RESUMEN
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.
Asunto(s)
Moléculas de Adhesión Celular/química , Ácido Hialurónico/química , Pliegue de Proteína , Aminoácidos/química , Aminoácidos/metabolismo , Moléculas de Adhesión Celular/metabolismo , Humanos , Ácido Hialurónico/metabolismo , Concentración de Iones de Hidrógeno , Ligandos , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
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.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Animales , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Ratones , Ratones Endogámicos AKRRESUMEN
Tumor necrosis factor-stimulated gene-6 (TSG-6) encodes a 35-kDa protein, which is comprised of contiguous Link and CUB modules. TSG-6 protein has been detected in the articular joints of osteoarthritis (OA) patients, with little or no constitutive expression in normal adult tissues. It interacts with components of cartilage matrix (e.g. hyaluronan and aggrecan) and thus may be involved in extracellular remodeling during joint disease. In addition, TSG-6 has been found to have anti-inflammatory properties in models of acute and chronic inflammation. Here we have mapped the human TSG-6 gene to 2q23.3, a region of chromosome 2 linked with OA. A single nucleotide polymorphism was identified that involves a non-synonymous G --> A transition at nucleotide 431 of the TSG-6 coding sequence, resulting in an Arg to Gln alteration in the CUB module (at residue 144 in the preprotein). Molecular modeling of the CUB domain indicated that this amino acid change might lead to functional differences. Typing of 400 OA cases and 400 controls revealed that the A(431) variant identified here is the major TSG-6 allele in Caucasians (with over 75% being A(431) homozygotes) but that this polymorphism is not a marker for OA susceptibility in the patients we have studied. Expression of the Arg(144) and Gln(144) allotypes in Drosophila Schneider 2 cells, and functional characterization, showed that there were no significant differences in the ability of these full-length proteins to bind hyaluronan or form a stable complex with inter-alpha-inhibitor.