Kinetic trapping on the surface of lymphoid cell lines identifies a prominent Trx1 interaction partner
Human Trx1 was first identified as an autocrine factor secreted by and acting on virally transformed lymphoid cell lines. We therefore applied cell surface trapping to a human EBV-transformed lymphoblastoid B-cell line (LCL-721.220) derived from the same parental cell line (LCL-721) as the 3B6 cell line, originally used in the description of the costimulatory factor ‘3B6-IL1', later identified as Trx1 (Wakasugi et al, 1990). Mixed disulfide complexes, which formed on the surface of LCL-721.220 cells were isolated and analyzed by Trx1-specific immunoblotting to visualize overall mixed disulfide conjugates. Interestingly, we found that Trx1 predominantly engages a single protein on the lymphoblastoid surface, suggesting a highly selective interaction (Figure 2A, lane 3). As expected, the trapping product, a mixed disulfide conjugate of about 160 kDa, was susceptible to reduction (Figure 2A, lane 4). The 160 kDa conjugate did not form on the surface of the EBV-negative Burkitts lymphoma cell line BL-41 (Figure 2B). In contrast, a conjugate of the same size was found to be formed on the surface of CCRF-CEM T cells (Figure 2C) and YT large granular lymphoma cells (data not shown). Pretreatment of the cell surface with the alkylating agent iodoacetamide (IAA) did not interfere with the formation of the 160 kDa mixed disulfide conjugate, thus confirming that the conjugate was formed by the expected disulfide exchange mechanism (rather than by de novo disulfide bond formation between two thiol groups).