title: E. coli sabotages the in vivo production of O-linked β-N-acetylglucosamine-modified proteins.
abstract: The O-linked β-N-acetylglucosamine (O-GlcNAc) post-translational modification is an important, regulatory modification of cytosolic and nuclear enzymes. To date, no 3-dimensional structures of O-GlcNAc-modified proteins exist due to difficulties in producing sufficient quantities with either in vitro or in vivo techniques. Recombinant co-expression of substrate protein and O-GlcNAc transferase in Escherichia coli was used to produce O-GlcNAc-modified domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2). Recombinant expression in E. coli is an advantageous approach, but only small quantities of insoluble O-GlcNAc-modified protein were produced. Adding β-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-dexoy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), to the culture media provided the first evidence that an E. coli enzyme cleaves O-GlcNAc from proteins in vivo. With the inhibitor present, the yields of O-GlcNAc-modified protein increased. The E. coli β-N-acetylglucosaminidase was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. The identity of the interfering β-N-acetylglucosaminidase was confirmed by testing a nagZ knockout strain. In E. coli, NagZ natively cleaves the GlcNAc-β1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm and cleaves the GlcNAc-β-O-linkage of foreign O-GlcNAc-modified proteins in vivo, sabotaging the recombinant co-expression system.
authors.affiliation: Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, United States.
authors.forename: Octavia Y
authors.initials: OY
authors.lastname: Goodwin
authors.forename: Maggie S
authors.initials: MS
authors.lastname: Thomasson
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Lin
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Sweeney
authors.forename: Megan A
authors.initials: MA
authors.lastname: Macnaughtan
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000118:Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism; D010091:Oximes / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D048448:Phenylcarbamates / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D010091:Oximes; D010455:Peptides; D048448:Phenylcarbamates; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D011505:Protein-Tyrosine Kinases; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine
keywords: 4,4-dimethyl-4-silapentane-1-sulfonic acid; 4-methylumbelliferyl-β-N-acetylglucosaminide; ABL2; CREB1; CXC; D(2)O; DSS; E. coli; E. coli β-N-acetylglucosaminidase; F-actin binding domain; FABD; FITC-WGA; Glycosidase; K(m); LB; LOQ; Luria broth; MU-GlcNAc; Michaelis–Menten constant; MurNAc; N-acetylmuramic acid; NMR; NaCl; NagZ; O-(2-acetamido-2-dexoy-d-glucopyranosylidene)amino-N-phenylcarbamate; O-GlcNAc; O-GlcNAc transferase; O-GlcNAcase; O-linked β-N-acetylglucosamine; OGA; OGT; PUGNAc; Q2; S/N; SDS-PAGE; UDP-GlcNAc; cation exchange chromatography; deuterated water; fluorescein-conjugated wheat-germ agglutinin; human Abelson tyrosine-kinase 2; human cAMP responsive element-binding protein; limit of quantitation; nagZ knockout; nuclear magnetic resonance; p-nitrophenyl-β-N-acetylglucosamine; pNP-GlcNAc; second glutamine-rich domain; signal-to-noise ratio; sodium chloride; sodium dodeyl sulfate polyacrylamide gel electrophoresis; tris; tris(hydroxymethyl)aminomethane; uridine 5′-diphospho-N-acetylglucosamine; ΔnagZ; β-N-Acetylglucosaminidase