Comparative immunogenicity and structural analysis of epitopes of different bacterial L-asparaginases

Abstract

Background: E.coli type II L-asparaginase is widely used for treatment of acute lymphoblastic leukemia. However, serious side effects such as allergic or hypersensitivity reactions are common for L-asparaginase treatment. Methods for minimizing immune response on L-asparaginase treatment in human include bioengeneering of less immunogenic version of the enzyme or utilizing the homologous enzymes of different origin. To rationalize these approaches we compared immunogenicity of L-asparaginases from five bacterial organisms and performed sequence-structure analysis of the presumable epitope regions.

Methods: IgG and IgM immune response in C57B16 mice after immunization with Wollinella succinogenes type II (WsA), Yersinia pseudotuberculosis type II (YpA), Erwinia carotovora type II (EwA), and Rhodospirillum rubrum type I (RrA) and Escherichia coli type II (EcA) L-asparaginases was evaluated using standard ELISA method. The comparative bioinformatics analysis of structure and sequence of the bacterial L-asparaginases presumable epitope regions was performed.

Results: We showed different immunogenic properties of five studied L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin as a second-line treatment. Studied L-asparaginases might be placed in the following order based on the immunogenicity level: YpA > RrA, WsA ≥ EwA > EcA. Most significant cross-immunogenicity was shown between EcA and YpA. We propose that a long N-terminus of YpA enzyme enriched with charged aminoacids and tryptophan could be a reason of higher immunogenicity of YpA in comparison with other considered enzymes. Although the recognized structural and sequence differences in putative epitope regions among five considered L-asparaginases does not fully explain experimental observation of the immunogenicity of the enzymes, the performed analysis set the foundation for further research in this direction.

Conclusions: The performed studies showed different immunogenic properties of L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin. The preferable enzymes for the second line treatment are WsA, RrA, or EwA.

Fig. 1

GMTs for serum anti-L-asparaginases-specific neutralizing antibodies, IgG response

Fig. 2

Structure-based multiple sequence alignment of five studied L-asparaginases. Four lines below the alignment show putative epitope region predicted by EPSVR, Discotope, and ElliPro methods, respectively, as well as the experimentally known epitopes projected from the Erwinia chrysanthemi L-asparaginase. Lines above the alignment represent the secondary structure and the estimation of solvent accessibility of EcA tetramer

Fig. 3

Three-dimensional structure of the EcA protein with a color mapping representing the example of the epitope prediction obtained by EPSRV method (blue – low probability, red – high probability). Following key structural elements of the enzyme are designated in the figure: three N-terminal active site loops, two C-terminal active site loops, multi-subunit interface regions and inter-domain linker. Inset: Tetrameric structure of Escherichia coli L-asparaginase EcA

Fig. 4

Superimposition of the predicted epitopes of EcA (red), EwA (yellow), WsA (green), YpA (blue) L-asparaginases including C-terminal active site loops (a), inter-domain linker (b), N-terminal active site loops (c), and multi-subunit interface regions (d)