A Novel Allelic Variant of the Human TSG-6 Gene Encoding an Amino Acid Difference in the CUB Module

Abstract

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 3 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-␣-inhibitor.

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

“…Preparation and Characterization of Full-length Human TSG-6 and Link_TSG6 -Full-length human TSG-6 (allelic variant TSG-6Q with Gln at amino acid position 144 in the pre-protein) was expressed in Drosophila Schneider 2 cells and purified by ion exchange chromatography and reverse-phase high performance liquid chromatography as described previously (43).…”

TSG-6 binds via its CUB_C domain to the cell-binding domain of fibronectin and increases fibronectin matrix assembly

“…Biotinylated-FN was prepared using sulfo-Nhydroxysuccinimidyl-biotin (Pierce Biotechnology, Rockford, IL), followed by a dialysis step to remove unconjugated biotin (Pankov and Yamada, 2004). Recombinant full-length TSG-6Q (with a glutamine at residue 144) was expressed in Drosophila S2 cells (Nentwich et al, 2002), and Link_TSG6 was expressed in E. coli as described previously (Day et al, 1996;Kahmann et al, 1997).…”

“…Day, unpublished observations). Briefly, the CUB_C coding sequence was amplified from human osteoblast mRNA, as described previously for the full-length protein (Nentwich et al, 2002), cloned into pRK172 and transfected into Rosetta-gami BL21(DE3)pLysS. Cells were harvested 4h after induction with IPTG and inclusion bodies were purified as described in (Day et al, 1996).…”

TNF?-stimulated gene product (TSG-6) and its binding protein, I?I, in the human intervertebral disc: new molecules for the disc

“…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]. It is detectable in the chondrocyte pericellular matrix of these mice, prior to development of osteoarthritic lesions, and thus might be an early marker of disease.…”

“…TSG-6 is known to form a stable, $120 kDa, complex with inter-a-inhibitor (IaI), a 180 kDa serine protease inhibitor found in serum [23,31]. IaI-TSG-6 complexes of this size have been detected in the synovial fluids of arthritis patients [30].…”