Abstract
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.
Highlights
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 [1,2,3]
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
TSG-6 has been implicated in the stabilization of extracellular matrix (ECM) structure, by supporting the formation of cross-linked hyaluronan (HA) networks [12]
Summary
Sources of Materials—Wild-type (WT) and mutant forms of Link_TSG6 were expressed in E. coli and purified as described previously [27]. The extent of folding of free and HA8AN-bound Link_TSG6 at a particular pH value was assessed by comparing the total volume of all the amide resonances in a 1H,15N-HSQC spectrum arising from folded material at that pH value that were not occluded by resonances resulting from unfolded material at any pH value (ϳ70 resonances) with the total volume of the entire spectrum at that pH value (i.e. the combination of both folded and unfolded material). The titrating residue responsible for the pH-induced change in chemical shift of a particular amide resonance was identified using both the determined pKa value and the location of the amide group in the structure (Protein Data Bank codes 1o7b and 1o7c for free and HA8AN-bound forms, respectively) [28]. Molecular models have been displayed using MOLMOL (hugin.ethz.ch/wuthrich/software/ molmol/)
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