Role of kappa II-A2 light chain CDR-3 junctional residues in human antibody binding to the Haemophilus influenzae type b polysaccharide.
Abs using the kappaII-A2 V gene segment predominate the human Ab repertoire to the Haemophilus influenzae b (Hib) polysaccharide (PS). All A2 anti-Hib PS Abs sequenced to date possess a 10-amino acid L chain complementarity-determining region-3 (CDR-3) having an insertional arginine (Arg) at position 95a, the V-J junction. These findings suggest an essential requirement for this conserved Arg residue in determining Hib PS-binding affinity. We examined this requirement by performing chain recombination experiments in which a series of A2 L chains, differing at position 95a, were combined individually with an Fd region known to generate a Hib PS-combining site when paired with an A2-Arg(95a)-Jkappa1 V region. Hib PS binding of the recombinant Fabs was evaluated quantitatively using a radioantigen-binding assay. Fabs having A2 L chains with either Arg or lysine in position 95a in combination with Jkappa1 gave equivalent and strongest binding to Hib PS. Fabs having A2-Jkappa1 L chains with either tyrosine, glycine, alanine, leucine, serine, or threonine in position 95a, or having an A2-Arg(95a)-Jkappa3 L chain, gave intermediate binding. Fabs having A2-Jkappa1 L chains with glutamate or aspartate at 95a or with no junctional residue showed little or no Hib PS binding. These results demonstrate the importance of L chain junctional residue, as well as Jkappa usage and CDR-3 length, in determining Hib PS-binding affinity. Contrary to expectation, an Arg junctional residue is not essential for generating either high or intermediate affinity-binding sites.
Children's Hospital Oakland Research Institute, CA 94609, USA.
A H
AH
Lucas
K D
KD
Moulton
D C
DC
Reason
D000328:Adult; D000907:Antibodies, Bacterial / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D001666:Binding Sites, Antibody / Q000235:genetics; D005260:Female; D006193:Haemophilus influenzae / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D007223:Infant; D016254:Mutagenesis, Insertional; D011135:Polysaccharides, Bacterial / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D000907:Antibodies, Bacterial; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins