Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme.
To experimentally examine the functional roles of somatically derived structural variation in the lysozyme-binding mAb HyHEL-10, we have introduced three different point mutations and one insertion at two different sites in HyHEL-10 by site-directed mutagenesis and expression of the mutant antibodies. Mutation of Asp----Ala at position 101 of the H chain returns a somatically mutated residue to its germline sequence for HyHEL-10, and reduces affinity for chicken lysozyme by approximately 9000-fold. Lengthening the third H chain hypervariable region by two amino acids reduces affinity by about 2000-fold. Two mutations, Asp----Thr at position 101 in the H chain and Lys----Thr at position 49 in the L chain, model somatic differences found in another structurally related but functionally distinguishable mAb and minimally decrease affinity for chicken lysozyme. The H chain mutation Asp101VVH----Thr has little effect on affinity for other avian lysozymes but does alter relative fine specificity for these lysozymes. The L chain mutation Lys49VK----Thr increases affinity for duck lysozyme by approximately fivefold. Neither of the positions mutated, 101 in the H chain nor 49 in the L chain, nor the residues near the insertion contact lysozyme in the x-ray structure of the HyHEL-10 F(ab)-HEL complex. The results suggest that these mutations, which model observed somatic mutations, produce functional variation by indirect or long-range effects.
Lavoie
T B
TB
Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Drohan
W N
WN
Smith-Gill
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity / Q000235:genetics*; D000918:Antibody Specificity / Q000235:genetics*; D001483:Base Sequence; D008969:Molecular Sequence Data; D009113:Muramidase / Q000276:immunology*; D009154:Mutation; D013329:Structure-Activity Relationship
D009113:Muramidase