for HC transfer. At the moment, we do not know the temporal sequence of this CS·bikunin-binding event relative to the metal ion-dependent interaction between the TSG-6 CUB module and an IαI HC (described in the present study). What seems certain is that these interactions are precisely coordinated in such a way as to correctly orient the TSG-6 molecule relative to IαI so that the ester bond connecting an HC to the CS chain can be transferred onto Ser-28 of TSG-6 (39.
). The fact that HC1·TSG-6 and HC2·TSG-6 complexes form in essentially equal amounts (37.
, 38.
) suggests a stochastic element to the process. A plausible mechanism would be for an initial Link module-mediated interaction between the TSG-6 and bikunin·CS, followed by a random “molecular collision” between the Glu-183 of the TSG-6 CUB module and the MIDAS of either HC1 or HC2. This would probably lead to a short lived, high affinity, intermediate involving both interactions, which is destabilized once the HC·CS bond has been transferred onto TSG-6 and the bikunin·CS by-product (37.
, 38.
) is released.Regardless of the precise sequence of the interactions, the non-covalent HC·TSG-6 complex formed must position the Ser-28 side chain of TSG-6 and the HC·CS ester bond in close proximity to the catalytic site, allowing the covalent HC·TSG-6 complex to form via a transesterification reaction (
39.
). However, currently, we do not know where the enzyme active site is located. Our analyses of the pH dependences of complex formation and HC transfer suggest that a histidine (which usually has pKa values between ∼6.0 and 6.5) is involved in both reactions (Fig. 8B); this functional residue(s) is likely to be present in TSG-6, given its role as the catalyst of HC·HA formation (37.
). In this regard, we observed that residues Asp-200 and His-203 of TSG-6 adopt relative conformations reminiscent of Asp-His-Ser catalytic triads within the CUB module structure, and the propKa software (94.
, 95.
) predicted that the pKa of His-203 might be elevated, as is the case for serine proteases (96.
). However, the H203S mutant of rhTSG-6 was found to have WT activity for HC·TSG-6 complex formation and HC transfer (data not shown), ruling out a role for this amino acid. Systematic mutagenesis will be required to determine whether a histidine residue of TSG-6 does form part of the catalytic site.Somewhat counterintuitively, we have found that the HA-binding site in “free” TSG-6 is not the same as that used for HA recognition in the context of HC transfer; this was based on a lack of correlation between the abilities of rhTSG-6 mutants to interact with HA and to form HC·HA complexes (Fig. 7). Furthermore, the observation that the Link_TSG6 protein does not inhibit HC·HA formation (Fig. 8A) provided further evidence that the HA-binding site in free TSG-6 is not utilized for HA recognition by HC·TSG-6. This conclusion is consistent with our recent studies showing that there was also no correlation between the substrate activities of various HA oligosaccharides in transfer assays and their affinities for Link_TSG6 (
45.
). Moreover, our previous biophysical experiments have revealed that the interaction of TSG-6 with IαI and the formation of HC·TSG-6 inhibit the binding of TSG-6 to HA, reversing TSG-6-mediated cross-linking of HA (- Higman V.A.
- Briggs D.C.
- Mahoney D.J.
- Blundell C.D.
- Sattelle B.M.
- Dyer D.P.
- Green D.E.
- DeAngelis P.L.
- Almond A.
- Milner C.M.
- Day A.J.
A refined model for the TSG-6 Link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.
J. Biol. Chem. 2014; 289: 5619-5634
44.
).It is noteworthy that the Tyr-47 and Tyr-94 residues are located close together within the HA-binding groove of the TSG-6 Link module (
45.
, - Higman V.A.
- Briggs D.C.
- Mahoney D.J.
- Blundell C.D.
- Sattelle B.M.
- Dyer D.P.
- Green D.E.
- DeAngelis P.L.
- Almond A.
- Milner C.M.
- Day A.J.
A refined model for the TSG-6 Link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.
J. Biol. Chem. 2014; 289: 5619-5634
49.
), yet despite their proximity (∼7 Å between hydroxyl oxygens, based on x-ray structure (50.
)) and the similar (greatly reduced) HA-binding phenotype of their phenylalanine mutants (Fig. 7A), they exhibit markedly different contributions to HC transfer activity (Fig. 7, B and C). This could be explained by the formation of a composite HA recognition site involving both TSG-6 and HC within the HC·TSG-6 complex, in which Tyr-47, but not Tyr-94 or Tyr-113, of TSG-6 plays a role (i.e. where HC probably occludes part of the binding surface used for HA in free TSG-6).Given that HC·TSG-6 (unlike free TSG-6) does not bind tightly to HA (
44.
), it seems plausible that during HC transfer, there is only a transient “interaction” of HA with the active site of this enzyme complex. It seems likely that residues from both TSG-6 and HC contribute to a composite active site (including a histidine, as discussed earlier) that is stabilized by the Glu-183- and Ca2+-dependent interaction of the CUB module with the MIDAS site of the HC (i.e. based on the requirement for divalent cations in the transfer of HC onto HA (37.
)). This active site is probably similar to that used in the initial transesterification reaction (i.e. within the non-covalent HC·TSG-6 complex). However, it is clearly not identical, as can be inferred from the different pH minima of the two reactions (Fig. 8B); this is to be expected because the formation of HC·HA has a different specificity requirement, with the transfer of the ester bond onto the C6 hydroxyl of HA (28.
) rather than onto the side chain hydroxyl of Ser-28 (39.
).Here we have demonstrated that the TSG-6-dependent transfer of HC onto HA is an absolute requirement for the organization/stabilization of the cumulus matrix, whereas the HA-binding properties of TSG-6 do not play an important role (i.e. based on the ability of rhTSG-6 mutants to rescue in vitro expansion of COCs from TSG-6−/− mice) (Fig. 9). These data are consistent with previous studies showing the involvement of HC·HA in COC expansion (
9.
, 12.
, 13.
, 91.
), where the formation of these complexes is mediated by TSG-6 (37.
, 39.
, 40.
). Furthermore, the present study indicates that TSG-6 is not a major participant in the structural stabilization of the cumulus matrix through its direct cross-linking of HA chains, as has been suggested previously (12.
, 13.
, 15.
, - Salustri A.
- Garlanda C.
- Hirsch E.
- De Acetis M.
- Maccagno A.
- Bottazzi B.
- Doni A.
- Bastone A.
- Mantovani G.
- Beck Peccoz P.
- Salvatori G.
- Mahoney D.J.
- Day A.J.
- Siracusa G.
- Romani L.
- Mantovani A.
PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization.
Development. 2004; 131: 1577-1586
31.
, 34.
, 53.
, 54.
). This is perhaps not surprising, given the recent findings that the interaction of TSG-6 with IαI impairs the binding of TSG-6 to HA (44.
) and that the full-length TSG-6 protein is unable to bridge between pentraxin-3 and HA (16.
), although this is a property of its isolated Link module domain (15.
, - Salustri A.
- Garlanda C.
- Hirsch E.
- De Acetis M.
- Maccagno A.
- Bottazzi B.
- Doni A.
- Bastone A.
- Mantovani G.
- Beck Peccoz P.
- Salvatori G.
- Mahoney D.J.
- Day A.J.
- Siracusa G.
- Romani L.
- Mantovani A.
PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization.
Development. 2004; 131: 1577-1586
16.
). Moreover, our recent biophysical studies have provided strong evidence that TSG-6, IαI, and pentraxin-3 cooperate to cross-link HA (16.
), where multiple HC·HA complexes probably associate with the octamer pentraxin-3 (31.
, 32.
- Inforzato A.
- Rivieccio V.
- Morreale A.P.
- Bastone A.
- Salustri A.
- Scarchilli L.
- Verdoliva A.
- Vincenti S.
- Gallo G.
- Chiapparino C.
- Pacello L.
- Nucera E.
- Serlupi-Crescenzi O.
- Day A.J.
- Bottazzi B.
- Mantovani A.
- De Santis R.
- Salvatori G.
Structural characterization of PTX3 disulphide bond network and its multimeric status in cumulus matrix organization.
J. Biol. Chem. 2008; 283: 10147-10161
33.
). However, this cross-linking process is tightly regulated, and, surprisingly, pentraxin-3 does not integrate into preformed HC·HA films but requires a prior encounter with IαI (- Inforzato A.
- Baldock C.
- Jowitt T.A.
- Holmes D.F.
- Lindstedt R.
- Marcellini M.
- Rivieccio V.
- Briggs D.C.
- Kadler K.E.
- Verdoliva A.
- Bottazzi B.
- Mantovani A.
- Salvatori G.
- Day A.J.
The angiogenic inhibitor long pentraxin PTX3 forms an asymmetric octamer with two binding sites for FGF2.
J. Biol. Chem. 2010; 285: 17681-17692
16.
). Why this should be the case is unclear, indicating that there is still much we do not understand regarding how the process of COC expansion is controlled (both temporally and spatially) and the way that these proteins organize HA in the cumulus matrix. In this regard, it seems probable that as well as the association of HC·HA with pentraxin-3, there are likely to be other interactions that play a role in stabilizing the HA network. Interestingly, the finding that the Y47F mutant of rhTSG-6 has the most impaired rescue activity (Fig. 9B), although it retains more HC transfer activity than E183S (Fig. 7C), is indicative that TSG-6 does play an additional role in COC expansion besides its catalysis of HC·HA formation.This research has provided important new insights into the mechanisms underlying TSG-6-mediated HC·HA formation and has clarified the role of divalent metal ions in this fundamental biological process. It has also identified that it is the transferase activity of TSG-6 that is essential for COC expansion rather than its HA-binding function. These studies therefore provide an excellent basis for additional work to further understand the molecular basis of HA cross-linking during ovulation and inflammation.
Author Contributions
D. C. B. was responsible for protein crystallization, crystallographic data collection, processing and refinement, heavy chain transfer assays, and drafting the paper. H. L. B. performed surface plasmon resonance, intrinsic fluorescence spectroscopy, and CUB_C expression and purification. T. A. carried out CUB_C expression/purification and NMR. M. S. R. conducted mutagenesis of rhTSG-6, heavy chain transfer, and HA-binding assays. J. P. W. aided in NMR data collection, processing, and interpretation. E. I. conducted the in vitro COC expansion assays. T. A. J. co-supervised H. L. B. and aided in fitting/interpretation of SPR data. J. J. E. provided purified IαI protein and contributed to writing of the paper. R. P. R. provided the bHA10 oligosaccharide and contributed to writing of the paper. A. S. supervised the in vitro COC expansion assays and contributed to writing of the paper. C. M. M. co-directed research and contributed to writing of the paper. A. J. D. directed research, supervised experiments, and coordinated writing of the paper.
Acknowledgments
We thank Katalin Mikecz for generously providing TSG-6-deficient mice, Erik Fries for provision of purified IαI used in some of the experiments, and David Knight and Tony Willis for mass spectrometry and amino acid analysis. We also thank Pat Bryant for help with crystallographic data collection and maintenance of local x-ray facilities.
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Article Info
Publication History
Published online: October 14, 2015
Received in revised form:
October 1,
2015
Received:
June 4,
2015
Footnotes
The atomic coordinates and structure factors (code 2WNO) have been deposited in the Protein Data Bank (http://wwpdb.org/).
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