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Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice.

Zito E, Hansen HG, Yeo GS, Fujii J, Ron D - Mol. Cell (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge, UK. ez235@medschl.cam.ac.uk

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A Subtle Kinetic Defect in Disulfide Bond Formation in Mice Compromised in ERO1 and PRDX4(A) Immunoblot of the indicated proteins in extracts of lipopolysaccharide-induced lymphoblasts produced from wild-type (WT), Ero1αm/m; Ero1βm/m compound double mutant (DM), and Ero1αm/m; Ero1βm/m; Prdx4m/y compound triple mutant (TM) mice.(B) Nonreducing and reducing immunoblot of IgM heavy chain extracted from lipopolysaccharide-induced lymphoblasts of the indicated genotypes. Where noted, the cells were exposed to a 30 min pulse of the reducing agent dithiothreitol (DTT, 10 mM) followed by a washout period. The migration of IgM monomers (μ), dimers (2μ), and pentamers (5μ) is noted, and their relative distribution in the various samples is depicted graphically in the central panel. Note that despite the minor kinetic defect, nearly all the reduced monomeric IgM is converted to oxidized higher-molecular-weight forms in the mutant cells over time.(C) Reducing immunoblot of insoluble high-molecular-weight complex (HMW) and total IgM heavy chain extracted from lipopolysaccharide-induced lymphoblasts of the indicated genotypes. The HMW complex (upper panel) was recovered as a pellet from the detergent lysates layered on a glycerol cushion, as previously described (Marciniak et al., 2004). The lower panel reports of the total content of IgM heavy chain in the sample and the relative content of insoluble HMW complex is noted below each lane.
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fig1: A Subtle Kinetic Defect in Disulfide Bond Formation in Mice Compromised in ERO1 and PRDX4(A) Immunoblot of the indicated proteins in extracts of lipopolysaccharide-induced lymphoblasts produced from wild-type (WT), Ero1αm/m; Ero1βm/m compound double mutant (DM), and Ero1αm/m; Ero1βm/m; Prdx4m/y compound triple mutant (TM) mice.(B) Nonreducing and reducing immunoblot of IgM heavy chain extracted from lipopolysaccharide-induced lymphoblasts of the indicated genotypes. Where noted, the cells were exposed to a 30 min pulse of the reducing agent dithiothreitol (DTT, 10 mM) followed by a washout period. The migration of IgM monomers (μ), dimers (2μ), and pentamers (5μ) is noted, and their relative distribution in the various samples is depicted graphically in the central panel. Note that despite the minor kinetic defect, nearly all the reduced monomeric IgM is converted to oxidized higher-molecular-weight forms in the mutant cells over time.(C) Reducing immunoblot of insoluble high-molecular-weight complex (HMW) and total IgM heavy chain extracted from lipopolysaccharide-induced lymphoblasts of the indicated genotypes. The HMW complex (upper panel) was recovered as a pellet from the detergent lysates layered on a glycerol cushion, as previously described (Marciniak et al., 2004). The lower panel reports of the total content of IgM heavy chain in the sample and the relative content of insoluble HMW complex is noted below each lane.

Mentions: To assess the impact of the triple mutant genotype on rate of disulfide bond formation in the ER of secretory cells, we prepared lipopolysaccharide stimulated splenocytes (so-called LPS blasts) from TM, DM, and wild-type animals and compared the rate at which disulfide bonds recovered in the cell-associated pool of immunoglobulin M (IgM) following a pulse of the reducing agent DTT. While a subtle kinetic delay was evident in both mutant samples (and was slightly more conspicuous in the TM compared with the DM cells), this experiment showed that disulfide bond formation proceeded rapidly even in cells deficient in all three ER thiol oxidases, ERO1α, ERO1β, and PRDX4 (Figures 1A and 1B).


Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice.

Zito E, Hansen HG, Yeo GS, Fujii J, Ron D - Mol. Cell (2012)

A Subtle Kinetic Defect in Disulfide Bond Formation in Mice Compromised in ERO1 and PRDX4(A) Immunoblot of the indicated proteins in extracts of lipopolysaccharide-induced lymphoblasts produced from wild-type (WT), Ero1αm/m; Ero1βm/m compound double mutant (DM), and Ero1αm/m; Ero1βm/m; Prdx4m/y compound triple mutant (TM) mice.(B) Nonreducing and reducing immunoblot of IgM heavy chain extracted from lipopolysaccharide-induced lymphoblasts of the indicated genotypes. Where noted, the cells were exposed to a 30 min pulse of the reducing agent dithiothreitol (DTT, 10 mM) followed by a washout period. The migration of IgM monomers (μ), dimers (2μ), and pentamers (5μ) is noted, and their relative distribution in the various samples is depicted graphically in the central panel. Note that despite the minor kinetic defect, nearly all the reduced monomeric IgM is converted to oxidized higher-molecular-weight forms in the mutant cells over time.(C) Reducing immunoblot of insoluble high-molecular-weight complex (HMW) and total IgM heavy chain extracted from lipopolysaccharide-induced lymphoblasts of the indicated genotypes. The HMW complex (upper panel) was recovered as a pellet from the detergent lysates layered on a glycerol cushion, as previously described (Marciniak et al., 2004). The lower panel reports of the total content of IgM heavy chain in the sample and the relative content of insoluble HMW complex is noted below each lane.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3473360&req=5

fig1: A Subtle Kinetic Defect in Disulfide Bond Formation in Mice Compromised in ERO1 and PRDX4(A) Immunoblot of the indicated proteins in extracts of lipopolysaccharide-induced lymphoblasts produced from wild-type (WT), Ero1αm/m; Ero1βm/m compound double mutant (DM), and Ero1αm/m; Ero1βm/m; Prdx4m/y compound triple mutant (TM) mice.(B) Nonreducing and reducing immunoblot of IgM heavy chain extracted from lipopolysaccharide-induced lymphoblasts of the indicated genotypes. Where noted, the cells were exposed to a 30 min pulse of the reducing agent dithiothreitol (DTT, 10 mM) followed by a washout period. The migration of IgM monomers (μ), dimers (2μ), and pentamers (5μ) is noted, and their relative distribution in the various samples is depicted graphically in the central panel. Note that despite the minor kinetic defect, nearly all the reduced monomeric IgM is converted to oxidized higher-molecular-weight forms in the mutant cells over time.(C) Reducing immunoblot of insoluble high-molecular-weight complex (HMW) and total IgM heavy chain extracted from lipopolysaccharide-induced lymphoblasts of the indicated genotypes. The HMW complex (upper panel) was recovered as a pellet from the detergent lysates layered on a glycerol cushion, as previously described (Marciniak et al., 2004). The lower panel reports of the total content of IgM heavy chain in the sample and the relative content of insoluble HMW complex is noted below each lane.
Mentions: To assess the impact of the triple mutant genotype on rate of disulfide bond formation in the ER of secretory cells, we prepared lipopolysaccharide stimulated splenocytes (so-called LPS blasts) from TM, DM, and wild-type animals and compared the rate at which disulfide bonds recovered in the cell-associated pool of immunoglobulin M (IgM) following a pulse of the reducing agent DTT. While a subtle kinetic delay was evident in both mutant samples (and was slightly more conspicuous in the TM compared with the DM cells), this experiment showed that disulfide bond formation proceeded rapidly even in cells deficient in all three ER thiol oxidases, ERO1α, ERO1β, and PRDX4 (Figures 1A and 1B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge, UK. ez235@medschl.cam.ac.uk

Show MeSH
Related in: MedlinePlus