| Full text | |
| Author(s): |
Lima, Guilherme Meira
[1]
;
Effer, Brian
[1, 2]
;
Biasoto, Henrique Pellin
[1]
;
Feijoli, Veronica
[3]
;
Pessoa, Adalberto
[1]
;
Palmisano, Giuseppe
[3]
;
Monteiro, Gisele
[1]
Total Authors: 7
|
| Affiliation: | [1] Univ Sao Paulo, Fac Ciencias Farmaceut, Dept Tecnol Bioquim Farmaceut, Sao Paulo - Brazil
[2] Univ La Frontera, Fac Engn & Sci, Dept Chem Engn, Temuco - Chile
[3] Univ Sao Paulo, Inst Biomed Sci, Dept Parasitol, Sao Paulo - Brazil
Total Affiliations: 3
|
| Document type: | Journal article |
| Source: | Biochemical Engineering Journal; v. 156, APR 15 2020. |
| Web of Science Citations: | 0 |
| Abstract | |
L-Asparaginase (L-ASNase) is a key component in the treatment of acute lymphoblastic leukemia (ALL), but several clinical disadvantages, such as immunogenicity and rapid clearance, are still present. We evaluated the possibility to synthesize a new L-ASNase from Escherichia coll. with human-like glycosylation and study the glycosylation effect on the biochemical properties of the enzyme. Six L-ASNase mutants were also created in which L-ASNase glycosylation sites were removed through site-directed mutagenesis. The WT L-ASNase was successfully expressed, secreted and glycosylated by an engineered P. pastoris strain and presented predominantly Man 5 G1cNAc 2 glycans on its structure, which were then able to decrease L-ASNase immunogenicity in vitro. The purified glycosylated L-ASNase has shown a 30-fold decrease in specific enzymatic activity compared to the non-glycosylated proteoform, but a triple mutant L-ASNase (3M) was able to restore L-ASNase biological activity to significant levels. 3M accumulated in the yeast periplasmic space and there presented a 28fold increase in enzymatic activity when compared to the fully glycosylated proteoform. Both WT and 3M L-ASNases presented increased stability in human serum compared to non-glycosylated L-ASNase. This study demonstrates the important effects of glycosylation on L-ASNase properties and opens up new possibilities to use glycosylated L-ASNases for the treatment of ALL. (AU) | |
| 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: | 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: | 14/06863-3 - Post-translational modifications in cancer and parasite infection diagnosis: methodological approaches and biological implications |
| Grantee: | Giuseppe Palmisano |
| Support Opportunities: | Research Grants - Young Investigators Grants |
| FAPESP's process: | 18/18257-1 - Multi-user equipment approved in grant 14/06863-3: HPLC system configured for analysis of carbohydrates, amino acidis, peptides and glycoproteins |
| Grantee: | Giuseppe Palmisano |
| Support Opportunities: | Multi-user Equipment Program |
| FAPESP's process: | 18/15549-1 - Post-translational modifications in Chagas Disease biological processes and diagnostics: novel methodological approaches and biological applications |
| Grantee: | Giuseppe Palmisano |
| Support Opportunities: | Research Grants - Young Investigators Grants - Phase 2 |
| 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 |