title: N-glycosylation-negative catalase: a useful tool for exploring the role of hydrogen peroxide in the endoplasmic reticulum.
abstract: Disulfide bond formation during protein folding of nascent proteins is associated with the generation of H2O2 in the endoplasmic reticulum (ER). Approaches to quantifying H2O2 directly within the ER failed because of the oxidative environment in the ER lumen, and ER-specific catalase expression to detoxify high H2O2 concentrations resulted in an inactive protein owing to N-glycosylation. Therefore, the N-glycosylation motifs at asparagine-244 and -439 of the human catalase protein were deleted by site-directed mutagenesis. The ER-targeted expression of these variants revealed that the deletion of the N-glycosylation motif only at asparagine-244 (N244) was associated with the maintenance of full enzymatic activity in the ER. Expression of catalase N244 in the ER (ER-Catalase N244) was ER-specific and protected the cells significantly against exogenously added H2O2. With the expression of ER-Catalase N244, a highly effective H2O2 inactivation within the ER was achieved for the first time. Catalase has a high H2O2-inactivation capacity without the need of reducing cofactors, which might interfere with the ER redox homeostasis, and is not involved in protein folding. With these characteristics ER-Catalase N244 is an ideal tool to explore the impact of ER-generated H2O2 on the generation of disulfide bonds or to study the induction of ER-stress pathways through protein folding overload and accumulation of H2O2.
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, 30623 Hannover, Germany. Electronic address: Lortz.Stephan@mh-hannover.de.
authors.forename: S
authors.initials: S
authors.lastname: Lortz
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, 30623 Hannover, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Lenzen
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, 30623 Hannover, Germany.
authors.forename: I
authors.initials: I
authors.lastname: Mehmeti
mesh_terms: D000818:Animals; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D002374:Catalase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology*; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D016086:Lentivirus / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding; D051381:Rats; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D004220:Disulfides; D017382:Reactive Oxygen Species; D001216:Asparagine; D006861:Hydrogen Peroxide; D002374:Catalase
keywords: Catalase; Disulfide bond formation; Endoplasmic reticulum; Free radicals; H(2)O(2); Protein folding