title: Reductive methylation and mutation of an anthrax toxin fusion protein modulates its stability and cytotoxicity.
abstract: We characterized an anti-cancer fusion protein consisting of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas exotoxin A by (i) mutating the N-terminal amino acids and by (ii) reductive methylation to dimethylate all lysines. Dimethylation of lysines was achieved quantitatively and specifically without affecting binding of the fusion protein to PA or decreasing the enzymatic activity of the catalytic moiety. Ubiquitination in vitro was drastically decreased for both the N-terminally mutated and dimethylated variants, and both appeared to be slightly more stable in the cytosol of treated cells. The dimethylated variant showed greatly reduced neutralization by antibodies to LF. The two described modifications offer unique advantages such as increased cytotoxic activity and diminished antibody recognition, and thus may be applicable to other therapeutic proteins that act in the cytosol of cells.
authors.affiliation: 1] Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA [2].
authors.forename: Christopher
authors.initials: C
authors.lastname: Bachran
authors.affiliation: 1] Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA [2].
authors.forename: Pradeep K
authors.initials: PK
authors.lastname: Gupta
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Silke
authors.initials: S
authors.lastname: Bachran
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Clinton E
authors.initials: CE
authors.lastname: Leysath
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Hoover
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Rasem J
authors.initials: RJ
authors.lastname: Fattah
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Leppla
mesh_terms: D036002:ADP Ribose Transferases / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics*; D000970:Antineoplastic Agents; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005098:Exotoxins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008745:Methylation; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity*; D054875:Ubiquitination; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000942:Antigens, Bacterial; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D000939:Epitopes; D005098:Exotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C030325:anthrax toxin
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