TSG-6 Is Concentrated in the Extracellular Matrix of Mouse Cumulus Oocyte Complexes Through Hyaluronan and Inter-Alpha-Inhibitor Binding1

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TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His 4 ) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pK a values of charged residues within the Link module were determined using NMR. These data indicated that His 4 makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp 89 ), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr 12 ). This molecular network transmits the change in ionization state of His 4 to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His 45 , is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments. TSG-6,5 the secreted product of tumor necrosis factor-stimulated gene 6, is not usually expressed constitutively in healthy adult tissues but is made in response to various inflammatory mediators and growth factors, acting as a potent anti-inflammatory and chondroprotective agent (1-3). The TSG-6 protein is produced in inflammatory diseases, for example rheumatoid arthritis, osteoarthritis, and asthma (4), as well as in normal physiological processes that have inflammation-like characteristics (e.g. ovulation and cervical ripening). A number of roles for TSG-6 have been determined, including inhibition of neutrophil migration (5-7) and down-regulation of the protease network (4, 5, 8), which could help explain its protective properties in, for example, joint tissues (9 -11). In addition, TSG-6 has been implicated in the stabilization of extracellular matrix (ECM) structure, particularly by supporting the formation of cross-linked hyaluronan (HA) networks (12). TSG-6-mediated HA cross-linking has a critical role in mammalian ovulation but also occurs in inflammatory diseases (4, 13-17).TSG-6 is composed mainly of contiguous Link and CUB modules (2). Although there are currently no known ligands for the CUB module, the Link module (expressed in Escherichia coli (18,19) and termed Link_TSG6) has been shown to interact with a large number of molecules commonly found in the ECM. Not only does Link_TSG6 bind ...

Section: Discussionmentioning

confidence: 99%

Determining the Molecular Basis for the pH-dependent Interaction between the Link Module of Human TSG-6 and Hyaluronan

Blundell

Mahoney

Cordell

et al. 2007

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“…TSG-6 is a 35-kDa secreted protein that is involved in both physiological and pathophysiological tissue responses. In addition to HA (23)(24)(25)(26)(27)(28), TSG-6 has been shown to interact with a wide range of ECM components such as the GAGs (29), chondroitin 4-sulfate (25), and heparan sulfate and heparin (30). TSG-6 hyaladherin properties are endowed by virtue of the link module (24) and have been shown to be important for mediating HA interaction with CD44, which is also a link module-containing hyaladherin (29,31).…”

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confidence: 99%

TSG-6 Protein Is Crucial for the Development of Pulmonary Hyaluronan Deposition, Eosinophilia, and Airway Hyperresponsiveness in a Murine Model of Asthma

Swaidani

Cheng

Lauer

et al. 2013

“…The precise roles of TSG-6 in inflammation are still unclear, although recent studies in mouse models of arthritis have revealed that its potent anti-inflammatory effects protect against cartilage matrix destruction (18 -20). In addition, TSG-6 is also produced during ovulation (21,22) and cervical ripening (23), processes that have many characteristics in common with inflammation. In this regard, TSG-6 has been shown to have a critical role in murine ovulation via its interactions with inter-␣-inhibitor (24,25) and pentraxin-3 (26), leading to stabilization of the HA-rich extracellular matrix that forms around the oocyte and that is required for fertilization in vivo (27).…”

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confidence: 99%

Towards a Structure for a TSG-6·Hyaluronan Complex by Modeling and NMR Spectroscopy

Blundell

Almond

Mahoney

et al. 2005

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123

The Link module from human TSG-6, a hyaladherin with roles in ovulation and inflammation, has a hyaluronan (HA)-binding groove containing two adjacent tyrosine residues that are likely to form CH-stacking interactions with sequential rings in the sugar. We have used this observation to construct a model of a protein⅐HA complex, which was then tested against existing experimental information and by acquisition of new NMR data sets of [ 13 C, 15 N]HA (8-mer) complexed with unlabeled protein. A major finding of this analysis was that acetamido side chains of two GlcNAc rings fit into hydrophobic pockets on either side of the adjacent tyrosines, providing a selectivity mechanism of HA over other polysaccharides. Furthermore, two basic residues have a separation that matches that of glucuronic acids in the sugar, consistent with the formation of salt bridges; NMR experiments at a range of pH values identified protein groups that titrate due to their proximity to a free carboxylate in HA. Sequence alignment and construction of homology models for all human Link modules in their HA-bound states revealed that many of these features are conserved across the superfamily, thus allowing the prediction of functionally important residues. In the case of cartilage link protein, its two Link modules were docked together (using bound HA as a guide), identifying hydrophobic residues likely to form an intra-Link module interface as well as amino acids that could be involved in supporting intermolecular interactions between link proteins and chondroitin sulfate proteoglycans. Here, we propose a mechanism for ternary complex formation that generates higher order helical structures, as may exist in cartilage aggregates.