Introduction
Disulfide bonds have long been recognized as structural elements stabilizing proteins in harsh extracellular environments. More recently, an additional concept has emerged: some disulfide bonds operate as dynamic scaffolds capable of regulated rearrangement into a variety of functional forms (Jordan and Gibbins, 2006). Consistent with this notion, various cell surface processes have long been known to depend on catalyzed thiol-disulfide exchange including cell adhesion (Essex, 2004), uptake of bacterial toxins (de Paiva et al, 1993) and viral fusion with the host membrane (Sanders, 2000). Moreover, a variety of cell surface signaling receptors appear to exist in more than one thiol-disulfide configuration, for example CD28 (Greene et al, 1996). However, in most cases, neither the catalyst driving thiol-disulfide exchange nor the functional differences between the redox forms have been elucidated.
A number of thiol-disulfide oxidoreductases are known to be secreted and to act on the cell surface. One of these redox catalysts is protein disulfide isomerase (PDI), a member of the thioredoxin (Trx) superfamily. Cell surface-PDI has been found to act on transmembrane and surface-associated proteins, including the envelope protein of HIV-1, to cause its fusogenic conformation (Markovic et al, 2004) and integrins, to mediate platelet adhesion (Lahav et al, 2003). Another thiol-disulfide oxidoreductase associated with extracellular functions is Trx1. Best known for its intracellular roles, Trx1 reduces transiently formed disulfide bonds of cytosolic and nuclear target proteins and thereby participates in a multitude of fundamental processes, ranging from oxidant scavenging and DNA synthesis to regulation of apoptosis and cell proliferation (Powis and Montfort, 2001). In addition, Trx1 is released to the extracellular environment by a variety of normal and neoplastic cells (Rubartelli et al, 1992). Human Trx1 was first purified as a cytokine-like factor from supernatants of virally transformed lymphocytes and initially named adult T-cell leukemia-derived factor (Tagaya et al, 1988), Tac-inducing factor (Tagaya et al, 1989), B-cell stimulatory factor or ‘B cell IL-1' (Wakasugi et al, 1990). Extracellular Trx1 is present in the circulation of healthy subjects and its levels increase under inflammatory conditions, including viral infection (Nakamura et al, 2001a). Circulatory Trx1 acts as a chemoattractant for monocytes, neutrophils and lymphocytes (Bertini et al, 1999), and inhibits neutrophil migration into inflammatory sites both in vitro and in vivo (Nakamura et al, 2001b). More recently, Trx1 was found to be secreted by dendritic cells upon cognate T-cell recognition and to contribute to subsequent T-cell activation (Angelini et al, 2002).
At present, the mechanism(s) and pathway(s) by which extracellular Trx1 influences cellular behavior remain unknown. As many of its reported extracellular activities depend on a functional active site, it appears likely that Trx1 catalyzes thiol-disulfide exchange in one or more cell surface target proteins through its enzymatic activity. However, thiol-disulfide exchange reactions, even if highly specific, are too transient to be detected by conventional techniques. To date, only a single cell surface receptor, CD4, a member of the immunoglobulin superfamily, has been shown to be susceptible to Trx1 redox activity (Matthias et al, 2002). Other cell surface proteins targeted by the enzymatic activity of Trx1 await identification.
In this study, we address the question as to which cell surface receptors expressed on lymphocytes specifically interact with extracellular Trx1 by way of disulfide bond exchange. Using a kinetic trapping technique that enables the detection and isolation of otherwise short-lived reaction intermediates on the surface of intact cells, we identify and validate the tumor necrosis factor receptor superfamily member CD30 (TNFRSF8) as the principal target molecule for Trx1 on infected and transformed lymphocytes. The cell surface activity of Trx1 is highly selective, discriminating between different members of the TNFR superfamily. Trx1-mediated thiol-disulfide exchange leads to a structural change in the CD30 ectodomain that can be detected with conformation-sensitive antibodies. We demonstrate that disulfide exchange between Trx1 and CD30 interferes with binding of the CD30 ligand (CD30L) to its cognate receptor and that Trx1 affects CD30-dependent changes in lymphocyte effector function. As CD30 is implicated in both stimulatory and apoptotic signaling, our findings suggest that Trx1 interacts with CD30 to modulate lymphocyte behavior and survival under conditions of infection and inflammation.