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

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]

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

• 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