A spontaneous variant of an antidigoxin hybridoma antibody with increased affinity arises from a heavy chain signal peptide mutation.
The A/J murine hybridoma cell line 40-150 secretes antidigoxin antibodies with high affinity for digoxin. A first-order spontaneous mutant (40-150 A2.4) produces antibodies containing a mutation at heavy chain position 94 resulting in reduced affinity for digoxin. A second-order mutant (40-150 A2.4 P.10) derived from 40-150 A2.4 produces two species of antibody: one identical to 40-150 A2.4 and the other with a two amino acid truncation at the heavy chain amino-terminus [Panka et al., Proc. natn. Acad. Sci. U.S.A. 85, 3080-3084 (1988)]. The truncated antibody has increased affinity for digoxin relative to the nontruncated variant. Direct nucleotide sequence analysis of polymerase chain reaction amplified heavy chain variable region cDNA derived from 40-150 A2.4 P.10 reveals a point mutation at the -2 position of the signal peptide, resulting in a glutamine to proline change. Southern blots of genomic DNA from all three cell lines gave identical patterns and were consistent with a single heavy chain mRNA derived from a single rearranged gene. The presence of proline at the heavy chain -2 position of antibody 40-150 A2.4 P.10 partially shifts the cleavage site of the signal peptidase to the +2 position, resulting in the production of both full-length and truncated antibody heavy chains. Signal peptide mutation resulting in a change in antibody affinity for antigen is a hitherto unidentified possible mechanism for antibody diversification.
Shaw
S Y
SY
Department of Medicine, Massachusetts General Hospital, Boston 02114.
Margolies
M N
MN
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000032:analysis; D004077:Digoxin / Q000276:immunology*; D015321:Gene Rearrangement; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008805:Mice, Inbred A; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals; D012689:Sequence Homology, Nucleic Acid
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D021382:Protein Sorting Signals; D004077:Digoxin; D004247:DNA