title: Mutations in subunit interface and B-cell epitopes improve antileukemic activities of Escherichia coli asparaginase-II: evaluation of immunogenicity in mice.
abstract: L-Asparaginase-II from Escherichia coli (EcA) is a central component in the treatment of acute lymphoblastic leukemia (ALL). However, the therapeutic efficacy of EcA is limited due to immunogenicity and a short half-life in the patient. Here, we performed rational mutagenesis to obtain EcA variants with a potential to improve ALL treatment. Several variants, especially W66Y and Y176F, killed the ALL cells more efficiently than did wild-type EcA (WT-EcA), although nonleukemic peripheral blood monocytes were not affected. Several assays, including Western blotting, annexin-V/propidium iodide binding, comet, and micronuclei assays, showed that the reduction in viability of leukemic cells is due to the increase in caspase-3, cytochrome c release, poly(ADP-ribose) polymerase activation, down-regulation of anti-apoptotic protein Bcl-XL, an arrest of the cell cycle at the G0/G1 phase, and eventually apoptosis. Both W66Y and Y176F induced significantly more apoptosis in lymphocytes derived from ALL patients. In addition, Y176F and Y176S exhibited greatly decreased glutaminase activity, whereas K288S/Y176F, a variant mutated in one of the immunodominant epitopes, showed reduced antigenicity. Further in vivo immunogenicity studies in mice showed that K288S/Y176F was 10-fold less immunogenic as compared with WT-EcA. Moreover, sera obtained from WT-EcA immunized mice and ALL patients who were given asparaginase therapy for several weeks recognized the K288S/Y176F mutant significantly less than the WT-EcA. Further mechanistic studies revealed that W66Y, Y176F, and K288S/Y176F rapidly depleted asparagine and also down-regulated the transcription of asparagine synthetase as compared with WT-EcA. These highly desirable attributes of these variants could significantly advance asparaginase therapy of leukemia in the future.
authors.affiliation: From the School of Biotechnology, KIIT University, Bhubaneswar, Odisha 751024, India.
authors.forename: Ranjit Kumar
authors.initials: RK
authors.lastname: Mehta
authors.forename: Shikha
authors.initials: S
authors.lastname: Verma
authors.forename: Rashmirekha
authors.initials: R
authors.lastname: Pati
authors.forename: Mitali
authors.initials: M
authors.lastname: Sengupta
authors.forename: Biswajit
authors.initials: B
authors.lastname: Khatua
authors.forename: Rabindra Kumar
authors.initials: RK
authors.lastname: Jena
authors.forename: Sudha
authors.initials: S
authors.lastname: Sethy
authors.forename: Santosh K
authors.initials: SK
authors.lastname: Kar
authors.forename: Chitra
authors.initials: C
authors.lastname: Mandal
authors.forename: Klaus H
authors.initials: KH
authors.lastname: Roehm
authors.forename: Avinash
authors.initials: A
authors.lastname: Sonawane
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000970:Antineoplastic Agents / Q000276:immunology / Q000494:pharmacology; D001215:Asparaginase / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D053148:Caspase 3 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018985:Epitopes, B-Lymphocyte / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016296:Mutagenesis; D020125:Mutation, Missense; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000276:immunology; D051020:bcl-X Protein / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C494811:BCL2L1 protein, human; C494812:Bcl2l1 protein, mouse; D018985:Epitopes, B-Lymphocyte; D029968:Escherichia coli Proteins; D051020:bcl-X Protein; D045304:Cytochromes c; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D001215:Asparaginase
keywords: Asparaginase; Cytotoxicity; E. coli; Enzyme Mutation; Escherichia coli; Immunogenicity; Leukemia; Leukemic Cells; Lymphoma; Mice; Microbiology