A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense

Abstract

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

Keywords: ALL; L-asparaginase; Penicillium cerradense; immunogenicity.

Figure 1

Maximum likelihood phylogenetic tree of L-asparaginases from several fungal species including P. cerradense. The consensus tree was inferred using IQ-TREE with 1000 ultrafast bootstrap replicates. The tree was rooted using the E. coli sequence as the outgroup and bootstrap support values are indicated below node edges. The Penicillium sp. representatives are highlighted.

Figure 2

Hinge region (HR), active-site llexible loop (ASFL), and active-site residues’ alignment. Alignment of the Penicillium and Aspergillus L-asparaginases including P. cerradense. HR is indicated in red, ASFL in green, the residues relevant to the catalytic activity are highlighted in orange, and variable N-terminus. Consensus is the identity percentage, and full alignment residue differences are shown (in blue) among the compared asparaginases.

Figure 3

Model of the monomeric three-dimensional structure of L-asparaginase from P. cerradense obtained using AlphaFold2 v1.5.5.

Figure 4

Structural overlap of L-asparaginase from P. cerradense (red) to L-asparaginase from E. coli (PDB: 3ECA) (blue) and D. chrysanthemi (PDB: 2JK0) (green).

Figure 5

Sequence-structure conservation mapping for L-asparaginases from Penicillium and Aspergillus genera (used in Figure 2), using the ASNase from P. cerradense as a model. Catalytic cavity is indicated with a black arrow. The figure was generated using the ConSurf server v.3.

Figure 6

Dot plot graph (median and interquartile ranges) representing the immunogenicity degree of the L-asparaginases by epitope density predicted for T-cell immunogenic epitopes for eight alleles (HLA-DRB1*01:01, HLA-DRB1*03:01, HLA-DRB1*04:01, HLA-DRB1*07:01, HLA-DRB1*08:01, HLA-DRB1*11:01, HLA-DRB1*13:01, and HLA-DRB1*15:01). The dots in the box represent the eight alleles evaluated. No statistically significant difference was observed (p < 0.05—Kruskal–Wallis with a posteriori Dunn’s test).

Figure 7

Bar graph representing the epitope density of different L-asparaginases, via predicting T-cell immunogenic epitopes for eight independent alleles.

Figure 8

Epitope density of T-cell allergenic epitopes for the HLA-DRB1*07:01 allele of L-asparaginase from the evaluated microorganisms.

Figure 9

Structural distribution of T-cell allergen epitopes for the HLA-DRB1*07:01 allele in the L-asparaginase monomer. Blue zones represent E. coli allergenic epitopes. Green zones represent D. chrysanthemi allergenic epitopes. Red zones represent P. cerradense allergenic epitopes. Gray zones represent non-allergenic zones.

Figure 10

Structural conservation mapping of T-cell allergen epitopes for the HLA-DRB1*07:01 allele in the L-asparaginases of P. cerradense, E. coli, and D. chrysanthemi. Gray zones represent non-allergenic regions. Blue zones represent E. coli allergenic epitopes. Green zones represent D. chrysanthemi allergenic epitopes. Red zones represent P. cerradense allergenic epitopes.

Figure 11

Bar graph representing the epitope density of B-cell epitopes of L-asparaginase from the evaluated microorganisms.

Figure 12

Structural distribution of B-cell epitopes for L-asparaginase monomers. Blue zones represent E. coli epitopes. Green zones represent D. chrysanthemi epitopes. Red zones represent P. cerradense epitopes. Gray zones represent non-allergenic zones.