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 kappa II-A2 V gene segment predominate the human Ab repe rtoire to the Haemophilus influenzae b (Hib) polysaccharide (PS), All A2 anti-Hib PS Abs sequenced to date possess a 10-amino acid L chain c omplementarity-determining region-3 (CDR-3) having an insertional argi nine (Arg) at position 95a, the V-J junction. These findings suggest a n essential requirement for this conserved Arg residue in determining Hib PS-binding affinity. We examined this requirement by performing ch ain recombination experiments in which a series of A2 L chains, differ ing at position 95a, were combined individually with an Fd region know n to generate a Hib PS-combining site when paired with an A2-Arg(95a)- J kappa 1 V region. Hib PS binding of the recombinant Fabs was evaluat ed quantitatively using a radioantigen-binding assay. Fabs having A2 L chains with either Arg or lysine in position 95a in combination with J kappa 1 gave equivalent and strongest binding to Hib PS. Fabs having A2-J kappa 1 L chains with either tyrosine, glycine, alanine, leucine , serine, or threonine in position 95a, or having an A2-Arg(95a)-J kap pa 3 L chain, gave intermediate binding. Fabs having A2-J kappa 1 L ch ains with glutamate or aspartate at 95a or with no junctional residue showed little or no Hib PS binding. These results demonstrate the impo rtance of L chain junctional residue, as well as J kappa usage and CDR -3 length, in determining Hib PS-binding affinity. Contrary to expecta tion, an Arg junctional residue is not essential for generating either high or intermediate affinity-binding sites.