The Link Module from Ovulation- and Inflammation-associated Protein TSG-6 Changes Conformation on Hyaluronan Binding

Blundell, Charles; Mahoney, David J.; Almond, Andrew; DeAngelis, Paul L.; Kahmann, Jan D.; Teriete, Peter; Pickford, Andrew R.; Campbell, Iain D.; Day, Anthony J.

doi:10.1074/jbc.m309623200

Cited by 88 publications

(278 citation statements)

References 43 publications

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“…The affinity of the interaction between H29K and an octasaccharide of HA (HA 8 ) was determined by isothermal titration calorimetry on a MicroCal VP-ITC instrument at 25°C in 5 mM Na-MES, pH 6.0, exactly as described for the other mutants (24). In this regard, HA 8 has been shown to bind to Link_TSG6 with optimum affinity (26).…”

Section: Methodsmentioning

confidence: 99%

“…The HA binding domains of TSG-6 and CD44 both contain a "Link module," a structural unit of about 100 amino acids that is present in many other members of the hyaladherin family including the proteoglycan aggrecan and cartilage link protein (17,18,26,27). Although the HA-interaction surfaces in CD44 and TSG-6 appear to be similar (27)(28)(29)(30), there are clearly significant differences in the molecular details of HA binding in these proteins (29).…”

mentioning

confidence: 98%

“…tion 6 may allow the formation of an additional ionic interaction between HA and the protein (26). The effects of Link_TSG6 mutants on the interactions between HA and CD44 on AKR1/CD44ϩ cells (i.e.…”

Section: Tsg-6 Modulates Hyaluronan Binding By Cd44mentioning

confidence: 99%

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

Lesley

Gál

Mahoney

et al. 2004

Journal of Biological Chemistry

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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%

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confidence: 98%

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

Lesley

Gál

Mahoney

et al. 2004

Journal of Biological Chemistry

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“…We have determined the solution structure of Link_TSG6 in the absence and presence of an HA octasaccharide (HA 8 AN ) (28) and recently have used these and other data to produce a model for the HA 8 AN -Link_TSG6 complex (see Fig. 1B) (29).…”

mentioning

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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“…Indeed, TSG-6 has been shown to associate with many matrix components. For instance, it interacts with the glycosaminoglycans, hyaluronan (HA) [3,13,14] and chondroitin-4-sulphate [25], as well as the hyaluronan-binding region of aggrecan [26], the most common proteoglycan found in disc. In addition, TSG-6 is up-regulated in the matrix surrounding osteoarthritis-like lesions in STR/ort mice, with decreased levels of aggrecan being seen in areas strongly expressing this protein [23].…”

Section: Introductionmentioning

confidence: 99%