Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice.
These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins.In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H(2)O(2)-generating system.Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.
Affiliation: University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge, UK. firstname.lastname@example.org
- Ascorbic Acid/metabolism*/pharmacology
- Endoplasmic Reticulum/metabolism*
- Cells, Cultured
- Connective Tissue/metabolism/pathology
- Disease Models, Animal
- Mice, Mutant Strains
- Protein Folding
- Protein Processing, Post-Translational/drug effects
- Sulfenic Acids/metabolism
- Transforming Growth Factor beta/metabolism
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fig6: Elevated Cysteinyl Sulfenic Acid in Proteins from Mutant Cells and Their Role in Ascorbate Depletion(A) Representative immunoblot of extracts from cells exposed to the sulfenic acid reactive probe dimedone with an antibody reactive to dimedone-conjugated cysteine residues. The bar diagram is the mean ± SEM of the dimedone signal (integrated across each lane and expressed in arbitrary units, au) from experiments as shown above (n = 3, ∗∗p < 0.01).(B) As in (A), but the cells were fractionated to a cytosolic fraction and a membrane (microsomal) fraction by digitonin permeabilization and cytosolic squeeze-out. Calnexin (CNX) and eIF2α serve as compartment markers for the ER and cytosol. The asterisk indicates a faster-migrating species reactive with the calnexin antiserum that is reproducibly detected in the membrane fraction of the mutant cells and may correspond to sulfenylated calnexin (Leonard et al., 2009).(C) Bar diagram of the total glutathione (GSH + GSSG) and oxidized glutathione (GSSG) content in picomols glutathione/μg of proteins of cells of the indicated genotypes (n = 3, ∗∗p < 0.01).(D) Schema of the experiment to test the effect of protein cysteinyl sulfenic acid in catalyzing ascorbic acid oxidation. The active site cysteine of papain (P) is found as both a thiol (−SH) and cysteinyl sulfenic acid (−SOH, due to air oxidation). H2O2 converts all the −SH to −SOH, which then reacts irreversibly with dimedone, blocking the active cysteine (−S−DM). Following gel filtration, to remove remaining H2O2 and dimedone, this generates an inactive control (Cont). Ascorbate reduces any −SOH back to −SH. Removal of the ascorbate by gel filtration generates the active (Test) protein. However, the free thiol can be blocked by NEM, generating an inactive alkylated (−S−M) protein (Cont). The test and control proteins are incorporated at substochiometric concentrations into an assay with an H2O2-generating system (glucose + glucose oxidase) and ascorbate, whose consumption is measured by reading the absorbance at 265 nm.(E) Time-dependent changes in absorbance of ascorbate (at 265 nm, initial concentration 100 μM) observed in the experimental system described in (B). “Papain,” trace of sample with no H2O2-generating system but with 0.2 μM papain. “glu/gluox,” trace of sample with no papain. “Papain, NEM, glu/gluox,” trace of sample with NEM-blocked papain (0.2 μM). “Papain, Dimedone, glu/gluox,” trace of sample with dimedone-blocked papain (0.2 μM). “Papain, glu/gluox,” trace of sample with active papain (0.2 μM).(F) The initial rate of ascorbate oxidation (in OD units/time) as a function of papain concentration (the initial rate of the uncatalyzed reaction, with only glucose and glucose oxidase, was subtracted).
Ascorbic acid can react with cysteinyl sulfenic acid side chains. The resulting transfer of two electrons converts ascorbate to an unstable oxidized derivative, dehydroascorbate (which is rapidly hydrolysed to 2,3 diketo l-gulonate), and reduces the sulfenic acid, regenerating the free thiol (Monteiro et al., 2007). Therefore, elevated levels of sulfenylated cysteines could accelerate the clearance of ascorbate from the mutant cell’s ER and contribute to the defect in collagen biogenesis. To compare the burden of sulfenylated proteins in MEFs of divergent genotypes, we exposed cells and lysates to dimedone, a chemical that selectively modifies sulfenylated cysteines, and detected the dimedone-modified proteins by immunoblot with an antibody to the chemically modified group (Seo and Carroll, 2009). In absence of dimedone, the antibody gave a weak background signal (Figure 6A, lane 1), and the dimedone-dependent signal was effaced by incubating cells with DTT, a direct reductant of sulfenic acid (Figure S4). Importantly, the sulfenic acid signal was notably higher in the DM and TM cells (Figure 6A, lanes 3 and 4) and was enriched in the ER-containing membrane fraction (Figure 6B). The quantity of sulfenylated proteins detected by dimedone modification decreased following exposure to ascorbate (Figure 6A, lane 5), an observation consistent with a role for ascorbate as a reductant of sulfenylated proteins.