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...

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Bibliographic Details
Published in:	Biochemical journal Vol. 302; no. 3; pp. 921 - 927
Main Authors:	MOOLA, Z. B, SCWAEN, M. D, ATKINSON, T, NICHOLLS, D. J
Format:	Journal Article
Language:	English
Published:	Colchester Portland Press 15-09-1994
Subjects:	Amino Acid Sequence Animals Antigenic determinants, haptens, artificial antigens Antigens Antigens, Bacterial - analysis Asparaginase - chemistry Asparaginase - genetics Asparaginase - immunology Asparaginase - metabolism Base Sequence Biological and medical sciences Crystallography, X-Ray Enzyme-Linked Immunosorbent Assay Epitope Mapping Escherichia coli - genetics Fundamental and applied biological sciences. Psychology Fundamental immunology Immunodominant Epitopes - analysis Molecular immunology Molecular Sequence Data Mutagenesis, Site-Directed Pectobacterium chrysanthemi - enzymology Pectobacterium chrysanthemi - immunology Proline - chemistry Threonine - chemistry Ligand binding Antigenicity Antigenic determinant Structure activity relation Enzyme Erwinia chrysanthemi Site directed mutagenesis Bacteria Hydrolases Asparaginase Enterobacteriaceae
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Summary:	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.
ISSN:	0264-6021 1470-8728
DOI:	10.1042/bj3020921