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

Ruth A. Ettinger and William W. Kwok
J Immunol March 1, 1998, 160 (5) 2365-2373;
Ruth A. Ettinger
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William W. Kwok
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  • FIGURE 1.
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    FIGURE 1.

    Binding of insulin A(1-15) (A), insulin A(7–21) (B), insulin B(1–15) (C), insulin B(9–23) (D), and insulin B(16–30) (E) to HLA-DQ alleles on B-LCLs. Biotinylated insulin peptides (10 μM) were incubated with 1.5 × 106 paraformaldehyde-fixed B-LCLs in whole cell peptide binding buffer. Cells were washed to remove unbound peptide and lysed. HLA-DQ was bound to a microtiter plate coated with SPVL3. Bound biotinylated peptide was detected by fluorescence using a europium-labeled streptavidin system. Data are the means ± SD of triplicate determinations. The HLA-DQ genotype of the homozygous B-LCL is indicated on the y-axis and was represented by BLS-1 (none), LG2 (0101/0501), AMAI (0102/0602), COX (0501/0201), JVM (0501/0301), DEU (0301/0301), BSM (0301/0302), and HAS-15 (0301/0401).

  • FIGURE 2.
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    FIGURE 2.

    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.

  • FIGURE 3.
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    FIGURE 3.

    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.

  • FIGURE 4.
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    FIGURE 4.

    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.

  • FIGURE 5.
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    FIGURE 5.

    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.

  • FIGURE 6.
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    FIGURE 6.

    DQA1*0102/DQB1*0602 peptide binding motif. The arrows indicate the amino acids critical for binding to DQA1*0102/DQB1*0602 and the designated relative position. Residues in parentheses are weakly tolerated.

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    Table I.

    Binding capacity of single Arg insulin B(5–15) analogues for DQA1*0102/DQB1*0602a

    PeptideSequenceIC50 (μM)
    Insulin B(5–15)HLCGSHLVEAL1.6
    Insulin B(5–15)5RRLCGSHLVEAL1.5
    Insulin B(5–15)6RHRCGSHLVEAL>100
    Insulin B(5–15)7RHLRGSHLVEAL2.5
    Insulin B(5–15)8RHLCRSHLVEAL25
    Insulin B(5–15)9RHLCGRHLVEAL>100
    Insulin B(5–15)10RHLCGSRLVEAL2.2
    Insulin B(5–15)11RHLCGSHRVEAL>100
    Insulin B(5–15)12RHLCGSHLREAL2.0
    Insulin B(5–15)13RHLCGSHLVRAL3.0
    Insulin B(5–15)14RHLCGSHLVERL52
    Insulin B(5–15)15RHLCGSHLVEAR1.4
    • a Binding was measured as described for the DQ0602 competition assay in Materials and Methods.

    • View popup
    Table II.

    Binding capacity of overlapping peptides from HSV-2 UL49(105–190) to DQA1*0102/DQB1*0602a

    PeptideSequenceMotifIC50 (μM)b
    UL49(105–126)GGPVGAGGRSHAPPARTPKMTR−>100
    UL49(115–136)HAPPARTPKMTRGAPKASATPA−>100
    UL49(125–146)TRGAPKASATPATDPARGRRPA−>100
    UL49(135–156)PATDPARGRRPAQADSAVLLDAc+25
    UL49(145–166)PAQADSAVLLDAPAPTASGRTK+8.6
    UL49(161–176)ASGRTKTPAQGLAKKLd−>100
    UL49(165–190)TKTPAQGLAKKLHFSTAPPSPTAPWT+27
    • a Binding was measured as described for the DQ0602 competition assay in Materials and Methods.

    • b The IC50 for insulin B(5–15) in this experiment was 1.7 μM.

    • c Region of peptide containing the DQA1*0102/DQB1*0602 peptide binding motif is underlined.

    • d UL49(161–176) is shorter than the other peptides in this series due to difficulties in extending the synthesis amino terminal to residue 161.

    • View popup
    Table III.

    Binding capacity of GAD65, proinsulin, and IA-2 peptides containing the deduced DQA1*0102/DQB1*0602 peptide binding motif for DQA1*0102/DQB1*0602a

    PeptideSequencebIC50 (μM)
    IA-2(586–596)GVAGLLVALAV0.70
    IA-2(499–509)MSSGSFINISV0.94
    Insulin B(5–15)HLCGSHLVEAL1.7
    GAD65(334–344)ATAGTTVYGAF1.7
    GAD65(91–101)FLHATDLLPAC1.9
    GAD65(396–406)KMMGVPLQCSA3.0
    Proinsulin(11–21)LVEALYLVCGE5.8
    IA-2(576–586)SVLLTLVALAG6.6
    GAD65(116–126)NILLQYVVKSF12
    IA-2(504–514)FINISVVGPAL13
    IA-2(543-553)AQTGLQILQTG13
    IA-2(544–554)QTGLQILQTGV23
    IA-2(584–594)LAGVAGLLVAL31
    Proinsulin(36–46)DLQVGQVELGG40
    IA-2(379–389)VNVGADIKKTM48
    GAD65(503–513)NVCFWYIPPSL58
    IA-2(229–239)MVSVGPLPKAE62
    IA-2(530–540)VTQQAGLVKSE64
    GAD65(365–375)AAWGGGLLMSR70
    GAD65 (86–96)DVNYAFLHATD90
    IA-2(361–371)LTLLQLLPKGA>100
    GAD65(378–388)KWKLSGVERAN>100
    GAD65(526–536)LSKVAPVIKAR>100
    IA-2(373–383)RNPGGVVNVGA>100
    IA-2(335–345)LQRLAAVLAGY>100
    • a Binding was measured as described for the DQ0602 competition assay in Materials and Methods.

    • b The putative DQ0602 peptide binding motifs are shown in bold letters.

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The Journal of Immunology
Vol. 160, Issue 5
1 Mar 1998
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A Peptide Binding Motif for HLA-DQA1*0102/DQB1*0602, the Class II MHC Molecule Associated with Dominant Protection in Insulin-Dependent Diabetes Mellitus
Ruth A. Ettinger, William W. Kwok
The Journal of Immunology March 1, 1998, 160 (5) 2365-2373;

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A Peptide Binding Motif for HLA-DQA1*0102/DQB1*0602, the Class II MHC Molecule Associated with Dominant Protection in Insulin-Dependent Diabetes Mellitus
Ruth A. Ettinger, William W. Kwok
The Journal of Immunology March 1, 1998, 160 (5) 2365-2373;
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