TSG-6 Protein Binding to Glycosaminoglycans

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: Discussionsupporting

confidence: 66%

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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

“…This unusual pH dependence appeared to be in marked contrast to that for the G1 domain of aggrecan, where the highest affinity was reported at neutral pH 7.0 -8.0 (31). However, it has been suggested that the unusual pH dependence we reported for the HA-Link_TSG6 interaction may have resulted from an artifact of the solid phase assay used in the analysis (32). In recent work, isothermal titration calorimetry (ITC) was used to show that the Link_TSG6 had a higher affinity at pH 6.0 compared with pH 7.5 for short oligosaccharides of HA in solution phase and a structural basis for the pH dependence was identified using a combination of site-directed mutagenesis, NMR and ITC (33).…”

mentioning

confidence: 85%

“…Therefore, it seems likely that HA 10 is the minimum length of HA able to occupy the HA-binding site on G1, but that HA 12 has slightly tighter binding. Earlier estimates of the footprint of aggrecan on HA suggested that at maximal packing density it occupied a longer stretch of HA (ϳHA 18 ) than was part of the minimal binding site (31) and more recent experiments comparing the binding of recombinant G1 to HA 32 and HA 40 also suggested a longer site occupied, which may result from negative cooperativity (13). Alternatively, this may be caused by the spatial constraints of how close G1 domains (which each have 2 Link modules and an Ig fold) can pack when bound to HA.…”

Section: Effect Of Ph On the Binding Of The G1 Domain Of Aggrecan Withmentioning

confidence: 99%

Hyaluronan Binding to Link Module of TSG-6 and to G1 Domain of Aggrecan Is Differently Regulated by pH

Heng

Gribbon

Day

et al. 2008

The physiological functions of hyaluronan (HA) in the extracellular matrix of vertebrate tissues involve a range of specific protein interactions. In this study, the interaction of HA with the Link module from TSG-6 (Link_TSG6) and G1 domain of aggrecan (G1), were investigated by a biophysical analysis of translational diffusion in dilute solution using confocal fluorescence recovery after photobleaching (confocal FRAP). Both Link_TSG6 and G1 were shown to bind to polymeric HA and these interactions could be competed with HA 8 and HA 10 oligosaccharides, respectively. Equilibrium experiments showed that the binding affinity of Link_TSG6 to HA was maximal at pH 6.0, and reduced dramatically above and below this pH. In contrast, G1 had maximum binding at pH 7.0 -8.0 and moderate to strong binding affinity over a much broader pH range (5.5-8.0). The K D determined for Link_TSG6 binding to HA showed a 100-fold increase in binding affinity between pH 7.4 and 6.0, whereas G1 showed a 75-fold decrease in binding affinity over the same pH range. The sharp difference observed in their pH binding suggests that pH controls the physiological function of TSG-6, with a low affinity for HA at neutral pH, but with increased affinity as the pH falls below pH 7. TSG-6 and aggrecan interact with HA through structurally homologous domains and the difference in pH-dependent binding can be understood in terms of differences in the presence and topographical distribution of key regulatory amino acids in Link_TSG6 and in the related tandem Link domains in aggrecan G1.

“…The 35-kDa protein is composed of a link and a CUB domain. The link domain mediates binding to glycosaminoglycans via two distinct binding sites (2,3). The CUB domain in TSG-6 is less studied, but it has been shown recently that the domain mediates interaction with fibronectin (4).…”

mentioning

confidence: 99%

The Transfer of Heavy Chains from Bikunin Proteins to Hyaluronan Requires Both TSG-6 and HC2

Sanggaard

Sonne-Schmidt

Krogager

et al. 2008

Self Cite

In the present study we purified the required component from human plasma and identified it as HC2. Proteins containing HC2 including I␣I, HC2⅐bikunin, and free HC2 promoted the formation of HC3⅐TSG-6 and subsequently HC3⅐hyaluronan complexes. HC1 or HC3 did not possess this activity. The presented data reveal that both HC2 and TSG-6 are required for the transesterification reactions to occur.