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Virginia Mason Research Center, Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The MHC class II molecules, HLA-DR and -DQ, are heterodimeric proteins that function as peptide receptors for presentation of antigenic peptide to T lymphocytes (8). The rules that govern peptide interaction with MHC class II molecules have been described for some HLA-DR and -DQ alleles through binding studies with synthetic peptides, biochemical isolation of naturally associated MHC class II peptides, and x-ray crystallography. The x-ray crystal structure of HLA-DRA/DRB1*0101 complexed with the influenza virus hemagglutinin peptide, HA(306–318), has provided a foundation for current knowledge on MHC class II/peptide interaction (9). The more recent elucidation of the x-ray crystal structure for HLA-DRA/DRB1*0301 complexed with the class II-associated invariant chain peptide, CLIP(81–104), demonstrated that CLIP(81–104) interaction with HLA-DRA/DRB1*0301 was almost identical with HA(306–318) interaction with HLA-DRA/DRB1*0101 (10), suggesting universal rules for peptide binding to MHC class II.
Peptide binding motifs have been described for many HLA-DR molecules and a few HLA-DQ molecules (8). These include peptide binding motifs for molecules encoded by HLA-DRA/DRB1*0401 (11), HLA-DRA/DRB1*0402 (12), HLA-DRA/DRB1*0405 (13), HLA-DRA/DRB1*0301 (14), HLA-DQA1*0301/DQB1*0302 (15), and HLA-DQA1*0501/DQB1*0201 (16, 17), MHC class II molecules found on haplotypes associated with susceptibility to IDDM. A peptide binding motif for HLA-DRA/DRB1*1501 and HLA-DRA/DRB5*0101 found on the DR2 protective haplotype in the Caucasian population has also been described (18, 19). However, a peptide binding motif for the MHC class II molecule most closely associated with IDDM protection, HLA-DQA1*0102/DQB1*0602 (DQ0602), has not been determined.
More than a dozen putative autoantigens for IDDM have been identified, including GAD65, IA-2, and (pro)insulin (20). Of these, proinsulin is unique in its tissue distribution, being expressed primarily in the pancreas, and at very low levels in the fetal and postnatal thymus (21, 22). In the thymus, a higher level of insulin mRNA expression correlates with the class III variable number of tandem repeats polymorphism found in the insulin gene promoter, an allele associated with protection in IDDM. The correlation of high thymic insulin expression with protection has raised the hypothesis that higher concentrations of (pro)insulin expression result in negative selection of (pro)insulin-specific T lymphocytes. In nonobese diabetic (NOD) mice, it was shown that mice made transgenic for proinsulin expression in thymus were protected from diabetes (23).
A number of additional lines of evidence are supportive of proinsulin as an important autoantigen in IDDM. In humans, insulin and proinsulin autoantibodies have been associated with an increased risk for IDDM development (24, 25). Insulin-specific T lymphocyte proliferation has been demonstrated in peripheral blood derived from prediabetics and diabetics (26), including low levels of proliferation to the proinsulin peptide B24–C36 (27). In NOD mice, insulin-specific T lymphocytes represent a predominant component of islet infiltrates (28). Adoptive transfer experiments have shown that insulin-specific T cell clones are capable of accelerating IDDM in NOD mice (29). A direct role for the insulin B(9–23) peptide in the development of IDDM in NOD mice has been suggested based on the observation that s.c. and intranasal administration of insulin B(9–23) resulted in a marked delay in the onset and a decrease in the incidence of diabetes relative to mice given the control peptide, tetanus toxin-(830–843) (30).
The potential role of HLA-DQ in presenting peptides to disease-mediating T lymphocytes in IDDM led us to examine binding of peptides derived from IDDM autoantigens to HLA-DQ. We report the identification of insulin B(5–15) as a peptide that binds well and with allelic specificity to DQ0602. A peptide binding motif for DQ0602 was elucidated and used to identify additional peptides from IDDM autoantigens that bind DQ0602 in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Homozygous EBV-transformed B-lymphoblastoid cell lines (B-LCL) from the Tenth International Histocompatibility Workshop include MGAR (DQA1*0102/DQB1*0602), AMAI (DQA1*0102/DQB1*0602), HOM-2 (DQA1*0101/DQB1*0501), KT3 (DQA1*0301/DQB1*0401), AMALA (DQA1*0501/DQB1*0301), JVM (DQA1*0501/DQB1*0301), DEU (DQA1*0301/DQB1*0301), BSM (DQA1*0301/DQB1*0302), and COX (DQA1*0501/DQB1*0201) (31). Other EBV-transformed B-LCLs used in this study include LG2 (DQA1*0101/DQB1*0501), HAS-15 (DQA1*0301/DQB1*0401), PF97387 (DQA1*0301/DQB1*0301), PRIESS (DQA1*0301/DQB1*0302), and MAT (DQA1*0501/DQB1*0201), and they were HLA typed by high resolution oligonucleotide typing (Puget Sound Blood Center, Seattle, WA). HLA class II-deficient BLS-1 was a gift from Dr. Janet Lee (32). Cells were grown in Iscove’s modified Dulbecco’s medium with L-glutamine and 25 mM HEPES buffer (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 1 mM sodium pyruvate, 50 U/ml penicillin, and 50 µg/ml streptomycin.
Peptides
Peptides were synthesized with an Applied Biosystems 432 Peptide Synthesizer (Foster City, CA) or purchased from GeneMed Synthesis, Inc. (South San Francisco, CA). Peptides were biotinylated as described (33). The m.w. of each peptide was analyzed by mass spectrometry. Mass spectrometry was performed by Anaspec, Inc. (San Jose, CA), GeneMed Synthesis, Inc., and the Protein and Carbohydrate Structure Facility at the University of Michigan (Ann Arbor, MI). The amino acid sequence of peptides that are not given elsewhere are: insulin A(1–15), GIVEQCCTSICSLYQ; insulin A(7–21), CTSICSLYQLENYCN; insulin B(1–15), FVNQHLCGSHLVEAL; insulin B(9–23), SHLVEALYLVCGERG; insulin B(16–30), YLVCGERGFFYTPKT.
Antibodies
SPVL3 (anti-DQ) hybridoma cells were kindly provided by Dr. Hans Yssel of DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA. L243 (anti-DR) hybridoma cells were purchased from American Type Culture Collection (Rockville, MD). Mouse IgG was purchased from Sigma BioSciences (St. Louis, MO). SPVL3 and L243 ascites were prepared at the University of Washington (Seattle, WA). SPVL3 and L243 were purified from ascites using protein A-Sepharose chromatography. Mouse IgG-, L243-, and SPVL3-Sepharose columns were prepared by coupling 20 mg of purified Ab with 5 ml of cyanogen bromide-activated Sepharose 4B (Sigma).
Purification of DQ0602
DQ0602 was purified from 1010 MGAR cells. All manipulations occurred at 4°C. Cells were lysed in 100 ml of 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 1 mM PMSF, 1 mM iodoacetamide by mixing for 1 h. The lysate was centrifuged at 100,000 x g for 60 min. The supernatant was precleared by sequential incubations on a rotator with mouse IgG-Sepharose (5 ml) for 1 h and L243-Sepharose (5 ml) for 3 h. The supernatant was than incubated with SPVL3-Sepharose (5 ml) overnight at 4°C. The SPVL3-Sepharose was poured into a column and the flowthrough was collected. The resin was than washed with Buffer 1 (10 mM Tris-HCl, pH 7.5, 0.1% deoxycholate), Buffer 2 (10 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% n-octyl-ß-D-glucopyranoside), and Buffer 3 (10 mM Tris-HCl, pH 7.5, 1% n-octyl-ß-D-glucopyranoside). DQ0602 was eluted with 100 mM Tris (pH 11.2), 1% n-octyl-ß-D-glucopyranoside and neutralized immediately with concentrated acetic acid. Purified DQ0602 was stored at -80°C.
Whole cell peptide binding assay
EBV-transformed B-LCLs (1.5 x 106 cells) were washed with HBSS and than incubated for 20 min in 1% paraformaldehyde. Fixed cells were washed with Iscove’s complete medium followed by PBS. Cells were resuspended in 200 µl of 150 mM citrate-phosphate (pH 5.4), 5 mM EDTA, 1 mM iodoacetamide, 1 mM benzamidine, 1 mM PMSF. Biotinylated peptide was added to the cells in 4 µl of a DMSO:ß-mercaptoethanol solution (1 part DMSO and 1 part ß-mercaptoethanol diluted 1:10 in whole cell peptide binding buffer) to a final concentration of 10 µM and incubated for 18 h at 37°C. Cells were washed with HBSS and lysed by resuspending in 100 µl of 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.15 M NaCl, 1% Nonidet P-40, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin A for 1 h on ice. The lysates were centrifuged at 20,000 x g for 10 min, and the supernatants were transferred to a 96-well microtiter plate coated with 10 µg/ml SPVL3, neutralized with 100 µl of 50 mM Tris-HCl (pH 8.0), 0.02% n-dodecyl-ß-D-maltoside, and incubated overnight at 4°C. The plate was washed with PBS containing 0.05% Tween-20. DELFIA europium-labeled streptavidin (Wallac, Turku, Finland) diluted 1:1000 in DELFIA assay buffer (Wallac) was added to the wells and incubated for 4 h at room temperature. The plate was washed with PBS containing 0.05% Tween-20. DELFIA enhancement solution (Wallac) was added to the wells and incubated for 1 h at room temperature. Fluorescence was measured using a DELFIA 1232 fluorometer (Wallac).
Purified DQ peptide binding assay
The reaction mixture consisted of 48 µl of affinity-purified DQ0602 in 150 mM citrate-phosphate (pH 5.4), 1 mM PMSF, 0.02% n-dodecyl-ß-D-maltoside at a final concentration of 25 nM and 2 µl of biotinylated peptide in a DMSO:ß-mercaptoethanol solution (1 part DMSO and 1 part ß-mercaptoethanol diluted 1:20 in purified peptide binding buffer) at a final concentration of 0.001 to 30 µM. Nonspecific binding was determined by omitting DQ0602 from the reaction mixture. DQ0602 was incubated with peptide for 48 h at 37°C. The reaction mixture was transferred to a 96-well microtiter plate coated with 10 µg/ml SPVL3, neutralized with 50 µl of 50 mM Tris-HCl (pH 8.0), 0.02% n-dodecyl-ß-D-maltoside, and incubated overnight at 4°C. The detection of bound biotinylated peptide was conducted as described above for the whole cell peptide binding assay. For the peptide saturation curve, nonspecific binding was determined by the addition of 200 µM nonbiotinylated peptide to the reaction mixture and was conducted under equilibrium conditions with a 96-h incubation time. For the time course experiment, the reaction was started by the addition of 10 µM biotinylated peptide and was stopped by the addition of 200 µM nonbiotinylated peptide. The reaction time varied from 15 min to 120 h.
DQ0602 competition assay
The reaction mixture consisted of 46 µl of affinity-purified DQ0602 in 150 mM citrate-phosphate (pH 5.4), 1 mM PMSF, 0.02% n-dodecyl-ß-D-maltoside at a final concentration of 25 nM, 2 µl of nonbiotinylated competitor peptide in DMSO:ß-mercaptoethanol solution (1 part DMSO and 1 part ß-mercaptoethanol diluted 1:20 in purified peptide binding buffer), and 2 µl of biotinylated insulin B(5–15) in DMSO:ß-mercaptoethanol solution at a final concentration of 0.25 µM. The competitor peptide was added first at 0.1, 0.3, 1.0, 3.0, 10, 30, and 100 µM final concentrations to the assay. The incubation was conducted at 37°C for 48 h. The reaction mixture was transferred to a microtiter plate coated with 10 µg/ml SPVL3, and the remainder of the assay was conducted as described above for the purified peptide binding assay. The concentration at which 50% inhibition occurs (IC50) was determined by plotting a curve for each peptide examined and extrapolating from the curve the concentration at which 50% inhibition occurs. Relative binding values were calculated by dividing the IC50 for insulin B(5–15) by the IC50 for the analogue peptide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Binding of the insulin peptides to HLA-DQ alleles was examined in
an assay that uses paraformaldehyde-fixed EBV-transformed B-LCLs
derived from individuals who are homozygous for different HLA-DQ
alleles. The insulin peptides chosen, insulin A(1–15), insulin
A(7–21), insulin B(1–15), insulin B(9–23), and insulin B(16–30),
represent the primary structure of insulin and were used previously for
mapping insulin T cell epitopes in NOD mice (29). Figure 1
shows the results of insulin
peptide binding to DQA1*0101/DQB1*0501, DQA1*0102/DQB1*0602,
DQA1*0501/DQB1*0201, DQA1*0501/DQB1*0301, DQA1*0301/DQB1*0301,
DQA1*0301/DQB1*0302, and DQA1*0301/DQB1*0401. These molecules are
representative of HLA-DQ serologic specificities and are also commonly
found in the Caucasian population with the exception of
DQA1*0301/DQB1*0401, which is rare in Caucasians but is prevalent in
Japanese (3). Figure 1 shows that insulin B(1–15) bound the best to
DQ0602, whereas the other peptides showed lower levels of binding to
the HLA-DQ alleles examined. In addition, binding of insulin B(1–15)
to DQ0602 occurred with high allelic specificity. Similar results were
obtained with a second panel of EBV-transformed B-LCLs (data not
shown).
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The minimal insulin B(1–15) peptide required for binding to
DQ0602 was determined by measuring binding of truncated biotinylated
insulin peptides to purified DQ0602. Figure 2 shows that insulin B(1–15) and insulin
B(5–15) bound to DQ0602 in a similar fashion. Further truncations at
the amino- and carboxyl-terminal end of insulin B(1–15) resulted in a
decrease in binding, suggesting that the minimal peptide for maximal
binding was insulin B(5–15). Insulin B(6–15) and insulin B(1–14)
were efficient binders compared with insulin B(7–15) and insulin
B(1–13) but bound less well than insulin B(1–15), suggesting that the
minimal required epitope for binding is insulin B(6–14).
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A. An apparent
KD calculated by Scatchard analysis
for insulin B(5–15) binding to DQ0602 was 0.7 to 1.0 µM. An apparent
KD of 0.01 to 3 µM has been
reported for other MHC class II molecules (34, 35, 36). Figure 3B shows the time course for binding of insulin B(5–15) to
purified DQA1*0102/DQB1*0602. A slow association time was exhibited,
with 96 h being required to reach equilibrium. The association of
peptides with purified MHC class II molecules has been shown to occur
slowly, ranging between 1 and 5 days for various HLA-DR, HLA-DQ, IA,
and IE alleles (34, 35, 36, 37). These results suggest that insulin B(5–15)
interaction with DQ0602 is occurring in a manner consistent with
conventional MHC class II peptide interaction.
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The peptide binding motif for DQ0602 was defined by examining the
effect of single amino acid substitutions in insulin B(5–15) on
binding to DQ0602. Arginine substitutions were chosen to map the
primary anchors, even though its effect could be pleiotropic, because
of the effectiveness of using positively charged substitutions to
determine peptide contact sites for HLA-DQ (15, 17, 38). Alanine
substitutions, which typically have been used to define motifs for
HLA-DR alleles, often have little effect on binding to HLA-DQ alleles
(17, 38). Figure 4 shows the effect of
single arginine (R) substitutions in biotinylated insulin B(5–15) on
binding to DQ0602 on AMAI B-LCLs. Binding of 6R, 8R, 9R, 11R, and 14R
insulin B(5–15) peptides to DQ0602 on AMAI B-LCLs was greatly reduced,
whereas 5R, 7R, 10R, 12R, 13R, and 15R insulin B(5–15) peptides bound
as well as the unsubstituted peptide. Comparable results were obtained
with MGAR B-LCL (data not shown). Table I
shows the results of analyzing nonbiotinylated arginine (R)-substituted
insulin B(5–15) peptides in a competition assay with biotinylated
insulin B(5–15) and purified DQ0602. In this assay, the binding of 6R,
8R, 9R, 11R, and 14R insulin B(5–15) peptides to DQ0602 was also
greatly reduced.
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and is expressed as relative binding.
The relative binding value was determined by dividing the
IC50 for insulin B(5–15) by the IC50 for
each insulin B(5–15) analogue peptide. At amino acids 6, 8, and 9 (P1,
P3, and P4), Arg (R)- and Pro (P)-substituted peptides had a binding
capacity of <0.1 (Fig. 5, A–C). In
addition, a Gly (G)-substituted peptide was not tolerated at amino acid
6; Cys (C)- and Asp (D)-substituted peptides were not tolerated at
amino acid 9. At amino acid 11 (P6), Arg (R)-, Cys (C)-, Phe (F)-, Asp
(D)-, Asn (N)-, and Gly (G)-substituted peptides had a relative binding
capacity of <0.1 and Thr (T)-, Pro (P)-, and Ala (A)-substituted
peptides had a relative binding capacity between 0.1 and 0.3 (Fig. 5D). Only the highly conservative Val- and
Ile-substituted peptides bound well to DQ0602 with relative binding
capacities of 0.63 and 1.0, respectively (data not shown), suggesting
that a large aliphatic amino acid at amino acid 11 (P6) is important
for binding. At amino acid 14 (P9), Arg (R)-, Cys (C)-, Asp (D)-, Asn
(N)-, and Phe (F)-substituted peptides had a relative binding capacity
of 0.1 or less, whereas Pro (P)- and Leu (L)-substituted peptides
exhibited relative binding capacities between 0.1 and 0.3, and Gly
(G)-, Thr (T)-, and Ser (S)-substituted peptides exhibited relative
binding capacities between 0.3 and 0.5 (Fig. 5E).
Insulin B(5–15) contains an Ala at amino acid 14, thus suggesting a
general trend toward smaller amino acids, which possess aliphatic and
hydroxyl side chains at this position. The deduced DQ0602 peptide
binding motif is shown in Figure 6.
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The predictive power of the deduced motif for DQ0602 was tested by
examining the binding capacity of a randomly chosen set of seven
overlapping peptides to DQ0602 in the DQ0602 competition assay. Binding
of overlapping peptides from a region of herpes simplex virus-2 (HSV-2)
UL49, amino acids 105 to 190, is shown in Table II. The correlation of motif with binding
was 100%. The four peptides that did not contain the motif bound to
DQ0602 with an IC50 of >100 µM. The three peptides
that did contain the motif bound to DQ0602 with IC50 values
between 8.6 and 27 µM. Each of the peptides that contained the motif
had one weakly tolerated anchor amino acid (relative binding between
0.1 and 0.3), whereas the four other anchor positions were tolerated
(relative binding between 0.3 and 1.0). The DQ0602 motif in
UL49(135–156) also lacked carboxyl-terminal flanking residues which
are predicted to increase binding (Fig. 2
). UL49(145–166), which
contains the same motif found in UL49(135–156), but located centrally
within the peptide, bound 2.9-fold better to DQ0602. These results
suggest that the DQ0602 motif identified is typical because the
presence of the motif correlates with binders and the absence of the
motif correlates with nonbinders.
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shows
the IC50 for competition with biotinylated insulin B(5–15)
for binding to DQ0602. Of these peptides, 79% (19 of 24) bound to
DQ0602 (IC50 >100 µM). The IC50 value
of the binders ranged from 0.7 to 90 µM, with insulin B(5–15)
binding with an IC50 of 1.7 µM. The range of binding may
be explained, in part, by a loose correlation between binding and the
number of highly preferred amino acids at anchor positions. These
results suggest that the DQ0602 motif, deduced within the context of
the insulin B(5–15) peptide, is able to select peptides which bind
DQ0602. However, it is also apparent that there are factors that have
not been elucidated by this study that will modify the DQ0602 peptide
binding motif.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The approach utilized in this report to identify a DQ0602 peptide ligand was to screen synthetic peptide ligands derived from human insulin, a putative autoantigen in IDDM, for binding to HLA-DQ on EBV-transformed B-LCLs. Insulin is considered to be an important autoantigen in IDDM because detection of insulin autoantibodies along with GAD65 and IA-2 contributes to accurate prediction of IDDM (20). Insulin is of particular interest as an autoantigen in IDDM because the expression of its precursor, proinsulin, occurs primarily in the pancreas along with low levels in thymus. Recently, a correlation between higher insulin expression level in thymus and the presence of the class III variable number of tandem repeats allele, associated with protection in IDDM, has been drawn (21, 22).
The peptide identified as a model peptide ligand for DQ0602 was insulin B(5–15). Insulin B(5–15) was shown to bind DQ0602 following the general rules that govern most MHC class II/peptide interactions and not in a fashion that would suggest peptide groove-independent interaction as was recently reported for insulin B(10–30) and HLA-DRA/DRB1*0101 (45). Interaction of insulin B(5–15) with the peptide binding groove of DQ0602 is supported by the allelic specificity of binding. A peptide binding motif suggestive of pockets at P1, P4, P6, and P9 is consistent with the x-ray crystal structure deduced for HLA-DRA/DRB1*0101 complexed with HA(306–318) and HLA-DRA/DRB1*0301 complexed with CLIP(81–104) (9, 10). In addition, the time course of insulin B(5–15) binding with purified DQ0602 requires days to reach equilibrium and not hours as was seen for insulin B(10–30) and HLA-DRA/DRB1*0101.
The peptide binding motif for DQ0602 deduced using insulin B(5–15) as a model peptide suggests that large aliphatic amino acids in relative position 6 and small aliphatic or hydroxyl amino acids in relative position 9 are most important for binding to DQ0602. A critical role for position 6 in allele-specific binding was previously demonstrated by Hammer et al. (46) by selection of peptides from a M13 phage display library with HLA-DRA/DRB1*0101, HLA-DRA/DRB1*0401, and HLA-DRA/DRB1*1101. Sequence analysis revealed peptide binding motifs for the three HLA-DR molecules that shared anchor residues at relative positions 1 and 4 while having an allele-specific anchor at position 6. This suggests that pocket 6 plays a critical role in peptide binding; however, the mechanism by which position 6 confers allelic specificity is not clear.
The preference for a small aliphatic or hydroxyl residue in position 9
of DQ0602-binding peptides is consistent with previous observations
that ß57-Asp containing MHC class II alleles bind peptides with Ala
in position 9. These studies showed that peptides with acidic residues
at position 9 bind well to non-Asp-containing alleles but do not bind
well to Asp-containing alleles (47, 48). By changing the acidic residue
at position 9 to Ala, peptides reverse their binding pattern, binding
well to Asp-containing alleles and not binding well to
non-Asp-containing alleles. This phenomenon has been attributed to a
salt bridge that forms between ß57-Asp in P9 and Arg76 of the
-chain in HLA-DRA/DRB1*0101. In HLA-DQ molecules, an analogous salt
bridge has been proposed to form between ß57-Asp in P9 and Arg79 of
the -chain.
Naturally processed DQ0602 peptides have not been described, and very
few DQ0602 binders are known. Therefore, examination of these peptides
for the DQ0602 peptide binding motif could not be used as a method for
validating the motif determined herein. In note, the p21 ras
oncogene peptide (VVGAAGVGKSA) previously identified to bind DQ0602
(44) does contain the deduced DQ0602 peptide binding motif. As a
result, the approach that was taken to address the validity of the
motif was to correlate the presence of the motif with binding to
overlapping peptides from HSV-2 UL49 and insulin. As is shown in Table II, the binding of overlapping peptides from HSV-2 UL49 correlated
completely (7 of 7) with the presence or absence of the motif. For
insulin, the whole cell peptide binding data (Fig. 1) suggested that
insulin A(1–15), insulin A(7–21), and insulin B(9–23) bound
moderately to DQ0602 whereas insulin B(16–30) bound poorly. The
presence or absence of the motif correlated with binding for insulin
A(1–15), insulin B(9–23), and insulin B(16–30), with insulin
A(7–21) being the exception. In addition, the DQ0602 peptide binding
motif was used to identify peptides from GAD65, proinsulin, and IA-2.
Of the peptides identified as containing the motif, 79% (19 of 24)
competed for binding with insulin B(5–15) to DQ0602. The correlation
of binding with motif in overlapping peptides from randomly selected
Ags as well as the ability to select peptides that bind to DQ0602
provides evidence that the preferences determined with the insulin
B(5–15) peptide define, at least in part, the requirements of
conventional DQ0602 binding peptides.
The inability of the deduced DQ0602 peptide binding motif to correctly predict all peptides that will bind to DQ0602 suggests that there are additional factors involved in determining motif. This has been observed in other studies determining peptide binding motifs for MHC class II molecules (11, 14). One explanation for the inadequacy of the motif may be the overall amino acid composition of the peptide which was not addressed by single amino acid substitutions in the insulin B(5–15) peptide. The amino acid composition may affect the ability of the peptide to interact with the peptide binding groove or simply its solubility in aqueous solution. This point is exemplified by three of the IDDM autoantigen peptides that did not bind and were somewhat unusual in their amino acid composition; IA-2(361–371) had five leucine amino acids, and GAD65(378–388) and GAD65(526–536) both had three positively charged residues. Another factor that was not addressed by the single amino acid substitutions in insulin B(5–15) is the contribution of each individual anchor to the overall motif. The wide range (0.7–90 µM) of IC50 values obtained for the peptides containing a DQ0602 peptide binding motif may partially result from different additive effects between the anchors.
The DQ0602 binding peptides identified from GAD65 (86–96, 91–101, 116–126, 334–344, 365–375, 396–406, 503–513), proinsulin (5–15, 11–21, 36–46, 66–80, 72–86), and IA-2 (229–239, 379–389, 499–509, 504–514, 530–540, 543–553, 544–554, 576–586, 584–594, 586–596) have not previously been implicated in the context of DQ0602. However, autoantibodies to GAD65, insulin, and IA-2 have been identified in DQ0602 individuals who are first-degree relatives of type I diabetics (49). Of the peptides found within regions identified as immunodominant T cell epitopes in NOD mice, insulin B(11–21) bound moderately well, GAD65(503–513) bound poorly, and GAD65(526–536) did not bind to DQ0602.
Insulin B(5–15) was found to bind well and with specificity to DQ0602 in vitro, but whether insulin B(5–15) will be presented by DQ0602 on APC in vivo remains to be determined. The only human insulin-specific T cell clones to be reported were restricted by HLA-DRA/DRB1*0406 and came from healthy donors and insulin autoimmune syndrome patients (50). The epitope specificity of these T cell clones was not determined. Jensen (51) determined that reduction of disulfide bonds is both necessary and sufficient for presentation of insulin to a major population of class II-restricted T cells from H-2d and H-2b mice. Insulin A(1–13), existing in an extended conformation with its A-loop disulfides reduced, was characterized as the major immunogenic determinant presented by I-Ad (52). However, the effect of different MHC class II molecules and APC on insulin processing and presentation is not known.
In conclusion, we have identified peptides that bind DQ0602 in vitro and determined a peptide binding motif for DQ0602. These results provide a stepping stone for understanding the biochemical properties of DQ0602 and its ligands in IDDM. However, these results do not address the physiologic relevance of the peptides being identified. This will be the objective of further study.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William W. Kwok, Virginia Mason Research Center, 1000 Seneca St., Seattle, WA 98101. E-mail address:
3 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; DQ0602, HLA-DQA1*0102/DQB1*0602; B-LCL, B-lymphoblastoid cell line; NOD, nonobese diabetic; IC50, concentration at which 50% inhibition occurs; HSV-2, herpes simplex virus 2.
Received for publication June 23, 1997. Accepted for publication November 11, 1997.
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References |
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. 1995. MHC ligands and peptide motifs: first listing. Immunogenetics 41:178.[Medline]
1*0501, ß1*0201) molecule. Immunogenetics 44:246.[Medline]
1*0501, ß1*0201) molecule. Int. Immunol. 6:453.1*0102,ß1*0602) and -DR(,ß1*0101) molecules. Scand. J. Immunol. 39:607.[Medline]
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M. Sospedra, P. A. Muraro, I. Stefanova, Y. Zhao, K. Chung, Y. Li, M. Giulianotti, R. Simon, R. Mariuzza, C. Pinilla, et al. Redundancy in Antigen-Presenting Function of the HLA-DR and -DQ Molecules in the Multiple Sclerosis-Associated HLA-DR2 Haplotype J. Immunol., February 1, 2006; 176(3): 1951 - 1961. [Abstract] [Full Text] [PDF]
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R. A. Ettinger, G. K. Papadopoulos, A. K. Moustakas, G. T. Nepom, and W. W. Kwok Allelic Variation in Key Peptide-Binding Pockets Discriminates between Closely Related Diabetes-Protective and Diabetes-Susceptible HLA-DQB1*06 Alleles J. Immunol., February 1, 2006; 176(3): 1988 - 1998. [Abstract] [Full Text] [PDF]
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I. A. Doytchinova and D. R. Flower In Silico Identification of Supertypes for Class II MHCs J. Immunol., June 1, 2005; 174(11): 7085 - 7095. [Abstract] [Full Text] [PDF]
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 C. Siebold, B. E. Hansen, J. R. Wyer, K. Harlos, R. E. Esnouf, A. Svejgaard, J. I. Bell, J. L. Strominger, E. Y. Jones, and L. Fugger Crystal structure of HLA-DQ0602 that protects against type 1 diabetes and confers strong susceptibility to narcolepsy PNAS, February 17, 2004; 101(7): 1999 - 2004. [Abstract] [Full Text] [PDF]
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M.-H. Jang, N. P. Seth, and K. W. Wucherpfennig Ex Vivo Analysis of Thymic CD4 T Cells in Nonobese Diabetic Mice with Tetramers Generated from I-Ag7/Class II-Associated Invariant Chain Peptide Precursors J. Immunol., October 15, 2003; 171(8): 4175 - 4186. [Abstract] [Full Text] [PDF]
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 M A Kelly, M L Rayner, C H Mijovic, and A H Barnett Molecular aspects of type 1 diabetes Mol. Pathol., February 1, 2003; 56(1): 1 - 10. [Abstract] [Full Text] [PDF]
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J. Sidney, M.-F. del Guercio, S. Southwood, and A. Sette The HLA Molecules DQA1*0501/B1*0201 and DQA1*0301/B1*0302 Share an Extensive Overlap in Peptide Binding Specificity J. Immunol., November 1, 2002; 169(9): 5098 - 5108. [Abstract] [Full Text] [PDF]
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 M. H. J. Vogt, J. W. van den Muijsenberg, E. Goulmy, E. Spierings, P. Kluck, M. G. Kester, R. A. van Soest, J. W. Drijfhout, R. Willemze, and J. H. F. Falkenburg The DBY gene codes for an HLA-DQ5-restricted human male-specific minor histocompatibility antigen involved in graft-versus-host disease Blood, April 15, 2002; 99(8): 3027 - 3032. [Abstract] [Full Text] [PDF]
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E. J. Novak, A. W. Liu, J. A. Gebe, B. A. Falk, G. T. Nepom, D. M. Koelle, and W. W. Kwok Tetramer-Guided Epitope Mapping: Rapid Identification and Characterization of Immunodominant CD4+ T Cell Epitopes from Complex Antigens J. Immunol., June 1, 2001; 166(11): 6665 - 6670. [Abstract] [Full Text] [PDF]
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 A. K. Moustakas, Y. van de Wal, J. Routsias, Y. M. C. Kooy, P. van Veelen, J. W. Drijfhout, F. Koning, and G. K. Papadopoulos Structure of celiac disease-associated HLA-DQ8 and non-associated HLA-DQ9 alleles in complex with two disease-specific epitopes Int. Immunol., August 1, 2000; 12(8): 1157 - 1166. [Abstract] [Full Text] [PDF]
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W. W. Kwok, A. W. Liu, E. J. Novak, J. A. Gebe, R. A. Ettinger, G. T. Nepom, S. N. Reymond, and D. M. Koelle HLA-DQ Tetramers Identify Epitope-Specific T Cells in Peripheral Blood of Herpes Simplex Virus Type 2-Infected Individuals: Direct Detection of Immunodominant Antigen-Responsive Cells J. Immunol., April 15, 2000; 164(8): 4244 - 4249. [Abstract] [Full Text] [PDF]
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 M. Mosorin, H.-M. Surcel, A. Laurila, M. Lehtinen, R. Karttunen, J. Juvonen, J. Paavonen, R. P. Morrison, P. Saikku, and T. Juvonen Detection of Chlamydia pneumoniae-Reactive T Lymphocytes in Human Atherosclerotic Plaques of Carotid Artery Arterioscler. Thromb. Vasc. Biol., April 1, 2000; 20(4): 1061 - 1067. [Abstract] [Full Text] [PDF]
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