Truncated and Full-Length Thioredoxin-1 Have Opposing Activating and Inhibitory Properties for Human Complement with Relevance to Endothelial Surfaces

Abstract

Thioredoxin (Trx)-1 is a small, ubiquitously expressed redox-active protein with known important cytosolic functions. However, Trx1 is also upregulated in response to various stress stimuli, is found both at the cell surface and secreted into plasma, and has known anti-inflammatory and antiapoptotic properties. Previous animal studies have demonstrated that exogenous Trx1 delivery can have therapeutic effects in a number of disease models and have implicated an interaction of Trx1 with the complement system. We found that Trx1 is expressed in a redox-active form at the surface of HUVEC and acts as an inhibitor of complement deposition in a manner dependent on its Cys-Gly-Pro-Cys active site. Inhibition occurred at the point of the C5 convertase of complement, regulating production of C5a and the membrane attack complex. A truncated form of Trx1 also exists in vivo, Trx80, which has separate nonoverlapping functions compared with the full-length Trx1. We found that Trx80 activates the classical and alternative pathways of complement activation, leading to C5a production, but the inflammatory potential of this was also limited by the binding of inhibitors C4b-binding protein and factor H. This study adds a further role to the known anti-inflammatory properties of Trx1 and highlights the difference in function between the full-length and truncated forms.

Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase

“…35 Our data show that C5 activation results in a marked decrease of cell surface PDI reductase activity requiring C5 activation. Because induction of TF PCA is furthermore dependent on intact lipid raft domains, we propose that, upon activation of the classical complement pathway by cell-bound ATG, rapid depletion of reduced Trx-1 from the cell surface causes oxidation of PDI, which in turn promotes TF activation by shifting the equilibrium from reduced to oxidized TF.…”

“…Formation of the initial membrane insertion complex C5b-7 is critical for TF activation and PS exposure (right), but full MAC assembly leading to lytic PS externalization is not required. PDIdependent thiol-disulfide exchange reactions occur following C5 conversion through engagement of complement regulatory proteins, 35 resulting in depletion of membrane reductive equivalents (ie, thioredoxin-1) with consecutive PDI and TF oxidation. 38 Rearrangements of the cell membrane by C5b-7 insertion may lead to PS exposure in raft domains and thus facilitate dissociation of TF from regulatory proteins and/or TF oxidation.…”

“…35 Thioredoxin (Trx)-1, a disulfide reductase controlling cellular redox potential, has recently emerged as a crucial cell surface regulator of complement activation.…”

“…surface reducing activity by complement activation and inhibition 35 and provides a mechanism by which the complement system can promote TF oxidation in a PDI-dependent thiol-disulfide exchange pathway.…”

Functional analyses of complement convertases using C3 and C5-depleted sera

“…CD55 (DAF) and Coxsackie adenovirus receptor (CAR) were cloned into the pTorsten vector (kind gift of Dr. Brad Spiller, Cardiff University) in frame with C-terminal human Fc tag and expressed in CHO cells, as described [29]. C4BP [30], FI [31] and FH [32] were purified from human plasma as described previously and thioredoxin-1 (Trx-1) as well as its active site mutant CC/SS variant (D Trx-1) were produced and purified as described [33]. Trx-1 and D Trx-1 as well as BSA (Fraction V, pH 5.2, Sigma) used in convertase formation assays were first preincubated with DTT at a final concentration of 25 mM for 15 min at 37uC.…”