TY - JOUR
T1 - A spontaneous variant of an antidigoxin hybridoma antibody with increased affinity arises from a heavy chain signal peptide mutation
AU - Shaw, Shyh Yu
AU - Margolies, Michael N.
N1 - Funding Information:
*This work was supported by NIH Grant POl-HL19259. $Author to whom correspondence should be addressed at: Jackson 14, Massachusetts General Hospital, Boston, MA 02114, U.S.A.
PY - 1992/4
Y1 - 1992/4
N2 - 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 ofantibody40-150A2.4P.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.
AB - 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 ofantibody40-150A2.4P.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.
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U2 - 10.1016/0161-5890(92)90010-U
DO - 10.1016/0161-5890(92)90010-U
M3 - Article
C2 - 1565100
AN - SCOPUS:0026535719
VL - 29
SP - 525
EP - 529
JO - Immunochemistry
JF - Immunochemistry
SN - 0161-5890
IS - 4
ER -