First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate

Abstract

The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (Ginglymostoma cirratum) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments.

Fig. 1.

Amino acid alignment for the five family clones against Fr1–Fr3 of their germ-line sequence. Numbers above the alignment indicate amino acid number, and underlining shows primer annealing sites. CDR1, HV2, HV4, and CDR3 are shown in bold. Residues in CDR3 contributed by D regions are indicated by shaded boxes.

Fig. 2.

Amino acid alignment for the extended family clones against that of the putative ancestral clone. Numbers above the alignment indicate the amino acid number, and underlining indicates primer annealing sites. CDR1, HV2, HV4, and CDR3 are shown in bold.

Fig. 3.

Type II V-region model, mapping the position of amino acid changes between clone PBLA8 and the putative ancestral clone. CDR1 is shown in blue, HV2 in magenta, HV4 in green, CDR3 in red, and mutated residues in yellow.

Fig. 4.

Type II V-region model mapping the position of amino acid changes A30V and S61R, which were postulated to be important in improving affinity for antigen. CDR1 is shown in blue, HV2 in magenta, HV4 in green, CDR3 in red, and mutated residues in yellow.