| Full text | |
| Author(s): |
Effer, Brian
;
Kleingesinds, Eduardo Krebs
;
Lima, Guilherme Meira
;
Costa, Iris Munhoz
;
Sanchez-Moguel, Ignacio
;
Pessoa, Adalberto
;
Santiago, Veronica Feijoli
;
Palmisano, Giuseppe
;
Farias, Jorge G.
;
Monteiro, Gisele
Total Authors: 10
|
| Document type: | Journal article |
| Source: | Biochemical Engineering Journal; v. 163, p. 11-pg., 2020-11-15. |
| Abstract | |
L-asparaginase (L-ASNase) is an important bacterial enzyme used as a biopharmaceutical to treat acute lymphoblastic leukemia (ALL). Side effects of L-ASNase therapy have been counteracted by polyethylene glycol (PEG)-modification of L-ASNase; however, immunogenicity of PEG has been observed in patients. Here we explore L-ASNase glycosylation as a biological alternative to the PEGylation industrial process. In our results, a recombinant Erwinase expressed in the Glycoswitch (R) Pichia pastoris strain occurred in three extracellular and glycosylated active variants; two tetrameric forms: Erw240 (240 kDa) and Erw160 (160 kDa), with a specific activity of 15.71 and 302.02 U mg(-1) respectively; and one monomeric version: Erw40 (40 kDa), with a specific activity of 48.45 U mg(-1). The lighter tetramer and the monomer showed a catalytic efficiency of 7.7 x 10(5) and 1.05 x 10(6) M-1 s(-1) respectively, while the heaviest form considerably lost its activity. Mass spectrometry analysis determined that Erw40 and Erw160 tetramer were N-glycosylated at Asn(170) mostly with GlcNAc(2)Man(7) N-glycans. These glycosylation sites are part of a predicted immunogenic T-cell epitope related to ASNase allergy in ALL patients. ELISA assay showed a significant reduction of antibody recognition of the glycosylated Erwinase suggesting that the glycans had a cloaking effect against antibodies. The new L-ASNase versions reported here could provide an alternative for the treatment of ALL. (AU) | |
| FAPESP's process: | 13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical |
| Grantee: | Adalberto Pessoa Junior |
| Support Opportunities: | Research Projects - Thematic Grants |
| FAPESP's process: | 15/07749-2 - Protein engineering and comparison of microbial expression systems of the biopharmaceutical L-asparaginase |
| Grantee: | Gisele Monteiro |
| Support Opportunities: | Regular Research Grants |
| FAPESP's process: | 17/20384-9 - Development of downstream process of humanized L-asparaginase and its characterization |
| Grantee: | Eduardo Krebs Kleingesinds |
| Support Opportunities: | Scholarships in Brazil - Doctorate |
| FAPESP's process: | 18/15104-0 - Preclinical assays of glycoprotein asparaginase proteoforms or resistant to serum proteases. |
| Grantee: | Gisele Monteiro |
| Support Opportunities: | Regular Research Grants |
| FAPESP's process: | 16/15787-4 - Cloning of L-Asparaginase from Escherichia coli in a Pichia pastoris strain with humanized glysosylation |
| Grantee: | Guilherme Meira Lima |
| Support Opportunities: | Scholarships in Brazil - Scientific Initiation |
| 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 |