To investigate the role of tumor necrosis factor stimulated gene 6 (TSG-6) and metal ions in the coupling of inter-alpha-trypsin inhibitor (ITI) to hyaluronan in human synovial fluid.
The concentration of ITI heavy chains bound to hyaluronan was determined by a two-step electrophoretic technique. Synovial fluid, TSG-6 depleted synovial fluid and metal chelated synovial fluid were tested for their ability to support the coupling of ITI heavy chains to hyaluronan.
When synovial fluid was mixed with an ITI-source (serum or purified ITI), coupling of ITI heavy chains to hyaluronan took place. TSG-6 immunodepleated synovial fluid lost the coupling activity, but addition of recombinant TSG-6 restored the activity. EDTA inhibited the coupling activity, but combinations of the metal-ion chelators Mg-EGTA and Ca-EGTA demonstrated, that Ca++ is essential for the coupling of ITI heavy chains to hyaluronan.
Tumor necrosis factor stimulated gene 6 (TSG-6) and calcium ions are both essential for the coupling of inter-alpha-trypsin inhibitor to hyaluronan in human synovial fluid.