Biochemical characterization and cytotoxic evaluation of mutant isoforms of L-asparaginase II from Dickeya chrysanthemi (Erwinia chrysanthemi)

Author(s)

Iris Munhoz Costa

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2022-07-08

Examining board members

Gisele Monteiro; Patricia Pereira Coltri

• Advisor: Gisele Monteiro

Abstract

Acute lymphoblastic leukemia (ALL) is the most frequent neoplasm in children and adolescents. This tumor is characterized by auxotrophy for the amino acid L-asparagine, which allows the treatment of the disease to be carried out with L-asparaginase (ASNase), an enzyme obtained from the bacteria Escherichia coli and Dickeya chrysanthemi (also called Erwinia chrysanthemi). ASNase hydrolyzes L-asparagine, and L-glutamine to a lesser extent, preventing tumor cells from obtaining L-asparagine from the bloodstream for protein synthesis, leading to cell death by apoptosis. However, the formulations available for therapy are associated with a high rate of adverse effects, such as toxicity and drug resistance caused by the production of antibodies and the action of proteases. In this way, the development of mutant proteoforms from enzymes already commercialized and the study of the action of proteases during treatment can contribute to the development of an enzyme with fewer adverse effects. Therefore, our research group created a library of mutants using the D. chrysanthemi ASNase, by error prone PCR, and from ten mutant clones studied a double mutant proteoform (DM) showed better kinetic parameters than the wild-type enzyme (WT), being highly active under physiological conditions tested in vitro. In addition, the DM enzyme showed less recognition by anti-asparaginase antibodies and the same cytotoxic potential as the WT enzyme, which may cause fewer adverse effects. In addition, we created REH ALL cells knockout for cathepsin B (CTSB) protease by CRISPR/cas9, and evaluated cell viability after treatment with E. coli and D. chrysanthemi WT ASNases, and with the DM enzyme. Our results suggest that the expression of CTSB by REH cells is very low and does not alter the response to treatment with ASNases when evaluated in vitro. (AU)

• FAPESP's process: 16/25896-5 - Biochemical characterization and cytotoxic evaluation of mutant isoforms of L-Asparaginase II from Dickeya chrysanthemi (Erwinia chrysanthemi)
• Grantee: Iris Munhoz Costa
• Support Opportunities: Scholarships in Brazil - Doctorate