Trx1 discriminates between CD30 and other members of the TNFR superfamily
Ectodomains of TNFR superfamily proteins typically are composed of one to four cysteine-rich domains (CRDs), each normally harboring three disulfide bonds (Bodmer et al, 2002). To exclude the possibility that Trx1 interacts with CRDs uniformly, we tested whether Trx1 discriminates between distinct members of the TNFR superfamily. As shown in Figure 4A, CD95 (TNFRSF6) did not form a mixed disulfide conjugate with Trx1(CSAAA) on the same cells under identical conditions. The same result was obtained for the epidermal growth factor receptor (EGFR), which contains a total of 25 ectodomain disulfide bonds, and is expressed at substantial levels on A431 cells (Gill and Lazar, 1981) (Figure 4B). These findings indicate that Trx1 reactivity of cell surface receptors is selective and is not determined by the presence of CRDs or the number of ectodomain disulfide bonds. To further exclude that Trx1 reactivity of receptors is determined or limited by surface expression levels, we ectopically overexpressed CD30 or other TNFR superfamily members under control of the same promoter in HeLa cells and analyzed Trx1 cell surface trapping by indirect immunofluorescence. While mock-transfected HeLa cells did not capture Trx1(CSAAA) on their surface (Figure 4C, lower row), expression of CD30 led to a strong Trx1 surface association and colocalization of both proteins (Figure 4C, upper row). In contrast, expression of CD95 (Figure 4D), TNFR1 or NGFR (data not shown) did not promote Trx1 interactions with the cell surface, further strengthening the notion that Trx1 reactivity is a specific property of CD30.
The domain structure of human CD30 differs from other members of the TNFR superfamily and from its murine homologue by the presence of two additional CRDs, arising from the internal duplication of two exons (Burgess et al, 2004). We asked whether this unusual feature might confer Trx1 reactivity to human CD30 and tested whether the shorter murine CD30 could also interact with Trx1. As shown in Figure 4E, murine CD30 expressed on the Rauscher murine leukemia virus-induced T-cell lymphoma line RMA is efficiently targeted by Trx1(CSAAA), thus demonstrating that the additional CRDs in human CD30 are not required for Trx1 reactivity. The result also suggests that the enzymatic affinity of Trx1 for CD30 has been conserved during mammalian evolution.