RESUMEN
Bacterial L-asparaginase has been a universal component of therapies for childhood acute lymphoblastic leukemia since the 1970s. Two principal enzymes derived from Escherichia coli and Erwinia chrysanthemi are the only options clinically approved to date. We recently reported a study of recombinant L-asparaginase (AnsA) from Rhizobium etli and described an increasing type of AnsA family members. Sequence analysis revealed four conserved motifs with notable differences with respect to the conserved regions of amino acid sequences of type I and type II L-asparaginases, particularly in comparison with therapeutic enzymes from E. coli and E. chrysanthemi. These differences suggested a distinct immunological specificity. Here, we report an in silico analysis that revealed immunogenic determinants of AnsA. Also, we used an extensive approach to compare the crystal structures of E. coli and E. chrysantemi asparaginases with a computational model of AnsA and identified immunogenic epitopes. A three-dimensional model of AsnA revealed, as expected based on sequence dissimilarities, completely different folding and different immunogenic epitopes. This approach could be very useful in transcending the problem of immunogenicity in two major ways: by chemical modifications of epitopes to reduce drug immunogenicity, and by site-directed mutagenesis of amino acid residues to diminish immunogenicity without reduction of enzymatic activity.
Asunto(s)
Asparaginasa/uso terapéutico , Proteínas Bacterianas/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Rhizobium etli/enzimología , Secuencia de Aminoácidos , Asparaginasa/química , Asparaginasa/genética , Asparaginasa/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Dickeya chrysanthemi/química , Dickeya chrysanthemi/enzimología , Dickeya chrysanthemi/genética , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Datos de Secuencia Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Rhizobium etli/química , Rhizobium etli/genética , Rhizobium etli/inmunología , Alineación de SecuenciaRESUMEN
The elucidation of the structure of the O-antigen of Rhizobium etli CE3 predicts that the R. etli CE3 genome must contain genes encoding acetyl- and methyltransferases to confer the corresponding modifications to the O-antigen. We identified three open reading frames (ORFs) upstream of wzm, encoding the membrane component of the O-antigen transporter and located in the lps alpha-region of R. etli CE3. The ORFs encode two putative acetyltransferases with similarity to the CysE-LacA-LpxA-NodL family of acetyltransferases and one putative methyltransferase with sequence motifs common to a wide range of S-adenosyl-L-methionine-dependent methyltransferases. Mutational analysis of the ORFs encoding the putative acetyltransferases and methyltransferase revealed that the acetyl and methyl decorations mediated by these specific enzymes are essential for O-antigen synthesis. Composition analysis and high performance anion exchange chromatography analysis of the lipopolysaccharides (LPSs) of the mutants show that all of these LPSs contain an intact core region and lack the O-antigen polysaccharide. The possible role of these transferases in the decoration of the O-antigen of R. etli is discussed.