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.
Moola
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Division of Biotechnology, Centre for Applied Microbiology and Research, Porton, Salisbury, Wiltshire, U.K.
Scawen
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Atkinson
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Nicholls
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D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis*; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D018360:Crystallography, X-Ray; D016974:Dickeya chrysanthemi / Q000201:enzymology* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D016056:Immunodominant Epitopes / Q000032:analysis*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry; D013912:Threonine / Q000737:chemistry
D000942:Antigens, Bacterial; D016056:Immunodominant Epitopes; D013912:Threonine; D011392:Proline; D001215:Asparaginase