J. Biol. Chem., Vol. 263, Issue 35, 18702-18715, 12, 1988
A study of fine specificity of monoclonal antibodies to yeast iso-1- cytochrome c
I Silvestri and H Taniuchi
Laboratory of Chemical Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Seven monoclonal antibodies, prepared to yeast holo- or apo-iso-1-
cytochrome c by the method of Kohler and Milstein (Goding, J. W. (1983)
Monoclonal Antibodies: Principles and Practice, Academic Press, Orlando,
FL) were characterized by cross-reaction with a panel of evolutionarily
related cytochromes c, apocytochromes c, fragments and homologous and
hybrid fragment complexes, inhibition, competitive inhibition, and
complementation and fluorescence titration. The results have permitted us
to assign the specifically recognized amino acids as follows. IgG1
monoclonals: 4-74-6, Leu-63 and/or Asn-67 and/or Asn-68; 4-128-6, Glu-93;
4-145-10, Thr-74; 2-96-12, Asp-65; 2-34-19, Lys-59; and 10-28-86,
trimethyl-lysine 77. IgM monoclonal 39-14, Pro-30 and His- 31. With mAb
4-128-6 substitution of glutamic acid 93 with alanine, as it occurs in
Candida cytochrome c, has resulted in a decrease in affinity by a factor of
10(4). A calculation appears to show that this value is too large to be
accounted for solely by the sum of energy losses due to disruption of
charge neutralization and changes of hydrophobic interaction including van
der Waals interaction. This and similar results with mAb 10-28-86 have led
us to the idea that some new extra interatomic interaction sensitive to
differences in configuration of atomic groups may be present and perturbed
in the substitution. Furthermore, the assumption of the presence of such
interaction can explain the striking similarity between the
antigen-antibody interaction (e.g. Geysen, H. M., Meloen, R. H., and
Barteling, S. J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3998-4002) and a
model system of horse cytochrome c three-fragment complex (Juillerat, M.
A., and Taniuchi, H. (1986) J. Biol. Chem. 261, 2697-2711) with respect to
the high specificity of interacting residues in the interface. Thus, by
analogy to the hypothesis developed in the model system (Fisher, A., and
Taniuchi, H. (1988) FASEB J. 2, A1338), we hypothesize that a closed
interaction loop would be formed on the basis of contacting groups
including glutamic acid 93 across or within the interface between the
antigen and mAb 4-128-6 and mediate delocalized interaction to generate
extra binding energy.
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