A Peptide Binding Motif for HLA-DQA1*0102/DQB1*0602, the Class II MHC Molecule Associated with Dominant Protection in Insulin-Dependent Diabetes Mellitus

Binding of truncated insulin B(1–15) peptides to DQA1*0102/DQB1*0602. Biotinylated insulin B(1–15), insulin B(5–15), insulin B(6–15), insulin B(7–15), insulin B(1–14), and insulin B(1–13) at concentrations from 0.001 to 10 μM were incubated with 25 nM purified DQA1*0102/DQB1*0602 in purified peptide binding buffer for 48 h at 37°C. HLA-DQ was bound to a microtiter plate coated with SPVL3, and samples were washed to remove unbound peptide. Bound biotinylated peptide was detected by fluorescence using a europium-labeled streptavidin system. Data are the means ± SD of triplicate determinations.

Peptide saturation curve (A) and time course (B) of insulin B(5–15) binding to DQA1*0102/DQB1*0602. A, Biotinylated insulin B(5–15) (0.001–30 μM) was incubated with 25 nM purified DQA1*0102/DQB1*0602 in purified peptide binding buffer for 96 h at 37°C. Nonspecific binding was determined by the addition of 200 μM nonbiotinylated insulin B(5–15). B, Biotinylated insulin B(5–15) (10 μM) was incubated with 25 nM purified DQA1*0102/DQB1*0602 in purified peptide binding buffer for 15 min to 120 h at 37°C. The reaction was stopped with 200 μM nonbiotinylated insulin B(5–15). Nonspecific binding was determined by omitting HLA-DQ from the reaction mixture. Bound peptide was determined in both A and B by transferring the samples to a microtiter plate coated with SPVL3 and washing to remove unbound peptide. Bound biotinylated peptide was detected by fluorescence using a europium-labeled streptavidin system. Data are the means ± SD of triplicate determinations.

Binding of single Arg-substituted insulin B(5–15) peptides to DQA1*0102/DQB1*0602. Biotinylated Arg analogue insulin B(5–15) peptides (10 μM) were incubated with 1.5 × 10⁶ paraformaldehyde-fixed B-LCLs in whole cell peptide binding buffer for 18 h at 37°C. Cells were washed to remove unbound peptide. Cells were lysed and DQA1*0102/DQB1*0602 was bound to a microtiter plate coated with SPVL3. Bound biotinylated peptide was detected by fluorescence using a europium-labeled streptavidin system. The HLA-DQ genotype of the homozygous B-LCLs is BLS-1 (none), AMAI (0102/0602). The fluorescence units for insulin B(5–15) binding are: BLS-1, 8,324 ± 822; AMAI, 576,001 ± 8,608. Data are means ± SD of triplicate determinations.

Relative binding capacity of insulin B(5–15) analogues for DQA1*0102/DQB1*0602. A, amino acid 6-substituted insulin B(5–15) analogues. B, amino acid 8-substituted insulin B(5–15) analogues. C, amino acid 9-substituted insulin B(5–15) analogues. D, amino acid 11-substituted insulin B(5–15) analogues. E, amino acid 14-substituted insulin B(5–15) analogues. Insulin B(5–15) analogues (0.1–100 μM) were incubated with 25 nM purified DQA1*0102/DQB1*0602 and biotinylated insulin B(5–15) (0.25 μM) in purified peptide binding buffer for 48 h at 37°C. The reaction mixture was transferred to a microtiter plate coated with SPVL3, and samples were washed to remove unbound peptide. Bound biotinylated peptide was detected by fluorescence using a europium-labeled streptavidin system. Relative binding values were calculated by dividing the IC₅₀ for insulin B(5–15) by the IC₅₀ for the analogue peptide.