Structure-function analysis of human IL-6. Epitope mapping of neutralizing monoclonal antibodies with amino- and carboxyl-terminal deletion mutants.
To study the active site(s) of IL-6 we combined mutagenesis of IL-6 with epitope mapping of IL-6 specific mAb. In addition to amino-terminal deletion mutants we described previously, carboxyl-terminal deletion mutants were prepared. Functional analysis showed that deletion of only five carboxyl-terminal amino acids already reduced the bioactivity 1000-fold. A panel of mAb to IL-6 was subsequently analyzed by antibody competition experiments and binding to the amino- and carboxyl-terminal deletion mutants. On the basis of the competition experiments the six neutralizing mAb were divided in two groups (I and II). The binding pattern with the deletion mutants suggested that the region recognized by the four mAb in group I is composed of residues of amino- and carboxyl-terminus: binding of two mAb was abolished after deletion of amino acid Ala I-Ile26, of the third mAb after deletion of the four carboxyl-terminal amino acids whereas the fourth mAb did not bind to either mutant. Group II mAb retained binding to these mutants. Taken together these data suggest that in the native IL-6 molecule amino acid residues of amino and carboxyl terminus are in close proximity and that together they constitute an active site. Furthermore our data suggest that the part of the molecule recognized by group II antibodies is a second site involved in biologic activity.
Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
J P
JP
Brakenhoff
M
M
Hart
E R
ER
De Groot
F
F
Di Padova
L A
LA
Aarden
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D004252:DNA Mutational Analysis; D000939:Epitopes; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015850:Interleukin-6; D011994:Recombinant Proteins