We analyzed the structural correlates underlying the insulin-dependent selection of the specific anti-insulin IgG1 κ mAb13-producing cell clone, derived from a patient with insulin-dependent diabetes mellitus treated with recombinant human insulin. First, we cloned the germ-line genes that putatively gave rise to the expressed V(H) and Vκ segments and used them to generate the full (unmutated) 'germ-line revertant' of the 'wild-type' (somatically mutated) mAb13, using recombinant PCR methods and an in vitro human Cγ1 and Cκ expression system. The full 'germ-line revertant' bound insulin specifically and in a dose-saturable fashion, but with a relative avidity (Av(rel)) more than three-fold lower than that of its wild-type counterpart (Av(rel), 1.69 x 10-8 vs 4.91 x 10-9 g/μl). Second, we established, by reassorting wild-type and germ-line revertant forms of the mAb13 V(H) and Vκ segments, that the increased AV(rel) for insulin of mAb13 when compared with its full 'germ-line revertant' counterpart was entirely dependent on the mutations in the V(H) not those in the Vκ chain. Third, we determined, by site-directed mutagenesis experiments, that of the three mutations in the mAb13 V(H) segment (Ser→Gly, Ser→Thr, and Ser→Arg at positions 31, 56, and 58, respectively), only Arg58 was crucial in increasing the mAb13 Av(rel) (from 1.44 x 10-8 to 5.14 x 10-9 g/μl) and affinity (K(d), from 189 to 59 nM) for insulin. The affinity enhancement mediated by the V(H) segment Arg58 residue reflected about a threefold decrease in dissociation rate constant (K(off), from 4.92 x 10-3 to 1.54 x 10-3 s- 1) but not an increase in association rate constant (K(on), from 2.60 x 104 to 2.61 x 104 M-1 s-1), and it contrasted with the complete loss of insulin binding resulting from the substitution of the V(H) segment Asn52 by Lys. The present findings suggest that human insulin, a self Ag, has the potential to recruit a natural autoantibody-producing cell precursor expressing a specific surface receptor for Ag in unmutated configuration, and drive it through affinity maturation. They also show that binding of insulin by such a receptor can be enhanced or completely abrogated by a single amino acid change.
|Original language||English (US)|
|Number of pages||13|
|Journal||Journal of Immunology|
|State||Published - 1995|
ASJC Scopus subject areas
- Immunology and Allergy