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

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.

Decreasing the immunogenicity of Erwinia chrysanthemi asparaginase via protein engineering: computational approach.

Immunogenicity of therapeutic proteins is one of the main challenges in disease treatment. L-Asparaginase is an important enzyme in cancer treatment which sometimes leads to undesirable side effects such as immunogenic or allergic responses. Here, to decrease Erwinase (Erwinia chrysanthemiL-Asparaginase) immunogenicity, which is the main drawback of the enzyme, firstly conformational B cell epitopes of Erwinase were predicted from three-dimensional structure by three different computational methods. A few residues were defined as candidates for reducing immunogenicity of the protein by point mutation. In addition to immunogenicity and hydrophobicity, stability and binding energy of mutants were also analyzed computationally. In order to evaluate the stability of the best mutant, molecular dynamics simulation was performed. Among mutants, H240A and Q239A presented significant reduction in immunogenicity. In contrast, the immunogenicity scores of D235A slightly decreased according to two servers. Binding affinity of substrate to the active site reduced significantly in K265A and E268A. The final results of molecular dynamics simulation indicated that H240A mutation has not changed the stability, flexibility, and the total structure of desired protein. Overall, point mutation can be used for reducing immunogenicity of therapeutic proteins, in this context, in silico approaches can be used to screen suitable mutants.

Allergic reactions and antiasparaginase antibodies in children with high-risk acute lymphoblastic leukemia: A children's oncology group report.

BACKGROUND: The objectives of this study were to assess the incidence of clinical allergy and end-induction antiasparaginase (anti-ASNase) antibodies in children with high-risk acute lymphoblastic leukemia treated with pegylated (PEG) Escherichia coli ASNase and to determine whether they carry any prognostic significance. METHODS: Of 2057 eligible patients, 1155 were allocated to augmented arms in which PEG ASNase replaced native ASNase postinduction. Erwinia chrysanthemi (Erwinia) ASNase could be used to replace native ASNase after allergy, if available. Allergy and survival data were complete for 990 patients. End-induction antibody titers were available for 600 patients. RESULTS: During the consolidation phase, 289 of 990 patients (29.2%) had an allergic reaction. There were fewer allergic reactions to Erwinia ASNase than to native ASNase (odds ratio, 4.33; P < .0001) or PEG ASNase (odds ratio, 3.08; P < .0001) only during phase 1 of interim maintenance. There was no significant difference in 5-year event-free survival (EFS) between patients who received PEG ASNase throughout the entire study postinduction versus those who developed an allergic reaction to PEG ASNase during consolidation phase and subsequently received Erwinia ASNase (80.8% ± 2.8% and 81.6% ± 3.8%, respectively; P = .66). Patients who had positive antibody titers postinduction were more likely to have an allergic reaction to PEG ASNase (odds ratio, 2.4; P < .001). The 5-year EFS rate between patients who had negative versus positive antibody titers (80% ± 2.6% and 77.7% ± 4.3%, respectively; P = .68) and between patients who did not receive any ASNase postconsolidation and those who received PEG ASNase throughout the study (P = .22) were significantly different. CONCLUSIONS: The current results demonstrate differences in the incidence rates of toxicity between ASNase preparations but not in EFS. The presence of anti-ASNase antibodies did not affect EFS.

Specificity of monoclonal antibodies to strains of Dickeya sp. that cause bacterial heart rot of pineapple.

During a severe outbreak of bacterial heart rot that occurred in pineapple plantations on Oahu, Hawaii, in 2003 and years following, 43 bacterial strains were isolated from diseased plants or irrigation water and identified as Erwinia chrysanthemi (now Dickeya sp.) by phenotypic, molecular, and pathogenicity assays. Rep-PCR fingerprint patterns grouped strains from pineapple plants and irrigation water into five genotypes (A-E) that differed from representatives of other Dickeya species, Pectobacterium carotovorum and other enteric saprophytes isolated from pineapple. Monoclonal antibodies produced following immunization of mice with virulent type C Dickeya sp. showed only two specificities. MAb Pine-1 (2D11G1, IgG1 with kappa light chain) reacted to all 43 pineapple/water strains and some reference strains (D. dianthicola, D. chrysanthemi, D. paradisiaca, some D. dadantii, and uncharacterized Dickeya sp.) but did not react to reference strains of D. dieffenbachiae, D. zeae, or one of the two Malaysian pineapple strains. MAb Pine-2 (2A7F2, IgG3 with kappa light chain) reacted to all type B, C, and D strains but not to any A or E strains or any reference strains except Dickeya sp. isolated from Malaysian pineapple. Pathogenicity tests showed that type C strains were more aggressive than type A strains when inoculated during cool months. Therefore, MAb Pine-2 distinguishes the more virulent type C strains from less virulent type A pineapple strains and type E water strains. MAbs with these two specificities enable development of rapid diagnostic tests that will distinguish the systemic heart rot pathogen from opportunistic bacteria associated with rotted tissues. Use of the two MAbs in field assays also permits the monitoring of a known subpopulation and provides additional decision tools for disease containment and management practices.

Erwinia chrysanthemi L-asparaginase: epitope mapping and production of antigenically modified enzymes.

This study shows that the antigenicity of Erwinia chrysanthemi L-asparaginase can be reduced by site-directed mutagenesis. Ten B-cell epitopes of the enzyme were identified using synthetic hexapeptides and polyclonal antisera from rabbits and mice. The region 282GIVPPDEELP292 near the C-terminus was an immunodominant epitope. Binding of two hexapeptides (283IVPPDE288 and 287DEELPG292) to the antibodies was dependent on Pro285, and Pro286, since their replacement by almost any other amino acid resulted in reduced binding. The other residues were less important for binding the antibodies, as binding was relatively unaffected by amino acid substitutions. Three site-directed mutant enzymes, P285T (proline-285-->threonine etc.), P286Q and E288A, were expressed in Escherichia coli. The purified enzymes had subunit M(r) values of 35,000. The pI values of P285T, P286Q and the wild-type enzymes were 8.6, and that for the mutant E288A was 9.2. The kcat. and Km values for the mutants P286Q and E288A with L-asparagine and L-glutamine were comparable with those of the wild-type enzyme. The Km values for the mutant P285T with both substrates was similar to that of the wild-type enzyme, whereas the kcat. was reduced by 2-fold with L-asparagine and by 4-fold with L-glutamine. The change proline-->threonine reduced the antigenicity of the enzyme by 8-fold, as shown in sandwich e.l.i.s.a.s. using monoclonal antibodies raised against the wild-type enzyme.

Verification of ELISA results by immunomagnetic isolation of antigens from extracts and analysis with SDS-PAGE and western blotting, demonstrated for Erwinia spp. in potatoes.

Isolation of antigens on immunomagnetic beads and subsequent analysis with SDS-PAGE and Western blotting (immunomagnetic isolation-Western blotting (IMI-WB)) was used to verify positive ELISA results for Erwinia chrysanthemi and Erw. carotovora subsp. atroseptica in potato peel extracts. Direct analysis of highly contaminated extracts by Western blotting without previous immuno-isolation resulted in background reactions, whereas immunomagnetic isolation resulted in distinct bands of specific antigens. Target cells as well as antigenic cell products were captured in IMI-WB. Band patterns on IMI-WB of cell-free culture filtrates and cell suspensions were highly similar, but the removal of cells lowered the detection level by 10- to 100-fold. Threshold levels of IMI-WB were generally comparable with those of ELISA. No differences in threshold levels and band patterns were found between a direct format and an indirect format of immuno-isolation. In IMI-WB, blotting patterns differed between Erw. chrysanthemi and Erw. carotovora subsp. atroseptica. The patterns were identical for 15 Erw. chrysanthemi strains, isolated from potato peel extracts in The Netherlands. However, one of 15 strains of Erw, carotovora subsp. atroseptica from potato peel extracts in The Netherlands gave an aberrant pattern. Target bacteria could be easily distinguished from those of cross-reacting strains on the basis of band patterns. Potato peel extracts naturally contaminated with Erw. chrysanthemi gave IMI-WB patterns that were similar to pure cultures of the homologous strains.