Tolerance of single, but not multiple, amino acid replacements in antibody VH CDR 2: a means of minimizing B cell wastage from somatic hypermutation?
Mutations in the heavy chain complementarity determining region 2 (CDR2) of the phosphocholine-specific T15 Ab can have a dramatic effect on the ability of the Ab to bind Ag. A panel of multisite mutants that had lost detectable binding to phosphocholine-containing Ags was previously created by saturation mutagenesis of the CDR2 region of T15. Based on the predicted importance of amino acid changes represented in the multisite mutants, we have created single-site mutations, yielding a panel of Abs with which to test 17 of the 19 CDR2 residues. Of the 17 positions examined, only one, Arg52, is intolerant to change, yielding a nonbinder phenotype even with conservative amino acid replacement. Mutation at two other sites, Ala50 and Tyr55, can yield a nonbinder phenotype depending on the amino acid replacement. Single-site mutations of the remaining 14 positions allowed retention of binding ability. Thus, except for positions 50, 52, and 55, multiple mutations must be introduced into the CDR2 region to create a nonbinder phenotype. We provide a newly refined model of T15, illustrating the structure and the interactions of the CDR2 region. Our results imply that introduction of point mutations would not normally delete Ag-binding ability until two or more mutations had accumulated. This would minimize potentially harmful effects of somatic mutation on Ig V region genes and improve the chance of survival for an Ab such as T15, which in its unmutated form is already well suited to bind Ag.
Brown
M
M
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland 97201, USA.
Rittenburg
M B
MB
Chen
C
C
Roberts
V A
VA
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000235:genetics; D007108:Immune Tolerance / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D018448:Models, Immunological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000276:immunology*; D009194:Myeloma Proteins / Q000235:genetics; D017354:Point Mutation / Q000276:immunology*
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D009194:Myeloma Proteins; C043925:myeloma protein TEPC15