The antigenic surface of staphylococcal nuclease. I. Mapping epitopes by site-directed mutagenesis.
The analysis of the antigenic surface of staphylococcal nuclease was begun by generating and characterizing a panel of mAb. Twelve mAb were selected from a large number of anti-nuclease mAb and characterized for affinity and isotype, by their ability to block enzyme activity, and by complementation and competitive inhibition assays for the relative location of epitopes. The mAb were placed in complementation groups based on their distinct binding patterns. These groups define a series of eight overlapping epitopes that are estimated to cover a large portion of the nuclease surface. Four mAb blocked the enzyme activity of nuclease. The epitopes defined by two of these four mAb were localized on the surface of nuclease using single amino acid variant Ag generated by site-directed mutagenesis of the cloned nuclease coding sequence. mAb-25 maps to residue 46 which is located at the edge of the enzyme active site consistent with its ability to inhibit enzyme activity. mAb-19, which also blocks enzyme activity and belongs to the same complementation group as mAb-25, was unaffected by the substitution at position 46. This suggests that mAb-19 and mAb-25, if they do react with the same epitope, have differences in fine specificity. mAb-22 blocks enzyme activity and belongs to an overlapping complementation group. The fourth mAb, mAb-1, which belongs to a distinct, nonoverlapping, complementation group, does not blocks enzyme activity, and is directed to a region of nuclease that includes the amino acid at position 133. This residue is located a short distance from the active site in a region that has been suggested to participate in binding of DNA, a substrate for nuclease. Therefore, the four epitopes defined by these mAb are localized at or near the enzyme active site.
Smith
A M
AM
Department of Microbiology, School of Medicine, University of Virginia, Charlottesville 22908.
Woodward
M P
MP
Hershey
C W
CW
Hershey
E D
ED
Benjamin
D C
DC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000939:Epitopes / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D008836:Micrococcal Nuclease / Q000276:immunology*; D016297:Mutagenesis, Site-Directed
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008836:Micrococcal Nuclease