Glycosylation of Erwinase results in active protein less recognized by antibodies

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

• 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