A mutational analysis of the epitopes of recombinant human H-ferritin.
Murine monoclonal antibodies were elicited by the recombinant human H-ferritin overexpressed in Escherichia coli. They had a specificity analogous to that of the antibodies elicited by natural human H-chain, and all of them showed low additivity in binding the recombinant ferritin. Four antibodies of each group were challenged with four H-ferritin mutants overexpressed in E. coli, altered in different accessible areas of the molecule. They consisted of deletions of the first 13 and last 22 amino acids, a duplication of an 18 amino acid sequence in the loop region, and a substitution of a 5 amino acid stretch in the three-fold symmetry axis region. Double diffusion, immunodot analyses and inhibition plots indicated that: (1) all the mutants were recognized by at least one antibody; (2) the deletion of the N-terminus and the duplication in the loop region had the strongest effect on antibody binding; and (3) epitope boundaries of the various antibodies could not be recognized. The antibodies were tested with H-containing ferritins from rat and hen hearts, and showed low or absent reactivities despite their high structural homology with human ferritin. Comparison of the amino acid sequences of human, mouse, rat and hen H-chains, together with mutational data, suggested that; (i) ferritin epitopes are large, probably encompassing a large portion of the subunit surface and (ii) Thr-5 and Cys-90 have a role in H-ferritin immunogenicity.
Department of Biomedical Science and Technology, University of Milan, San Raffaele Hospital, Italy.
P
P
Arosio
A
A
Cozzi
R
R
Ingrassia
S
S
Levi
A
A
Luzzago
G
G
Ruggeri
C
C
Iacobello
P
P
Santambrogio
A
A
Albertini
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D002645:Chickens; D000939:Epitopes / Q000032:analysis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005293:Ferritins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005293:Ferritins