Identification of protein-disulfide isomerase activity in fibronectin

Assembly and degradation of fibronectin-containing extracellular matrices a re dynamic processes that are up-regulated during wound healing, embryogene sis, and metastasis. Although several of the early steps leading to fibrone ctin deposition have been identified, the mechanisms leading to the accumul ation of fibronectin in disulfide-stabilized multimers are largely unknown, Disulfide-stabilized fibronectin multimers are thought to arise through in tra- or intermolecular disulfide exchange. Several proteins involved in dis ulfide exchange reactions contain the sequence Cys-X-X-Cys in their active sites, including thioredoxin and protein-disulfide isomerase, The twelfth t ype I module of fibronectin (I-12) contains a Cys-X-X-Cys motif, suggesting that fibronectin may have the intrinsic ability to catalyze disulfide bond rearrangement. Using an established protein refolding assay, we demonstrat e here that fibronectin has protein-disulfide isomerase activity and that t his activity is localized to the carboxyl-terminal type I module I-12. I-12 was as active on an equal molar basis as intact fibronectin, indicating th at most of the protein-disulfide isomerase activity of fibronectin is local ized to I-12. Moreover, the protein-disulfide isomerase activity of fibrone ctin appears to be partially cryptic since limited proteolysis of I-10-I-12 increased its isomerase activity and dramatically enhanced the rate of RNa se refolding, This is the first demonstration that fibronectin contains pro tein disulfide isomerase activity and suggests that cross-linking of fibron ectin in the extracellular matrix may be catalyzed by a disulfide isomerase activity contained within the fibronectin molecule.