Glycosylation of Erwinase results in active protein less recognized by antibodies

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.