Trx1-mediated disulfide exchange interferes with CD30 receptor–ligand interactions
The observation that antibody binding to CD30 is influenced by Trx1 suggested a redox-dependent conformational change within the CD30 ectodomain. To test whether Trx1-mediated reduction of CD30 influences binding of CD30 to its ligand (CD30L), we analyzed the interaction between CD30 and recombinant soluble CD30L (sCD30L) on the cell surface by flow cytometry. A brief incubation of CD30+Hodgkin's lymphoma HDLM-2 cells with wild-type Trx1 led to a substantial loss in CD30L binding to the cell surface (Figure 6A). A similar result was obtained for the large granular lymphoma cell line YT (Supplementary Figure S4). The same effect was evident in the absence of an exogenously added reducing system (Supplementary Figure S5), thus demonstrating that under the given conditions Trx1–substrate interactions are not limited by oxidative inactivation.
As shown by fluorescence microscopy, sCD30L binds to the surface of CD30-transfected HeLa cells and colocalizes with its receptor (Figure 6B, upper row). Treatment of HeLa cells with Trx1(CCAAA) (Figure 6B, middle row) but not a redox-inactive mutant (Figure 6B, lower row) strongly interferes with CD30L binding and colocalization.