Glycosylation of L-asparaginase from E. coli throu... - BV FAPESP
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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Glycosylation of L-asparaginase from E. coli through yeast expression and site-directed mutagenesis

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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