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ß Protein Dimer, the Class II MHC Allele Associated with Protection from Insulin-Dependent Diabetes Mellitus1
*
Virginia Mason Research Center and
Department of Immunology, University of Washington School of Medicine, Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß dimers and
insulin-dependent diabetes mellitus (IDDM) susceptibility. The MHC
class II ß dimer encoded by HLA-DQA1*0102/DQB1*0602 (DQ0602),
which is a dominant protective allele in IDDM, exhibits the greatest
SDS stability among HLA-DQ molecules in EBV-transformed
B-lymphoblastoid cells and PBLs. DQ0602 is also uniquely SDS stable in
the HLA-DM-deficient cell line, BLS-1. We addressed the molecular
mechanism of the stability of DQ0602 in BLS-1. A panel of mutants based
on the polymorphic differences between HLA-DQA1*0102/DQB1*0602 and
HLA-DQA1*0102/DQB1*0604 were generated and expressed in BLS-1. An Asp
at ß57 was found to be critical for SDS stability, whereas Tyr at
ß30, Gly at ß70, and Ala at ß86 played secondary roles.
Furthermore, the level of class II-associated invariant chain peptide
bound to HLA-DQ did not correlate with SDS stability, suggesting that
class II-associated invariant chain peptide does not play a direct role
in the unique SDS stability of DQ0602. These results support a role for
DQB1 codon 57 in HLA-DQ ß dimer stability and IDDM
susceptibility. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A correlation between SDS stability of HLA-DQ ß protein dimers and
IDDM susceptibility has been observed, such that the most SDS-stable
molecules are encoded by IDDM-protective alleles and the least
SDS-stable molecules are encoded by IDDM-susceptible alleles
(5). A similar observation was made for the mouse I-A
ß protein dimers such that the I-A molecule in the nonobese
diabetic mouse is least SDS-stable compared with other I-A molecules
(6). These observations suggest the hypothesis that the
stability of the ß protein dimer encoded by HLA-DQ and its mouse
homolog I-A plays a molecular role in dictating IDDM
susceptibility.
MHC class II ß dimer stability is dependent on loss of invariant
chain and binding of antigenic peptides. This process is dependent on
HLA-DM such that in HLA-DM-deficient cells, the SDS-resistant property
of various HLA-DR, -DQ, and -DP molecules is generally lost (5, 7, 8, 9). Peptides eluted from DR3 on HLA-DM-deficient cells are
predominantly derived from invariant chain (10, 11). These
peptides are called class II-associated invariant chain peptides
(CLIPs). The SDS stability of the MHC class II ß dimers in
HLA-DM-deficient cells can be induced by the addition of antigenic
peptides (10, 12). In assays with purified proteins,
HLA-DM has been shown to replace CLIP with peptides that have slower
intrinsic rates of dissociation to HLA-DR (13, 14, 15).
Recently, we reported that the HLA-DQA1*0102/DQB1*0602 ß protein
dimer (DQ0602) is SDS stable in the HLA-DM-deficient cell line, BLS-1,
whereas all other HLA-DQ and HLA-DR molecules examined, including the
structurally similar HLA-DQA1*0102/DQB1*0604 ß protein dimer
(DQ0604) (5), were not SDS stable. We now address the
molecular mechanism of the unique SDS stability of DQ0602 in BLS-1 by
generating a panel of mutants based on the polymorphic differences
between DQ0602 and DQ0604. The polymorphic residues essential for the
SDS stability of DQ0602 are determined, and the role of CLIP in the SDS
stability of DQ0602 is examined. We find that an Asp at ß57 plays a
primary role in the SDS stability of DQ0602.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The DQB1*0604 cDNA (a gift from Carolyn Hurley,
Georgetown University Medical Center, Washington, DC) was cloned into
the intermediate PCR cloning vector pCRII (Invitrogen, San Diego, CA).
Double-stranded site-directed mutagenesis was performed according to
the method of Deng and Nickoloff (16) using a transformer
site-directed mutagenesis kit (Clontech, Palo Alto, CA). Primers were
synthesized to make DQB1*0602-like changes to codons in
DQB1*0604 at codons 30, 57, 70, and 86 (Table I
). Primers
were designed to account for all 15 possible combinations of the amino
acids at codons 30, 57, 70, and 86. Generation of the desired mutations
was determined by sequencing with an ABI Prism dye terminator cycle
sequencing kit (Perkin-Elmer, Foster City, CA). The mutant clones were
subcloned into the pLNCX retroviral vector (Clontech). Two additional
swap mutants were generated by using two ScaI sites in the
pLNCX-DQB1*0604 and pLNCX-DQB1*0602 constructs.
ScaI cuts between codons 59 and 60 of the mature protein and
within the pLNCX vector generating a 3.1- and 3.9-kb fragment. These
fragments were swapped to generate DQB1*0602 (codons
1–59)/DQB1*0604 (codons 60–230) and DQB1*0604
(codons 1–59)/DQB1*0602 (codons 60–230).
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BLS-1 was a gift from Janet Lee (Memorial Sloan Kettering Cancer
Center, New York, NY) (18). BLS-1 is a HLA class II-null
EBV-transformed B-lymphoblastoid cell line generated from the
cells of a patient with bare lymphocyte syndrome (BLS),
complementation group B (18, 19). BLS-1 cell lines
expressing DQA1*0102/DQB1*0602, DQA1*0102/DQB1*0604, the 15
DQA1*0102/DQB1*0604 site-directed mutants, and the two swap mutants
(Table II) were generated by retroviral-mediated gene transfer as
previously described (5). The presence of the desired DQB1
sequence in each cell line was confirmed by sequencing. Briefly,
genomic DNA was extracted (Isoquick Kit; Microprobe, Bothell, WA), and
DQB1 was PCR amplified using primers that amplified the region from the
start of the 5' signal sequence to the stop codon in the presence of
TaqStart Ab (Clontech). The PCR product was sequenced using ABI Prism
dye terminator cycle sequencing. Stable surface expression of HLA-DQ
was confirmed by flow cytometric analysis with anti-HLA-DQ mAbs.
Cells were grown in IMDM with L-glutamine and 25 mM HEPES
buffer (Life Technologies, Gaithersburg, MD) supplemented with 10%
FBS, 1 mM sodium pyruvate, 50 U/ml penicillin, and 50 µg/ml
streptomycin.
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GS200.1 hybridoma cells were provided by Susan Radka (NexStar Pharmaceutical, Boulder, CO). GS200.1 mAb (IgG2a) is an HLA-DQ dimer-specific Ab (20). SPVL3 hybridoma cells were obtained from DNAX Research Institute of Molecular and Cellular Biology (Palo Alto, CA). SPVL3 mAb (IgG2a) recognizes a monomorphic epitope specific to HLA-DQ dimers (21). Purified 1a3 mAb (IgG2a) was purchased from BioDesign International (Kennebunk, ME). 1a3 mAb recognizes monomorphic epitopes on HLA-DQ dimers (22). L243 hybridoma cells were purchased from American Type Culture Collection (Manassas, VA). L243 mAb (IgG2a) recognizes a monomorphic epitope on HLA-DR dimers (23) and was used as an isotype control Ab for analysis of HLA-DQ with GS200.1, SPVL3, and 1a3 on BLS-1 cells (HLA-DR-negative). Purified CerCLIP (IgG1) was purchased from PharMingen (San Diego, CA). CerCLIP reacts with CLIP bound to HLA class II molecules (24).
Flow cytometric analysis
BLS-1 HLA-DQ cell lines (0.5 x 106 cells) were washed with 1% FBS in PBS (staining buffer), resuspended in 30 µl of L243, SPVL3, 1a3, GS200.1, or CerCLIP at a concentration of 10 µg/ml in staining buffer, and incubated on ice for 45 min. Cells were washed with staining buffer, resuspended in 15 µl of 10 µg/ml fluorescein FITC goat anti-mouse IgG, F(ab')2 (Jackson ImmunoResearch, West Grove, PA), and incubated on ice for 45 min. The cells were washed with staining buffer and resuspended in 500 µl of staining buffer for analysis on a Becton Dickinson FACSort using CellQuest Software (San Jose, CA).
SDS stability assay
Cell lysates were prepared from BLS-1 HLA-DQ cell lines (1 x 106 cells). Briefly, cells were washed in PBS, resuspended in 50 µl cell lysis buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 5 mM EDTA, 1% Nonidet P-40, 1 mM PMSF, 1 µg/ml pepstatin A, 1 µg/ml leupeptin), incubated on ice for 30 min with vortexing, and microfuged at 14,000 rpm for 10 min, then supernatants were collected. Supernatants (20 µg protein) were diluted 1:1 in 2x sample buffer with 0.4% SDS (0.125 M Tris-HCl, pH 6.8, 20% glycerol, 0.4% SDS, 0.005% bromophenol blue) and incubated at room temperature for 30 min. Samples were loaded on a 4–20% Tris-glycine gel (Novex, San Diego, CA), electrophoresed in running gel buffer (25 mM Tris, 190 mM glycine, 0.1% SDS), and transferred to Immobilon-P (Millipore, Bedford, MA). The membrane was blocked in 5% nonfat dry milk in TBS-0.05% Tween 20 (TBST) at room temperature for 1 h and washed three times in TBST. The membranes were incubated with GS200.1 hybridoma supernatant diluted 1:3 in TBST. The membranes were washed three times in TBST and incubated in 0.1 µg/ml goat anti-mouse HRP (Jackson ImmunoResearch) for 1 h at room temperature. The membranes were washed three times in TBST, and proteins were detected by enhanced chemiluminescence (ECL+Plus; Amersham, Arlington Heights, IL). Membranes treated with ECL+Plus were placed on an exposure pad in a GS-363 Loading Dock unit (Bio-Rad, Hercules, CA). The membrane was exposed to a chemiluminescence imaging screen (Imaging Screen-CH; Bio-Rad) for 18 h. The signal on the screen was extracted on a GS-525 scanner (Bio-Rad), and the digitized image was analyzed using Molecular Analyst Software (Bio-Rad).
Peptides
Peptides were synthesized with an Applied Biosystems 432 Peptide Synthesizer (Perkin-Elmer). Peptides were biotinylated as described (25). The identity of each peptide was confirmed by mass spectrometry (Protein and Carbohydrate Structure Facility at the University of Michigan, Ann Arbor, MI). The peptides used were CLIP (CLIP (81–104)) or fragments of CLIP. The amino acid sequence of CLIP (81–104) is LPKPPKPVSKMRMATPLLMQALPM.
Whole-cell peptide binding assay
BLS-1 HLA-DQ cell lines (1.5 x 106 cells) were washed with HBSS and than incubated for 20 min in 0.5% 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, and 1 mM PMSF. Biotinylated peptide was added to the cells in 2 µl of DMSO to a final concentration of 1 or 10 µM and incubated for 18 h at 37°C in a shaking water bath. 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, and 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).
General
Protein concentration was determined using the Bradford microassay with BSA as the standard (26). Protein m.w. markers for Western analysis were prestained low range SDS-PAGE standards (Bio-Rad).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, we showed that DQ0602 was uniquely SDS stable in the
HLA-DM-deficient cell line BLS-1, whereas other HLA-DQ and -DR ß
dimers were not stable, including the structurally similar DQ0604
(5). DQ0604 differs from DQ0602 at seven amino acids, all
of which are in the ß-chain of the molecule (Table I
). To examine the structural basis for
the unique SDS stability of DQ0602 in BLS-1, a panel of mutants was
generated based on the polymorphic differences between DQ0602 and
DQ0604. The amino acids at codons 30, 57, 70, and 86 were mutated in
DQB1*0604 to the corresponding codons in
DQB1*0602, to induce the SDS stability of DQ0604. Codons 9,
87, and 130, which are also polymorphic between DQB1*0604
and DQB1*0602, were not included in these analyses because
the polymorphisms at 9 and 87 are conservative (Tyr to Phe), and the
polymorphism at 130 is in the generally nonpolymorphic region of the
molecule and is not unique to DQB1*0602. The panel of
mutants that represent every combination at codon 30, 57, 70, and 86,
are listed in Table II. Two additional
mutants were prepared, which took advantage of a ScaI site
between codons 59 and 60 in DQB1 to swap codon 1–59 and 60–230 in
DQB1*0604 and DQB1*0602 (Table II).
The mutants listed in Table II were transfected into BLS-1 cells
containing DQA1*0102. Two cell lines were prepared for each
mutation indicated as A and B in Table II. The identity of each cell
line was confirmed by sequencing of DQB1 and characterized by flow
cytometry with three HLA-DQ dimer mAbs SPVL3, GS200.1, and 1a3. The
results of analysis of HLA-DQ cell surface expression with SPVL3
(A), GS200.1 (B), and 1a3 (C) are
shown in Fig. 1. The three HLA-DQ dimer
mAbs recognized all of the mutants, suggesting that the general
structure of the HLA-DQ mutants was unaffected by the mutations. Also,
the expression levels of the DQ0604 mutants in BLS-1 were similar,
varying no more than 3-fold.
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The SDS stability of the DQ0604 mutants was examined in BLS-1 cell
lysates incubated in the presence of 0.2% SDS. This system was
previously shown to result in detection of SDS stable dimer for DQ0602
but showed no or reduced levels of dimer for DQ0604 with the HLA-DQ
dimer mAbs SPVL3, GS200.1, and 1a3 (5). Representative
gels showing the SDS stability of the HLA-DQ mutants relative to
DQ0602, DQ0604, and DQA1*0102 are shown in Fig. 2. The gels are divided according to the
number of sites changed in DQB1*0604. In the single mutant
gel (Fig. 2A), a faint HLA-DQ dimer band was observed for
mutant 2, which contains the ß57 mutation from DQB1*0602.
Upon longer exposure, a more prominent dimer band for mutant 2 was
observed, whereas no dimer bands were detected for the other single
mutants (data not shown). In the double mutant gel (Fig. 2B), dimer bands similar in intensity to DQ0602 were
observed for mutants 5 and 8, which contain ß30 and ß57, and ß57
and ß70, respectively, from DQB1*0602. Less intense dimer
bands were observed for mutants 7 and 9, which contain ß30 and ß86,
and ß57 and ß86, respectively, from DQB1*0602. Dimer
bands were not detected for mutants 6 and 10, which contain ß30 and
ß70, and ß70 and ß86, respectively, from DQB1*0602. In
the triple and quadruple mutant gel (Fig. 2C), strong dimer
bands were observed for all of the mutants except mutant 12, which
contains ß30, ß70, and ß86, from DQB1*0602 and lacks
ß57. In the swap mutant gel (Fig. 2D), mutant 16 in which
ß1–59 comes from DQB1*0602 and ß60–230 from
DQB1*0604 is much more SDS stable than mutant 17 in which
ß1–59 comes from DQB1*0604. Collectively, these results
suggest that ß57 plays a critical role in inducing SDS stability of
HLA-DQ. This is clearly demonstrated in Fig. 2, A and
C, in which the only single mutant that is SDS stable is the
one with ß57 Asp (mutant 2), and the only triple mutant that is not
SDS stable is the one lacking ß57 Asp (mutant 12). ß57 from
DQB1*0602 is not essential to obtain some SDS-stable dimer
as observed for mutant 7 (ß30 and ß86) and mutant 17 (amino acids
1–59 from DQB1*0604) but it is essential for maximal
restoration of SDS stable dimer (Fig. 2, B–D). Similar
results to that shown in Fig. 2 were obtained in three additional
experiments and for the other cell line representing each mutant (data
not shown). The amount of HLA-DQ SDS-stable dimer was also quantitated
using a Molecular Imager, and results were generally consistent but
lacked the detection sensitivity observed on film (data not shown).
Thus in conclusion, ß57 Asp plays a primary role in the SDS stability
of DQ0602 and ß30, ß70, and ß86 play secondary roles in
increasing SDS stability of DQ0602 in BLS-1.
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Traditionally, CLIP is associated with SDS instability of HLA class II molecules. This is thought to be the case because CLIP is the predominant peptide found associated with HLA class II molecules in HLA-DM-deficient cells, and in these same cells HLA class II molecules are not SDS stable (10, 11). However, because of the unique SDS stability of DQ0602 in the HLA-DM-deficient cell line BLS-1, we hypothesized that DQ0602 was uniquely interacting with CLIP, resulting in the SDS stability of DQ0602. This notion was tested first by examining the amount of CLIP associated with the DQ0604 mutants and second by testing the binding of CLIP to the DQ0604 mutants in BLS-1. These results were than compared with the SDS stability of the various DQ0604 mutants.
First, the amount of CLIP associated with HLA-DQ on the BLS-1 DQ0604
mutant cell lines was examined with the CerCLIP Ab in flow cytometric
analysis. Fig. 3 shows the amount of CLIP
normalized for HLA-DQ expression for DQ0602, DQ0604, and the DQ0604
mutant cell lines. These results show that 5-fold more CLIP was
associated with DQ0602 than DQ0604, which was consistent with the
notion that CLIP interacts uniquely with DQ0602. However, the results
obtained with the BLS-1 DQ0604 mutant cell lines did not follow the
same trend observed with DQ0602 and DQ0604, such that a number of
DQ0604 mutants that showed no detectable SDS-stable-dimer, in
particular mutant 1 (codon 30 from DQB1*0602), mutant 6
(codons 30 and 70 from DQB1*0602), and mutant
12 (codons 30, 70, and 86 from DQB1*0602), showed levels of
CLIP comparable to DQ0602. Asterisks above the bars in Fig. 3 indicate
the cell lines with detectable SDS stable dimer levels. Clearly there
is no correlation between the level of HLA class II-associated CLIP and
amount of SDS-stable dimer.
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shows the binding of CLIP (81–104) (A), CLIP (90–100)
(B), and CLIP (93–103) (C) to one of each of the
BLS-1 DQ0604 mutant cell lines at 1 µM peptide concentration. Similar
results were obtained with the other cell line for each mutant and each
peptide tested at 10 µM peptide concentration (data not shown).
DQ0602 bound 2- to 3-fold better to CLIP (81–104) at 1 µM than
DQ0604 in the whole-cell assay, which is consistent with results
observed in a purified peptide binding assay (data not shown). One
mutant stands out in Fig. 4, A–C. This is mutant 6 (codons
30 and 70 from DQB1*0602), which binds considerably better to all three
peptides than any other mutants. This mutant is SDS unstable (Fig. 2)
and associated with high amounts of CLIP as detected by CerCLIP
staining (Fig. 3). Mutant 1 (codon 30 from DQB1*0602) also binds well
to CLIP and is SDS unstable and associated with high amounts of CLIP.
Thus, the ability to bind CLIP well does not result in SDS stability.
Additional, there is no correlation between the amount of CLIP binding
to HLA-DQ and SDS stability (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß dimers (HLA-DQ and I-A) and IDDM susceptibility has been
observed (5, 6). The HLA-DQ allele associated
with dominant protection in IDDM was the most stable HLA-DQ molecule,
even being uniquely SDS stable in the HLA-DM-deficient cell line,
BLS-1. We took advantage of this result to address the mechanistic
basis of the exceptional SDS stability of DQ0602. Optimal SDS stability of HLA-DQ in BLS-1 occurred with codon 57 from DQB1*0602 in combination with one or more additional codons from DQB1*0602 at ß30, ß70, and ß86. The greatest contribution was clearly played by ß57 Asp as only its absence in the triple mutant abolished stability and only its presence alone without any other changes induced SDS stability at a detectable level. IDDM susceptibility was initially proposed to be largely dependent on the amino acid at ß57 (28). An Asp at codon 57 of the ß-chain was found to protect from IDDM. However, additional studies have shown this to be an oversimplification (29, 30). Nevertheless, the Asp/non-Asp (Ala, Val, Ser) dimorphism is expected to play a functional role as it is an evolutionarily conserved feature. Also, the amino acid at ß57 has been observed to regulate peptide binding to two HLA-DQ alleles, HLA-DQA1*0301/DQB1*0302 and HLA-DQA1*0301/DQB1*0303, which differ only at ß57 (31, 32). HLA-DQA1*0301/DQB1*0302 (Ala at ß57) is positively associated with IDDM susceptibility, whereas HLA-DQA1*0301/DQB1*0303 (Asp at ß57) is not associated with IDDM (2). Thus, a critical role for the codon at ß57 in IDDM susceptibility has previously been suggested.
DQ0602 and DQ0604 have been molecularly modeled based on the crystal
structure of HLA-DR1 (29). A crystal structure of HLA-DQ
is currently lacking. The molecular model shows that amino acid 30, 57,
70, and 86 are found within the peptide-binding groove of the molecule.
The model predicts that the polymorphism at ß57 produces a
significant change in an anchor pocket in the peptide-binding groove.
When Asp is present, as is found in DQ0602, a salt bridge is formed
with the Arg at 79. When Val is present, as is found in DQ0604, the
salt bridge is broken and Arg 79 adopts a different conformation
resulting in a significant increase in anchor pocket size and
hydrophobicity. The model also predicts that the polymorphism at ß70,
Gly in DQ0602 and Arg in DQ0604, is significant, as it is positioned at
the top of a helical segment. There the residue at ß70 is likely to
form hydrogen bonds with the peptide ligand backbone and interact
directly with the TCRs. The other polymorphisms between DQ0602 and
DQ0604 were predicted to not have any significant effects due to their
conservative nature. The results presented herein are consistent with
the predictions of the model in that ß57 is critical for SDS
stability, which is generally a function of peptide binding. However,
the model does not reveal the role of the other polymorphisms in SDS
stability that is apparent from this study.
Previous studies have indicated that HLA class II proteins in HLA-DM-deficient cells are loaded with CLIP (10, 11). Removal of CLIP and the addition of antigenic peptides are important for SDS stability of HLA class II proteins (7, 8, 9, 10, 11, 12, 13, 14, 15). The unique stability of DQ0602 in the HLA-DM-deficient cell line BLS-1 suggested the possibility that DQ0602 interacted with CLIP in a unique way that resulted in SDS stability. To address this hypothesis, CLIP was analyzed in the context of DQ0602, DQ0604, and the HLA-DQ mutants. Two assays were used to examine CLIP. The first assay used the CerCLIP Ab in a flow cytometric assay to determine the amount of CLIP associated with HLA-DQ on the cell surface of the BLS-1 DQ0604 mutant cell lines. CerCLIP is a mouse mAb produced against CLIP (81–104) (24) and recognizes an epitope on CLIP between amino acids 81 and 92 (R. Ettinger, G. Nepom, and W. Kwok, unpublished data). The second assay examined the binding of CLIP to HLA-DQ in a whole-cell peptide binding assay and determined how well the DQ0604 mutants could bind CLIP. These assays showed that the amount of CLIP associated with the DQ0604 mutants on the BLS-1 HLA-DQ cell lines, as well as the amount of CLIP that binds to the DQ0604 mutants, does not correlate with SDS stability of the DQ0604 mutants. Also, the epitope of CLIP that DQ0602 and DQ0604 preferentially bind to is very similar. Thus, CLIP most likely does not uniquely interact with DQ0602 resulting in its unique SDS stability in BLS-1.
The results presented herein contribute to our understanding of HLA-DQ
ß dimer stability. An Asp at ß57 is critical for SDS stability
of HLA-DQ ß dimers. Other polymorphic residues in HLA-DQ modulate
stability, in this case, Tyr at ß30, Gly at ß70, and Ala at ß86,
significantly increased SDS stability of DQ0602. The importance of an
Asp at ß57 for the stability of DQ0602 is consistent with the primary
role the Asp/non-Asp dimorphism at ß57 plays in dictating IDDM
susceptibility, thus suggesting a potential mechanism by which ß57
Asp protects against IDDM.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ruth A. Ettinger, University of Washington, Department of Medicine, Box 357710, HSB K-165, 1959 NE Pacific Avenue, Seattle, WA 98195.
3 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; CLIP, class II-associated invariant chain peptide; DQ0602, protein encoded by HLA-DQA1*0102/DQB1*0602; DQ0604, protein encoded by HLA-DQA1*0102/DQB1*0604; BLS, bare lymphocyte syndrome.
Received for publication March 22, 2000. Accepted for publication July 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß protein dimer, the class II MHC molecule associated with protection from insulin-dependent diabetes mellitus. J. Immunol. 161:6439.This article has been cited by other articles:
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