TSG‐6 interacts with hyaluronan and aggrecan in a pH‐dependent manner via a common functional element: implications for its regulation in inflamed cartilage

Parkar, Ashfaq A; Kahmann, Jan D.; Howat, Sarah; Bayliss, Michael T.; Day, Anthony J.

doi:10.1016/s0014-5793(98)00523-7

Cited by 63 publications

(80 citation statements)

References 31 publications

(68 reference statements)

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“…Parkar et al (46) observed that Link_TSG6 binding to HA was optimal at pH 6.0 and significantly reduced at pH 7.0. In preliminary experiments we found that preincubation of HA and Link_TSG6 at either pH 6.0 or 7.0 resulted in similar binding to CD44.…”

Section: Methodsmentioning

confidence: 99%

TSG-6 Modulates the Interaction between Hyaluronan and Cell Surface CD44

Lesley

Gál

Mahoney

et al. 2004

Journal of Biological Chemistry

161

203

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Interactions between CD44 and hyaluronan are implicated in the primary adhesion of lymphocytes to endothelium at inflammatory locations. Here we show that preincubation of hyaluronan with full-length recombinant TSG-6 or its Link module domain (Link_TSG6) enhances or induces the binding of hyaluronan to cell surface CD44 on constitutive and inducible cell backgrounds, respectively. These effects are blocked by CD44-specific antibodies and are absent in CD44-negative cells. Enhancement of CD44-mediated interactions of lymphoid cells with hyaluronan by TSG-6 proteins was seen under conditions of flow at shear forces that occur in post-capillary venules. Increases in the number of rolling cells were observed on substrates comprising TSG-6-hyaluronan complexes as compared with a substrate containing hyaluronan alone. In ligand competition experiments, cell surface-bound TSG-6-hyaluronan complexes were more potent than hyaluronan alone in inhibiting cell adhesion to immobilized hyaluronan. Link_TSG6 mutants with impaired hyaluronan binding function had a reduced ability to modulate ligand binding by cell surface CD44. However, some mutants that exhibited close to wild-type hyaluronan binding were found to have either reduced or increased activity, suggesting that some amino acid residues outside of the hyaluronan binding site might be involved in protein self-association, potentially leading to the formation of cross-linked hyaluronan fibers. In turn, cross-linked hyaluronan could increase the binding avidity of CD44 by inducing receptor clustering. The ability of TSG-6 to modulate the interaction of hyaluronan with CD44 has important implications for CD44-mediated cell activity at sites of inflammation, where TSG-6 is expressed.

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Section: Methodsmentioning

confidence: 99%

TSG-6 Modulates the Interaction between Hyaluronan and Cell Surface CD44

Lesley

Gál

Mahoney

et al. 2004

Journal of Biological Chemistry

161

203

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confidence: 95%

“…1A) (22). In marked contrast, the pH dependences of both Link protein and the G1 domain of aggrecan (two constitutively expressed HA-binding proteins) for HA have no drop in affinity between pH 6.0 and 8.0 (22). Recently, the pH dependence of the HA-Link_TSG6 interaction has been questioned (35), with suggestions that the earlier observation results from an artifact of the solid-phase assays used (i.e.…”

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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

Journal of Biological Chemistry

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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 ...

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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%