Structural requirements for a specificity switch and for maintenance of affinity using mutational analysis of a phage-displayed anti-arsonate antibody of Fab heavy chain first complementarity-determining region.
We previously showed that a single mutation at heavy (H) position 35 of Abs specific for p-azophenylarsonate (Ars) resulted in acquisition of binding to the structurally related hapten p-azophenylsulfonate (Sulf). To explore the sequence and structural diversity of the H chain first complementarity-determining region (HCDR1) in modulating affinity and specificity, positions 30-36 in Ab 36-65 were randomly mutated and expressed as Fab in a bacteriophage display vector. Ab 36-65 is germline encoded, lacking somatic mutations. Following affinity selection on Sulf resins, 55 mutant Fab were isolated, revealing seven unique HCDR1 sequences containing different amino acids at position H:35. All Fab bound Sulf, but not Ars. Site-directed mutagenesis in a variety of HCDR1 sequence contexts indicates that H:35 is critical for hapten specificity, independent of the sequence of the remainder of HCDR1. At H:35, Asn is required for Ars specificity, consistent with the x-ray crystal structure of the somatically mutated anti-Ars Ab 36-71, while Sulf binding occurs with at least seven different H:35 residues. All Sulf-binding clones selected following phage display contained H:Gly33, observed previously for Ars-binding Abs that use the same germline V(H) sequence. Site-directed mutagenesis at H:33 indicates that Gly plays an essential structural role in HCDR1 for both Sulf- and Ars-specific Abs.
Wong
Y W
YW
Department of Obstetrics and Gynecology, The Ohio State University, Columbus 43120, USA.
Gill
D S
DS
Parhami-Seren
B
B
Short
M K
MK
Sompuram
S R
SR
Margolies
M N
MN
D000818:Animals; D000940:Antigenic Variation / Q000276:immunology*; D001152:Arsenicals / Q000276:immunology*; D001435:Bacteriophages; D002460:Cell Line; D004252:DNA Mutational Analysis; D015723:Gene Library; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping
D001152:Arsenicals; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; C054806:arsonic acid