Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice.
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.
Affiliation: University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge, UK. firstname.lastname@example.orgShow MeSH
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Mentions: Ascorbic acid maintains the activity of ER-localized proline 4-hydroxylases. Failure of proline 4 hydroxylation blocks procollagen secretion and leads to its retention in the ER (Juva et al., 1966). To determine if the procollagen retained in the ER of the mutant cells had abnormally low levels of 4-hydroxyproline, we used BFA to trap procollagen in the ER of MEFs of all three genotypes and assayed the cellular content of 4-hydroxyproline by hydrolysing proteins to amino acids and converting 4-hydroxyproline to a pyrrole by sequential oxidation and decarboxylation, in a colorigenic reaction (Prockop and Udenfriend, 1960). To validate this approach to trapping procollagen in wild-type cells, we confirmed that the 4-hydroxyproline content of wild-type MEFs increased following BFA treatment (Figure S3A). Procollagen 4-hydroxyproline content was 3-fold higher in the BFA-treated wild-type MEFs compared to the DM and 6-fold higher than in the TM cells (Figure 5A).
Affiliation: University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge, UK. email@example.com