Link modules are hyaluronan-binding domains found in proteins involved in the assembly of extracellular matrix, cell adhesion, and migration. The solution structure of the Link module from human TSG-6 was determined and found to consist of two alpha helices and two antiparallel beta sheets arranged around a large hydrophobic core. This defines the consensus fold for the Link module superfamily, which includes CD44, cartilage link protein, and aggrecan. The TSG-6 Link module was shown to interact with hyaluronan, and a putative binding surface was identified on the structure. A structural database search revealed close similarity between the Link module and the C-type lectin domain, with the predicted hyaluronan-binding site at an analogous position to the carbohydrate-binding pocket in E-selectin.

Background & Aims The gold standard in assessing liver fibrosis is biopsy despite limitations like invasiveness and sampling error and complications including morbidity and mortality. Therefore, there is a major unmet medical need to quantify fibrosis noninvasively to facilitate early diagnosis of chronic liver disease and provide a means to monitor disease progression. The goal of this study was to evaluate the ability of several magnetic resonance imaging (MRI) techniques to stage liver fibrosis. Methods A gadolinium (Gd)-based MRI probe targeted to type I collagen (termed EP-3533) was utilized to noninvasively stage liver fibrosis in a carbon tetrachloride (CCl4) mouse model and the results were compared to other MRI techniques including relaxation times, diffusion and magnetization transfer measurements. Results The most sensitive MR biomarker was the change in liver:muscle contrast to noise ratio (ΔCNR) after EP-3533 injection. We observed a strong positive linear correlation between ΔCNR and liver hydroxyproline (i.e. collagen) levels (r=0.89) as well as ΔCNR and conventional Ishak fibrosis scoring. In addition, the area under the receiver operating curve (AUR0C) for distinguishing early (Ishak ≤3) from late (Ishak ≥ 4) fibrosis was 0.942±0.052 (p<0.001). By comparison, other MRI techniques were not as sensitive to changes in fibrosis in this model. Conclusions We have developed a MRI technique using a collagen-specific probe for diagnosing and staging liver fibrosis, and validated it in the CCl4 mouse model. This approach should provide a better means to monitor disease progression in patients.

Link modules are hyaluronan-binding domains that are involved in the formation and stability of extracellular matrix and cell migration. We have examined the glycosaminoglycan specificity of the Link module from the arthritis-associated protein, human TSG-6, by microtitre plate-based assays employing biotinylated-hyaluronan or mono-biotinylated Link module. This domain was found to interact specifically with chondroitin-4-sulphate (C4S), with similar affinity to hyaluronan, but not with chondroitin-6-sulphate or heparin. Competition experiments indicate that C4S and hyaluronan have overlapping binding surfaces on the TSG-6 Link module. Disease-associated changes in C4S expression may influence the localisation and biological role of TSG-6.

Cartilage matrix is stabilised by the interactions of proteins with hyaluronan (HA). We compare the pH dependences of HA binding by aggrecan, link protein and TSG-6. Aggrecan and link protein exhibit maximal binding across a wide pH range (6.0^8.0). TSG-6, a protein that is only produced during inflammation, binds maximally at about pH 6.0 but shows a dramatic loss of function with increasing pH. TSG-6 also interacts with aggrecan, with a similar pH dependence, and this can be inhibited by HA. Thus, a common binding surface on TSG-6 may be involved in HA and aggrecan binding. We propose that TSG-6 is involved in matrix dissociation and that this is regulated by pH gradients in cartilage.z 1998 Federation of European Biochemical Societies.

Endothelial dysfunction is a hallmark of tissue injury and is believed to initiate the development of vascular diseases. Sphingosine-1 phosphate receptor-1 (S1P1) plays fundamental physiological roles in endothelial function and lymphocyte homing. Currently available clinical molecules that target this receptor are desensitizing and are essentially S1P1 functional antagonists that cause lymphopenia. They are clinically beneficial in autoimmune diseases such as multiple sclerosis. In patients, several side effects of S1P1 desensitization have been attributed to endothelial damage, suggesting that drugs with the opposite effect, namely, the ability to activate S1P1, could help to restore endothelial homeostasis. We found and characterized a biased agonist of S1P1, SAR247799, which preferentially activated downstream G protein signaling to a greater extent than β-arrestin and internalization signaling pathways. SAR247799 activated S1P1 on endothelium without causing receptor desensitization and potently activated protection pathways in human endothelial cells. In a pig model of coronary endothelial damage, SAR247799 improved the microvascular hyperemic response without reducing lymphocyte numbers. Similarly, in a rat model of renal ischemia/reperfusion injury, SAR247799 preserved renal structure and function at doses that did not induce S1P1-desensitizing effects, such as lymphopenia and lung vascular leakage. In contrast, a clinically used S1P1 functional antagonist, siponimod, conferred minimal renal protection and desensitized S1P1. These findings demonstrate that sustained S1P1 activation can occur pharmacologically without compromising the immune response, providing a new approach to treat diseases associated with endothelial dysfunction and vascular hyperpermeability.