Mutation of a single conserved residue in V-H complementarity-determining region 2 results in a severe Ig secretion defect

During an immune response, somatic mutations are introduced into the V-H an d V-L regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile(51) is present in 91% of muri ne V-H complementarity-determining region 2 sequences, and we demonstrate t hat single Ile(51)-Arg or Lys substitutions in the PCG1-1 Ab are sufficient to severely reduce Ig secretion (1-3% of wild-type (WT) levels). Mutant H chains, expressed in the presence of excess L chain, associate with Ig bind ing protein (BiP) and GRP94 and fail to form HL and H2L assembly intermedia tes efficiently. The mutations do not irreversibly alter the V-H domain as the small amount of mutant H chain, which assembles with L chain as H2L2, i s secreted. The secreted mutant Ab binds phosphocholine-protein with avidit y identical with that of WT Ab, suggesting that the combining site adopts a WT conformation. A computer-generated model of the PCG1-1 variable region fragment of Ig (Fv) indicates that Ile(51) is buried between complementarit y-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile(51)--> Arg or Ile(51)-Lys mutations impair association with the PCG1-1 L chain via indirect interactions. These interactions are in part dependent on the nature of the L chain as the PCG1-1 V-H single Ile (51) Arg or Ile(51)--> Lys mutants were partially rescued when expressed wi th the J558L At L chain. These results represent the first demonstration th at single somatic mutations in VH residues can impair Ig secretion and sugg est one reason for the conservation of Ile(51) in so many Ig V-H.