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 and V_L regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile⁵¹ is present in 91% of murine V_H complementarity-determining region 2 sequences, and we demonstrate that single Ile⁵¹→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 binding protein (BiP) and GRP94 and fail to form HL and H₂L assembly intermediates 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, is secreted. The secreted mutant Ab binds phosphocholine-protein with avidity 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 Ile⁵¹ is buried between complementarity-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile⁵¹→Arg or Ile⁵¹→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 Ile⁵¹→Arg or Ile⁵¹→Lys mutants were partially rescued when expressed with the J558L λ1 L chain. These results represent the first demonstration that single somatic mutations in Va residues can impair Ig secretion and suggest one reason for the conservation of Ile⁵¹ in so many Ig V_H.