HLA-DMA Polymorphisms Differentially
Affect MHC Class II Peptide Loading
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J Immunol 2015; 194:803-816; Prepublished online 10
December 2014;
doi: 10.4049/jimmunol.1401389
http://www.jimmunol.org/content/194/2/803
http://www.jimmunol.org/content/suppl/2014/12/10/jimmunol.140138
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Supplementary
Material
Miguel Álvaro-Benito, Marek Wieczorek, Jana Sticht,
Claudia Kipar and Christian Freund
The Journal of Immunology
HLA-DMA Polymorphisms Differentially Affect MHC
Class II Peptide Loading
Miguel Álvaro-Benito,*,1 Marek Wieczorek,*,†,1 Jana Sticht,* Claudia Kipar,* and
Christian Freund*,†
T
he canonical function of MHC class II proteins in adaptive
immunity depends critically on their ability to stimulate
CD4+ T cells. Although the classical MHCII proteins
(HLA-DR, -DQ, and -DP) present peptides on the surface of APC,
the nonclassical MHC class II (MHCII) molecules (HLA-DM and
-DO) perform accessory functions during the process of Ag
loading. Classical and nonclassical MHCII proteins are heterodimers encoded by the HLA class II locus, one of the most
polymorphic regions in the human genome. Although there are
2604 polymorphisms annotated for genes encoding classical
MHCII proteins, which would result in1894 different protein
chains, the nonclassical HLA-DM and HLA-DO molecules show
only limited variability, with 20 and 25 annotated variants at the
nucleic acid level that would result in 11 and 8 different protein
chains, respectively (1). Of interest, most of the polymorphisms
found in the MHCII locus affect positions forming the peptidebinding groove of classical MHCII proteins. Thereby, HLA-DR,
-DQ and -DP alleles encode for proteins presenting distinct peptide repertoires. As a consequence, the ability to tightly bind
pathogenic or potentially autoreactive self-peptides also varies
*Institut f€
ur Chemie und Biochemie, Freie Universität Berlin, 14195 Berlin, Germany; and †Leibniz Institute for Molecular Pharmacology, 13125 Berlin, Germany
1
M.Á.-B. and M.W. contributed equally.
Received for publication June 4, 2014. Accepted for publication November 11, 2014.
This work was supported by Deutsche Forschungsgemeinschaft Grants FR 1325/11-1,
SFB765, SFB854, and SFB958 (to C.F.).
Address correspondence and reprint requests to Prof. Christian Freund, Protein Biochemistry, Institute of Chemistry and Biochemistry, Thielallee 63, 14195 Berlin,
Germany. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: DRB1-N82A, HLA-DR1 heterodimer (HLADRA*0101/HLA-DRB1*0101) containing the mutation N82A in the b-chain; FP,
fluorescence polarization; MBP, myelin basic protein; MHCII, MHC class II; MS,
multiple sclerosis; pMHCII, peptide–MHC class II; RA, rheumatoid arthritis; SPR,
surface plasmon resonance; T1D, type 1 diabetes.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401389
between the different MHCII alleles. An additional key determinant
of peptides presented by particular MHC alleles is the peptide
exchange catalyst HLA-DM, a nonclassical MHCII molecule. HLADM is structurally related to MHCII molecules, but is itself incapable
of interacting with Ags. Instead, it binds transiently to MHCII, facilitating peptide release and fostering the loading of high-affinity
binders. This DM-catalyzed exchange can also be modulated by
the competitive inhibitor HLA-DO, another nonclassical MHCII
molecule. HLA-DO binds with high affinity to DM and is particularly
expressed in immature dendritic and B cells (2–4).
Polymorphisms of classical MHCII molecules (HLA-DR, -DQ,
and -DP) have been studied in great detail and their role as genetic
susceptibility factors contributing to autoimmunity has been firmly
established (5). Known examples of MHCII proteins containing
alleles linked to specific diseases are HLA-DR1 (HLA-DRA*0101HLA-DRB1*0101) related to rheumatoid arthritis (RA), HLA-DR2
(HLA-DRA*0101-HLA-DRB1*1501) associated with multiple sclerosis (MS), and HLA-DR4 (HLA-DRA*0101–DRB1*0401) associated with RA, MS, and type 1 diabetes (T1D). Of note, because
these HLA-DR alleles share the same HLA-DRA chain, their ability
to present self-antigens specific for each disease depends on the
polymorphisms of the b-chains.
Although a clear link exists between classical MHCII polymorphisms and disease, genetic association studies of HLA-DM
variants and autoimmunity remain controversial. Several investigations were unable to show HLA-DM polymorphisms to be an
additional risk factor in RA, MS, and lupus erythematosus in
certain populations (6–8). Other studies, however, have supported
a genetic link between DM and several autoimmune diseases,
including T1D, RA, and psoriasis (9–15). Presumably, the haplotypic combination of the MHCII and HLA-DM alleles expressed
in a particular individual will influence the peptide exchange kinetics and the presentation of self-peptides, thereby contributing
to autoimmune conditioning. Because the biochemical properties
of HLA-DM variants other than the HLA-DMA*0101/HLADMB*0101 heterodimer have not been investigated so far, there is
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During the adaptive immune response, MHCII proteins display antigenic peptides on the cell surface of APCs for CD4+ T cell
surveillance. HLA-DM, a nonclassical MHCII protein, acts as a peptide exchange catalyst for MHCII, editing the peptide repertoire.
Although they map to the same gene locus, MHCII proteins exhibit a high degree of polymorphism, whereas only low variability has
been observed for HLA-DM. As HLA-DM activity directly favors immunodominant peptide presentation, polymorphisms in HLADM (DMA or DMB chain) might well be a contributing risk factor for autoimmunity and immune disorders. Our systematic
comparison of DMA*0103/DMB*0101 (DMA-G155A and DMA-R184H) with DMA*0101/DMB*0101 in terms of catalyzed peptide
exchange and dissociation, as well as direct interaction with several HLA-DR/peptide complexes, reveals an attenuated catalytic
activity of DMA*0103/DMB*0101. The G155A substitution dominates the catalytic behavior of DMA*0103/DMB*0101 by decreasing peptide release velocity. Preloaded peptide–MHCII complexes exhibit ∼2-fold increase in half-life in the presence of DMA*0103/
DMB*0101 when compared with DMA*0101/DMB*0101. We show that this effect leads to a greater persistence of autoimmunityrelated Ags in the presence of high-affinity competitor peptide. Our study therefore reveals that HLA-DM polymorphic residues
have a considerable impact on HLA-DM catalytic activity. The Journal of Immunology, 2015, 194: 803–816.
804
surface plasmon resonance (SPR), we identified G155 as a critical
residue for efficient peptide release catalysis. While probing several autoimmune relevant self-peptides, we observed DMA*0103
to be less efficient in catalyzing peptide release, especially under
acidic conditions. These results establish the basis for understanding
differences in autoimmune susceptibilities for individuals positive for DMA*0103.
Materials and Methods
Chemical reagents, peptides, and buffers
Peptides used in this study were described previously [HA306–318: PKYVKQNTLKLAT and myelin basic protein (MBP)83–101: DENPVVHFFKNIVTPRTPP], or are listed when used. All peptides were purchased
from Peptides & Elephants (Berlin, Germany) or synthesized at the
Forschungsinstitut f€ur Molekulare Pharmakologie Berlin (Germany).
When required, an FITC label was covalently linked to the exposed P5
position (shown in the above peptides underlined and in bold). Coupling
was achieved by reaction of the N-hydroxysuccinimide ester with the
primary side-chain amide group in each case. Generally, peptide stocks
were prepared in PBS buffer (pH 5.8) at 5.0 or 0.5 mM and diluted
as required. Stocks of FITC-labeled peptides were first diluted in DMSO.
Unless otherwise indicated, all experiments were performed using 50 mM
citrate phosphate buffer containing 150 mM NaCl. When stated, the dipeptide FR (Phe-Arg) was used to increase the binding of peptides to empty
MHCII molecules. FR favors the transition of empty and nonreceptive
MHCII molecules to a receptive state (23), thereby accelerating peptide
loading.
HLA-DM reconstitution in HeLa cells
Full-length cDNA constructs encoding DMA*0101-HA-tagged, DMA*0103HA-tagged, DMB*0101-myc tagged, HLA-DRA1*0101, and -DRB1*0101
were based on the pCDNA3.1 vector, whereas the invariant chain (Li, CD74)
construct was based on the pCMV6 vector.
HeLa cells were grown on DMEM supplemented with 10% FBS and
2mM L-glutamine at 37˚C and 5% CO2. Cells growing in 6-cm plates were
cotransfected with a total of 4 mg DNA using FuGENE HD (Promega)
according to the manufacturer’s specifications. Stoichiometric concentrations of each construct were used to prepare the DNA mixtures, and
empty pCDNA3.1 or pCMV6 was added to keep total DNA amounts
constant when required. After 48 h, cells were washed with PBS, resuspended with Accutase and finally spun down. Cell pellets were washed
twice with PBS and lysed as previously described (24).
Protein expression was analyzed by Western blot. The samples were
loaded on 10% acrylamide SDS-PAGE gels and blotted on a nitrocellulose
membrane (30 V for 90 min), and the individual protein chains were
detected using Abs and a commercial chemoluminiscent reagent. We used
the following primary Abs: anti-DR (1B5), anti-CD74, and anti-HLA-DM
(Abcam); anti–c-myc (Pharmingen); anti-actin and anti-HA (Santa Cruz
Biotechnology). Appropriate secondary Abs coupled to HRP were used in
each case.
To verify both the expression of functional HLA-DR molecules and the
functionality of HLA-DM molecules, we assessed the presence of SDS
stable dimers as previously described (25). Cell pellets were lysed, divided
into two aliquots, and mixed with 23 SDS-PAGE loading buffer. One of
the samples was kept at room temperature, and the other one was heated
for 5 min at 95˚C. Finally, the presence of SDS-stable dimers was detected
after Western blot, using the 1B5 Ab.
HLA-DR and -DM cloning, mutagenesis, expression, and
purification
HLA-DR constructs used to produce recombinant proteins were based on
the pFastBacDual vector (Invitrogen) and baculovirus–insect cell expression system. These constructs include the extracellular domains of the
proteins (HLA-DRA*0101, DRB1*0101; HLA-DRA*0101, DRB15*0101
and HLA-DRA*0101, DRB1*0401) and the transmembrane domains
replaced by leucine zippers (DR1, DR2, and DR4). In addition, a cDNA
sequence encoding a flexible Gly-Ser linker and the CLIP103–117 fragment
spaced by a thrombin cleavage site was fused to the N-terminus of the
b-chains of the same constructs (DR1C, DR2C, and DR4C). HLA-DM
(HLA-DMA1*0101 and HLA-DMB1*0101), described in Nicholson et al.
(26), was also cloned into the pFastBacDual vector (DMA*0101 and
DMB*0101). The original C-terminal protein C tag in the b-chain was
replaced with a biotin acceptor sequence, and the a-chain FLAG-tag was
kept and used for purification. On the basis of this construct, HLA-DMA
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no clear molecular basis for understanding potential differences in
their biological function.
Recent biophysical and structural studies have shed light on the
general mechanism of DM peptide exchange catalysis (16–18), as
well as on DM inhibition by DO (19, 20). Studies using hydrogendeuterium exchange in combination with mass spectrometry have
shown that the dynamic properties of the 3/10-helix in MHCII
dictate its susceptibility to DM. In the reported DM/DR1 crystal
structures, the interaction surface is dominated by the a-subunits
of both molecules. In the DM/DR1 complex, HLA-DRA-F51
stabilizes the peptide-free P1 pocket by a structural rearrangement of the HLA-DRA 46–55 segment. This rearrangement seems
to be driven by the repositioning of a key residue, HLA-DRAW43, away from the peptide P1 region. This flipping of the
tryptophan side chain likely depends on the dissociation of the
N-terminal part of the peptide from MHCII (16, 17, 21). Of interest,
the interface between DO and DM is very similar to that of DR
and DM, supporting the idea that DO acts as a competitive inhibitor of DM.
HLA-DM heterodimers are encoded by HLA-DMA and HLADMB genes. Only four DMA alleles have been described to date,
and each of them contains one or two single nucleotide polymorphisms compared with the reference sequence of DMA*0101
(22). These single nucleotide polymorphisms result in amino acid
changes at positions 140, 155, and 184 of the mature protein. Of
these changes, positions 155 and 184 map to the MHCII interface
(Fig. 1A). DMA-155 is localized to a loop between the fifth and
sixth b-strand of the a-chain, close to the 3/10-helix, which is
located directly at the DM/DR interface; this region has been
proposed to dictate DR susceptibility for DM. Moreover, DMA155 may also directly affect positioning of crucial residues for the
DM/DR interaction, as, for example, DMA-D154, that get into
proximity to DRA-R50 (Fig. 1B). DMA-184 is positioned in
a turn of the seventh and eighth b-strands of the Ig-like domain.
Of interest, the conformation of this loop is significantly altered
upon DM/DR1 complex formation, leading to an interaction of
DMA-R184 and DMA-E181, which might go along with altered
dynamic properties (Fig. 1C, 1D).
The only DMA residue that is strictly conserved among different
species is DMA-N125 (17) (Fig. 1E, red boxes). Of note, the
polymorphic residue DMA-G155 seems to be partially conserved,
even among evolutionary distant species (e.g., mammals and
marsupials), whereas the two positions DMA-140 and DMA-184
are significantly less conserved (Fig. 1E, blue boxes). Such partial
conservation of DMA-G155 may reflect its relevance for the
catalytic function of the protein. In humans, the only allele
encoding a change of DMA-155 is the HLA-DMA*0103 allele,
which additionally carries a DMA-R184H substitution. Although
the frequency of HLA-DMA*0103 is not high when compared
with HLA-DMA*0101 among populations in different studies
(,2% versus .40% respectively), it is one of the alleles that have
been connected, albeit controversially, to immune disorders via
genetic association studies (8, 10, 12–15).
We hypothesize that a conceivable connection between HLA-DM
polymorphisms and immune disorders must be due to the altered
function of its peptide exchange and/or chaperone activities. In this
study, we provide evidence for impaired peptide exchange functionality of HLA-DM molecules carrying the DMA*0103 polymorphic a-chain. HLA-DM consisting of DMA*0101/DMB*0101
(“DMA*0101”) and DMA*0103/DMB*0101 (“DMA*0103”), as
well as two single mutations (G155A and R184H), were assayed
for their efficiency to bind different HLA-DR proteins, release the
prebound peptide, and catalyze the peptide exchange against higher
affinity binders. Using fluorescence polarization (FP) together with
HLA-DMA POLYMORPHISMS
The Journal of Immunology
Protein methods: linker cleavage and biotinylation
DR1C, DR2C, and DR4C proteins were treated with thrombin (20 U/mg in
PBS; Sigma-Aldrich), and cleavage was verified by the shift of b-chain
mobility on SDS-PAGE. The reaction mixtures were gel filtrated using
Superdex S200 (GE Healthcare); multimeric complexes and aggregates
were discarded. Fractions containing the proteins of the correct size were
pooled and concentrated using Vivaspin 30-kDa MWCO spin filters to
render purified DR1/C, DR2/C, and DR4/C (which would have the CLIP
peptide bound to the MHCII molecules, but not covalently linked).
HLA-DM protein variants were biotinylated using a commercial biotinylation kit (Avidity Biotechnology). HLA-DM and BirA ligase were
mixed at a 20:1 molar ratio in bicine buffer, including 100 mM biotin, 10
mM ATP, and 10 mM sodium acetate, then incubated for 16 h at room
temperature. Biotinylation was confirmed using native polyacrylamide gels
after incubation of the reaction mixtures with streptavidin, as described in
Day et al. (31). Biotin was removed from the reaction mixtures by extensive dialysis.
FP-based peptide–MHCII binding and release experiments
The use of FP to study MHCII peptide exchange and release has been widely
described (26, 28). In this study, FP was used to assess the ability of HLADM variants to catalyze peptide exchange under different conditions, and
to estimate kinetic parameters of this exchange. Fluorescently labeled
peptides (FITC-HA306–318 and FITC-MBP83–101) were used as probes to
detect their real-time binding to MHCII molecules. For the specific case of
HLA-DM–mediated peptide exchange, MHCII molecules preloaded with
a peptide (CLIP in this case) were incubated with substoichiometric
concentrations of labeled peptide. Binding of the reporter peptide was
measured in mP units, and the catalyst contribution to peptide exchange
was determined by subtracting the uncatalyzed exchange rates from the
rates of the catalyzed reactions (26). For peptide-dissociation experiments,
the labeled peptide was bound to the MHCII molecules, and the MHCIIlabeled peptide complexes were incubated in the presence of an excess of
competing, nonlabeled peptide. In this case the dissociation rates are obtained by fitting an exponential decay function to the release curves. We
used conditions identical to those previously described for peptide exchange determinations in experiments with DR1/C (28, 32) and extended
them to DR4/C, which includes 1 mM DR1/C, 100 nM FITC-HA, and 100 nM
HLA-DM at pH 5.2 (measured at 37˚C). For DR2/C, we used a similar
set-up as described by Nicholson et al. (26), with minor modifications:
200 nM DR2/C was incubated with or without 20 nM HLA-DM and 50 nM
MBP. DMSO was added to all the reactions to the same final concentration. For peptide dissociation investigations, we used 150 nM DR/FITC–
peptide complexes in the presence of 0–300 nM DM and 50 mM unlabeled
MBP to prevent rebinding of the labeled peptide. Each reaction was performed in at least two independent experiments, including triplicates for
each measurement (the reaction volume per measurement was 40 ml). FP
was recorded in a Victor 3V reader (PerkinElmer).
To study the catalytic efficiency of the different HLA-DM proteins over
the pH range 4.6–6.4, we used DR1/C and DR4/C in concentrations
ranging from 125 nM to 1.5 mM, and from 125 nM to 1.5 mM for DR2/C.
Reporter peptide and the different DM variants were added in the same
amounts as described for the standard conditions. Reactions were prepared
on ice and measured directly upon addition of the reporter peptides for
300–500 min.
For kinetic determinations, FP values were transformed into anisotropy
(A) units (Eq. 1), as these values are strictly linear in regard to labeled free:
bound peptide amounts (26). A single-phase exponential association curve
was used to estimate the binding rates (Eq. 2). The slope of the first 10% of
the binding curve was used to define the initial velocity of the reaction, in
units of mA/min, and these values were plotted against MHCII concentration. The resulting data points were used to fit a hyperbolic function,
providing Vmax and Km values (Eq. 3).
A¼
2 3 FP
ð3 2 F P Þ
ð1Þ
Anisotropy (A) is related to the FP value.
At ¼ Aeq 3 1 2 e2Kob 3 t
þ At¼0 ;
ð2Þ
where At is anisotropy (A) at a given time (t), t is the time in minutes, Aeq is
the anisotropy value at equilibrium, and A0 is the anisotropy value for the
free peptide at the assay temperature. Kob is the observed rate constant
(min21; kon[peptide] + koff).
A0 values were 90 mA for HA and 102 for MBP. Aeq values were 210
mA for DR1/HA, 230 mA in the case of DR4/HA, and 255 mA in the case
of DR2/MBP.
v¼
Vmax 3 ½DR=C
;
Km þ ½DR=C
ð3Þ
where Vmax is the maximum reaction velocity for a given amount of catalyst, [DR/C] stands for the molar concentration of the DR complex, and
Km is the Michaelis–Menten constant, representing the substrate (DR/C)
concentration required to reach 1/2Vmax. All kinetic constants were obtained using SigmaPlot software.
Endpoint peptide release assays
To investigate the ability of the different DM protein variants to release
peptides with different affinities in the presence of a higher affinity model
Ag, DR–biotinylated peptide complexes were produced as described previously (33). Excess peptide was removed from the loading reactions by
diafiltration and microspin columns (Bio-Rad; SP30) equilibrated with
PBS containing 1% BSA (34). Model Ags were the same peptides used for
the FP assay lacking the label; HA was used as the Ag for DR1 and DR4–
peptide complexes, and MBP was used for DR2–peptide complexes.
Peptide release was analyzed by measuring the remaining biotinylated
peptide on DR molecules in the absence and in the presence of DM proteins using time-resolved fluorometry (dissociation-enhanced lanthanide
fluorescence immunoassay), as described previously (33). The DR–biotinylated peptide complexes were incubated at a concentration of 20–40
nM with 200 nM HLA-DM in the presence of 100 mM nonbiotinylated
peptide in citrate phosphate buffer (pH 5.2) at 37˚C. At the indicated time
points, an aliquot was removed and the reaction stopped by addition of
cold PBS containing 1% BSA. The reaction mixtures were incubated in
microtiter plates coated with specific conformational Ab (L243), and the
remaining biotinylated peptides were detected by Eu3+-labeled streptavidin. Finally, fluorescence was detected by addition of enhancement solution (15 mM b-naphthoyltrifluoroacetone, 50 mM tri-n-octylphosphine
oxide, 6.8 mM potassium hydrogen phthalate, 100 mM acetic acid, 0.1%
Triton X-100) in a Victor fluorescence reader (PerkinElmer). For each
experiment, the remaining biotinylated peptide was normalized versus the
total biotinylated peptide at t = 0, if not otherwise indicated.
Surface plasmon resonance
Biotinylated DM variants (DMA*0101, DMA*0101 G155A, DMA*0101
R184H and DMA*0103) were subjected to size-exclusion chromatography, and fractions containing the protein of interest were pooled and filtrated versus 10 mM Tris-HCl, pH 8, using Amicon ultrafiltration tubes
(30 kDa MWCO). The resulting proteins were coupled to streptavidin chips,
and sample or control proteins (DR/C complexes or control DR) were
assayed for interactions, as previously described (16). We normalized the
amount of coupled protein [500–700 RU (response units)] to facilitate
further comparisons. Experiments were carried out at 30˚C in 50 mM
citrate buffer, 150 mM NaCl, pH 5.35, and 0.06% C12E9 detergent, with
a flow rate of 15 ml/min (if not stated otherwise) in a Biacore 3000
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mutants were prepared according to Zheng et al. (27). Sequencing was
used to verify each mutation.
All proteins were produced using the above-mentioned baculovirus–
insect cell expression system (pFastBacDual-Sf9). First, the pFastBacDualbased constructs were used to produce bacmids according to the manufacturer’s
specifications. Next, the virus was amplified upon infection of Sf9 cells.
For protein expression, Sf9 cells in the exponential growth phase (1–2 3 106
cells per milliliter) were infected at a multiplicity of infection of 10, then
kept at 27˚C for 3–4 d. Proteins were purified from the supernatants by
immunoaffinity chromatography. The L243 Ab coupled to Fast Flow
Sepharose was used for HLA-DR purification and M2-Sepharose (SigmaAldrich) was used for HLA-DM (28).
For control surface plasmon resonance (SPR) experiments, DR1 proteins
in complex with the peptides indicated in each case [DR1/HA, DRA-G49S
(HLA-DR1 heterodimer [HLA-DRA*0101/HLA-DRB1*0101] containing
the mutation G49S in the a-chain)/C102–120 M107W, DRA-F54S (HLA-DR1
heterodimer [HLA-DRA*0101/HLA-DRB1*0101] containing the mutation F54S in the a-chain)/C102–120 M107W, and DRB1-N82A (HLA-DR1
heterodimer [HLA-DRA*0101/HLA-DRB1*0101] containing the mutation F54S in the a-chain/C102–120 M107W)] were obtained from refolding
single subunits expressed in E. coli as inclusion bodies, as previously
described (29, 30). Empty DR molecules were loaded with the different
peptides after refolding (10:1 to 20:1 peptide/DR molar ratio) in the
presence of 2 mM FR-loading enhancer (dipeptide) for 48 h at 37˚C.
Peptide–MHC complexes were then separated from the peptide excess by
size-exclusion chromatography.
805
806
HLA-DMA POLYMORPHISMS
(GE Healthcare) device. The signal of nonspecific binding to the reference
flow cells was subtracted from the signal of the DM-coupled flow cells. In
a standard experiment, peptide–MHC complexes were injected at a flow
rate of 15 ml/min for 300 s on the flow cells containing the immobilized
DMs or controls followed by buffer (300 s) and peptide consecutively
(2 mM MBP or 50 mM HA for a further 450 s). The chip was regenerated
by injecting high-affinity peptides for the respective HLA-DR alleles in
the flow cell at concentrations ranging from 20 to 100 mM. DR1–peptide
complexes with different degrees of DM susceptibility were tested for DM
binding to ensure the specificity of the coupled proteins.
change of the different DR/C molecules. Although differences at
these conditions are small, they nevertheless show that DMA*0103
and DMA*0101 G155A display reduced exchange activity relative
to DMA*0101 and DMA*0101 R184H. Comparing the different
DR alleles used in this study, the rate enhancement indicated by the
slopes in Fig. 2E–G follow the order DR1/C . DR2/C . DR4/C,
and can be correlated to the stability of the DR/CLIP complexes
(Supplemental Fig. 1B).
ThermoFluor assays
DMA*0103 exhibits a lower catalytic efficiency than does
DMA*0101
The hydrophobic dye SyPro Orange (Life Technologies) was used to determine the thermal stability of each HLA-DM variant. The protein/dye
mixture (0.2–0.5 mg/ml monomeric protein with 53 dye) was heated
constantly from 25 to 95˚C with a temperature increase of 2˚C/min.
Samples were excited at 490 nm, and the fluorescence signal was detected
at 575 nm. Change in fluorescence intensity versus the temperature was
plotted, and a sigmoidal function was fit to determine the midpoint temperature of the unfolding reaction (Tm). Stability measurements were
performed in 50 mM phosphate/citrate buffer with 150 mM NaCl.
HLA-DMA polymorphisms affect DM stability and peptide
exchange catalysis
The allelic variant DMA*0103, carrying the mutations G155A and
R184H (relative to the most abundant allelic variant DMA*0101)
(Fig. 1) and its single-mutant variants, was expressed as soluble
HLA-DM heterodimers bearing the indicated DMA molecule in
complex with the HLA-DMB*0101 chain (Fig. 2A). SDS-PAGE
analysis and size-exclusion chromatography confirmed the existence of a 50-kDa heterodimer (Fig. 2B). Protein stability assays
showed a marked decrease in the denaturation temperature of
∼3˚C for DMA*0103 (∼56.5˚C) in comparison with DMA*0101
(∼59.5˚C) in a pH range of 4.6–6.4 (Fig. 2C). Interestingly, the
analysis of the single-mutant proteins revealed that such decreased
thermal stability is caused by the G155A substitution (56.2 6
0.2˚C), as DMA*0101 R184H (59.9 6 0.6˚C) exhibits a thermal
stability similar to that of DMA*0101 (Supplemental Fig. 1A).
Given the slightly reduced thermal stability of the HLA-DM
polymorphic variants carrying the G155A substitution, we wanted
to show that DM heterodimers constituting this mutation are formed in
cells. HeLa cells were used to reconstitute an MHC class II–like
compartment using HLA-DR1, CD74, and the two HLA-DM variants DMA*0101-DMB*0101 and DMA*0103-DMB*0101 (35).
Western blot analysis revealed similar expression levels for each individual chain (data not shown). The presence of SDS-resistant
MHCII heterodimers relies on efficient DM-mediated selection of
kinetically stable complexes and indirectly demonstrates effective
DM/DR interaction (25). We observed the formation of HLADR1 SDS-stable dimers in the presence of either DMA*0101 or
DMA*0103, DMB*0101, clearly indicating that the latter variant acts
as an exchange catalyst under cellular conditions (Fig 2D).
To investigate the catalytic activity of the DM variants we
measured peptide exchange by FP, using distinct DR allelic variants
preloaded with the placeholder peptide CLIP (DR/C). The exchange of CLIP for higher affinity reporter peptides in the presence
of the different DM protein variants was determined as a gain in FP
values detected under standard conditions. In the case of DR1 and
DR4, the influenza virus-derived peptide HA was used as a competitor peptide, whereas a well described high-affinity self-peptide
derived from MBP was chosen as reporter for DR2 (26, 36).
Because in this case uncatalyzed exchange of the MBP peptide for
CLIP was significant at 37˚C, all experiments measuring MBPFITC binding to DR2/C were performed at 25˚C. As depicted in
Fig. 2E–G, DM variants differentially catalyzed the peptide ex-
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Results
The mechanism of DM-mediated exchange is thought to be
a multistep reaction comprising the dissociation of at least the
N-terminal part of the peptide, DM recognition of the relatively rare
(partially) empty MHCII conformation accompanied by structural
rearrangements of the two molecules in the complex, and finally
competitive binding of a high-affinity peptide with subsequent
release of DM from the MHCII–DM complex (21, 37). Considering that DM activity is pH dependent (26, 38), we next asked
whether this observation holds true for different DMA variants.
We specifically sought to address whether the slower peptide exchange rates catalyzed by DMA*0103, when compared with those
by DMA*0101, are caused by reduced substrate recognition and/
or less efficient peptide release activity. In an approach similar to
that described by Nicholson et al. (26), we determined the initial
velocities of CLIP exchange against reporter Ags at different DR/C
concentrations and over a range of pH, from 4.6 to 6.4. HLA-DM
contribution to the peptide binding reaction was calculated by
subtracting the rates for the spontaneous peptide binding, from the
HLA-DM–catalyzed reactions. The resulting initial velocities for
peptide exchange were plotted versus the DR/C concentration for
every pH. As expected, the data displayed hyperbolic Michaelis–
Menten behavior (Fig. 3A–C). Plots were then used to estimate
the kinetic parameters Km and Vmax, shown for DMA*0101 and
DMA*0103 (Table I) and DMA*0101 G155A and DMA*0101
R184H (Table II). DMA*0101 and DMA*0103 show a clearly
different behavior, and again we observed that although the
DMA*0101 R184 mutant behaves similarly to DMA*0101,
DMA*0101 G155A resembles DMA*0103, indicating a predominant role of the G155A mutation in DMA*0103 activity. Of interest, all the tested DM variants display Km values within the
nanomolar to low micromolar range, consistent with previous
reports (26, 39). Increasing the pH from 4.6 to 6.4 resulted in
a consistent increase in the Km values for all DM-DR combinations. The only observed exception was DMA*0103 with respect
to DR2/C versus MBP exchange, which showed elevated Km
values at pH 4.6 and 5.8 (Table I). In this case, noncatalyzed
binding of the reporter peptide at high pH hindered us from
completing the peptide exchange experiments with DMA*0103
and DR2/C at pH 6.4.
The value of Vmax in our loading experiments represents the
maximum reaction velocity, quantified as the binding of the reporter peptide, which requires peptide dissociation as a prerequisite. It is clearly seen that for all HLA-DM variants Vmax values
are more dramatically affected by pH than is Km. The most dramatic difference observed was the 500-fold decrease in Vmax for
the DM-catalyzed reactions, with DR2/C between pH 4.6 and 6.4
(as quantitated from the two cases that were measured, namely,
DMA*0101 and DMA*0101 R184H). For DR1/C- and DR4/Ccatalyzed peptide exchange, Vmax was reduced 12-fold and 8-fold,
respectively. Differences in loading rates using DR1/C, DR2/C,
and DR4/C over a similar pH range have been previously described (36). When comparing Vmax values for DMA*0101- and
DMA*0103-catalyzed reactions, the first one generally shows
The Journal of Immunology
807
slightly higher values for DR1/C and DR4/C, whereas reporter
binding velocities are higher for DR2/C in the presence
DMA*0103 rather than DMA*0101. Moreover, the different Vmax
values (estimated using initial reaction conditions) indicate that
differences observed in the catalysis of the different DM variants
are related to their catalytic features rather than to the decrease in
thermal stability.
Next, Vmax/Km was used as a measure of the catalytic efficiency
of the different DMA variants, which includes CLIP dissociation
and binding of the reporter peptides (26). We calculated this coefficient from the kinetic data (Tables I and II) and plotted the
values versus pH for each DM and DR/C complex (Fig. 3D–F).
Our results show that the activity of DMA*0103 on DR1/C
exhibits an ∼2-fold decrease in the Vmax/Km coefficient when
compared with DMA*0101 in the pH range tested (Fig. 3D).
However, in the case of DR2/C and DR4/C peptide exchange, the
catalytic efficiency is reduced only by a factor of 1.3 and 1.2,
respectively. Of interest, differences in the Vmax/Km values for
DMA*0101 and DMA*0103 are paralleled by the DR-CLIP affinity of the individual DR/C complexes (40, 41), and the same
trend is observed in the thermal stabilities of the DR/C complexes
(Supplemental Fig. 1B).
In comparing the single-site mutant variants, DMA*0101
G155A showed a lower catalytic efficiency, whereas the catalytic
potential of DMA*0101 R184H depended on the DR/C complex
under study. Thus, DMA*0101 R184H has a markedly enhanced
activity for DR2/C at pH 6.4 when compared with DMA*0101,
whereas this was not observed for either DR1/C or DR4/C. Be-
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 1. HLA-DM polymorphism in the three-dimensional structure and conservation of these positions in different species: (A) Structural alignment
of free HLA-DM (PDB: 2bc4) and HLA-DM in complex with HLA-DR1 (PDB: 4fqx) shows that glycine 155 is close to the DR1 surface and that arginine
184 moves significantly upon interaction. The free DMA chain is colored green, and DMA in complex with DR1 is colored yellow. DR1A is colored
orange. (B) Magnification of the G155 region indicating the highly conserved residues DMA-N125 in proximity to G155. The small and flexible glycine at
the tip of the loop allows Asp154 in DMA and R50 in DR1A to get in proximity in the complex. (C) Zoom into the R184 region in the free DM structure. (D)
Zoom into the R184 region showing the interaction between E181 and R184 in the DM/DR1 complex. Note that the free DMA chain is depicted in green,
and the DR-complexed DMA is shown in yellow. (E) HLA-DM sequences from different taxonomic groups reveal different degrees of residue conservation
at polymorphic positions. Organisms are indicated at the left, positions relative to the HLA-DM (DMA*0101 chain) are indicated on top of the sequence,
and the polymorphic positions found in humans are boxed in blue. Position 140 (valine in DMA*0101) and 184 (arginine in DMA*0101) are highly
variable across the different taxonomic groups. Position 155 (glycine 155 in humans) is highly conserved in mammalians and marsupials but differs in birds
and amphibians.
808
HLA-DMA POLYMORPHISMS
cause all DR molecules used in this assay have the same a-chain,
differences in the b-chain and/or model reporter peptides should
be the source for such differential effects. Overall, our kinetic data
suggest that, again, DMA*0101 G155A dictates the catalytic
properties of DMA*0103.
Mechanistic insights into the impaired catalytic efficiency of
HLA-DMA*0103 and the role of G155A and R184H
substitutions
Our previous kinetic measurements gave insight into the enzymatic
parameters that govern peptide exchange (26, 39). However, individual parameters have to be measured to answer the question of
whether the on/off rates of DM–DR complex formation—and
hence its affinity—or peptide dissociation itself is affected by the
two mutations present in DMA*0103. To address this question, we
decided to investigate DM/DR interactions by SPR and peptide
dissociation by FP. First, our SPR experiments initially focused on
the interaction of DM with DR2/C, as this interaction has been
previously reported to be accessible by SPR analysis (16). The
background binding of DR2 to the sensor surface was negligible
when using freshly purified DR complexes (Supplemental Fig. 2).
As additional controls, we also used DM-susceptible and nonsusceptible complexes previously described in the context of
sensor-immobilized DM (16, 18) (Supplemental Fig. 3). When
injecting DR2/C into the flow cell, we observed a dose-dependent
response for all DM variants (Fig. 4A–D). Subsequent injection of
buffer led only to a slight dissociation of the empty DR2 molecule
from the sensor surface, indicating a low kd value, whereas injection of a high-affinity peptide (MBP83–101) resulted in the
complete release from the chip of DR2 bound to DM (Fig. 4A–D).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 2. Initial comparison of DMA*0101 and DMA*0103. (A) Scheme of HLA DM constructs used in this study to investigate the peptide exchange
catalysis and interaction with different DR–peptide complexes. (B) SDS-PAGE after affinity and size-exclusion chromatography of the four purified
proteins. (C) Thermal stability of DMA*0101 and DMA*0103 at different pH values. Data result from at least two independent experiments with at least
three replicates in one experiment. The effect of each single mutation present in DMA*0103 on thermal stability is shown in Supplemental Fig. 1A. (D)
Western blot analysis indicates that DM heterodimers are functional in mammalian cells. Constructs bearing the cDNA encoding for the different subunits
of the indicated proteins were cotransfected or substituted by empty vectors. Cells were harvested and lysed to prepare the samples that were subjected to
a SDS-stability assay. The 1B5 Ab was used for detection of the DR a-chain as a monomer or in SDS-stable dimers (arrows). Molecular weight markers are
indicated on the left in kDa. Similar results were obtained in three independent experiments. (E–G) Real-time peptide binding assay (FP) for the exchange of
CLIP on DR1/C (E), DR2/C (F), and DR4/C (G) against model antigenic peptides [HA-FITC in (D) and (F) and MBP-FITC in (F)] indicates significantly
reduced rates on DMA*0103 catalyzed reactions. Data are representative from two independent experiments with at least three replicates in each experiment.
The Journal of Immunology
809
The affinities of the different DMA variants for DR2/C were
obtained by plotting Req values versus the concentration of DR2/C
and curve fitting to a steady-state binding model. Although all
calculated parameters lie in the high-nanomolar range, we observed significant differences. DMA*0101 R184H showed the
highest affinity for DR2/C (KD = 110 nM), whereas DMA*0103
and DMA*0101 G155A showed a moderate reduction in their
affinities for DR2/C molecules (KD ∼650 nM and KD ∼500 nM,
respectively; Fig. 4A–D). The DMA*0101 variant showed an intermediate affinity, as indicated by a KD of 350 nM (see also
Table III). Owing to the very slow dissociation of empty DR2
from surface-immobilized DM, we were not able to determine
kinetic rates. To determine kinetic parameters such as ka and kd,
we had to use a peptide–MHC class II (pMHCII) complex displaying faster on and off rates.
We chose a DR1 mutant (DRB1-N82A) that is unable to form the
two conserved hydrogen bonds positioned near the P2 site, usually
formed between the peptide backbone of the ligand and the N82
side chain of DRB1*0101. The N82A mutation has been shown
to lead to a higher peptide release rate (32, 42). Moreover, the
Table I. Comparison of kinetic constants for the catalyzed loading of model Ags on DR1/C, DR2/C, and
DR4/C by DMA*0101 and DMA*0103
DR1/C
Allele
pH
Km (nM)
Vmax (mA*min21)
Vmax/Km
DMA*0101
4.6
5.2
5.8
6.4
4.6
5.2
5.8
6.4
4.6
5.2
5.8
6.4
4.6
5.2
5.8
6.4
4.6
5.2
5.8
6.4
4.6
5.2
5.8
6.4
188 6 22
370 6 34
630 6 60
774 6 116
301 6 34
543 6 91
691 6 213
863 6 82
405 6 105
855 6 284
1052 6 355
1292 6 275
1187 6 430
1395 6 440
1300 6 200
ND
618 6 54
666 6 174
491 6 54
1804 6 307
708 6 71
496 6 57
420 6 65
1291 6 154
2.94 6 0.08
2.03 6 0.06
0.81 6 0.03
0.23 6 0.01
3.32 6 0.12
1.71 6 0.12
0.38 6 0.05
0.10 6 0.00
8.7 6 0.87
4.5 6 0.6
0.42 6 0.7
0.016 6 0.02
17.3 6 0.2
5.8 6 2.5
0.4 6 0.01
ND
10.0 6 0.4
8.2 6 0.7
2.4 6 0.1
1.2 6 0.1
9.5 6 0.4
5.6 6 0.2
1.5 6 0.08
0.7 6 0.05
0.0157
0.00546
0.00127
0.00029
0.011
0.0031
0.00059
0.00011
0.021
0.0053
0.0004
1.24E-05
0.014574
0.004150
0.0003
ND
0.016
0.012
0.005
0.0006
0.013
0.011
0.003
0.0005
DMA*0103
DR2/C
DMA*0101
DMA*0103
DR4/C
DMA*0101
DMA*0103
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FIGURE 3. DMA*0103 shows a reduced catalytic efficiency in peptide exchange on pMHCII compared with DMA*0101. (A) Initial velocities for DM
catalyzed exchange of CLIP against HA-FITC on DR1 at different substrate concentrations. (B) Initial velocities for DM-catalyzed exchange of CLIP in the
DR2 complex (DR2/C) against MBP-FITC at different substrate concentrations. (C) As in (A) but with DR4/C. (D) Vmax/Km values of DM-catalyzed
(DMA*0101, DMA*0101 G155A, DMA*0101 R184H and DMA*0103) peptide exchange of preloaded CLIP on DR1 against HA-FITC (pH 4.6–6.4). (E)
Vmax/Km values of DM-catalyzed (DMA*0101, DMA*0101 G155A, DMA*0101 R184H, and DMA*0103) peptide exchange of preloaded CLIP on DR2
against MBP-FITC in the pH range from 4.6 to 6.4). (F) As in (D), but with DR4/C.
810
HLA-DMA POLYMORPHISMS
Table II. Comparison of kinetic constants for the catalyzed loading of model Ags on DR1/C, DR2/C, and
DR4/C by DMA*0101 G155A and DMA*0101 R184H
DR1/C
pH
Km (nM)
DMA*0101 G155A
4.6
5.2
5.8
6.4
4.6
5.2
5.8
6.4
4.6
5.2
5.8
6.4
4.6
5.2
5.8
6.4
4.6
5.2
5.8
6.4
4.6
5.2
5.8
6.4
572 6 135
579 6 111
685 6 76
670 6 197
200 6 13
426 6 63
1091 6 148
1394 6 193
922 6 200
1020 6 251
1468 6 291
ND
254 6 55
715 6 218
592 6 162
616 6 142
631 6 94
499 6 61
594 6 96
1009 6 161
673 6 48
425 6 48
562 6 48
1396 6 163
DMA*0101 R184H
DR2/C
DMA*0101 G155A
DMA*0101 R184H
DR4/C
DMA*0101 G155A
DMA*0101 R184H
truncation of hydrogen bonds of the DR1-P2 site results in increased DM binding affinities (43). We loaded DRA/DRB1-N82A
with a modified CLIP fragment that contains a tryptophan in P1
(CLIP M107W, “CM107W”). This peptide mutation enhances the
affinity for DR1 molecules owing to a more optimal occupation of
the P1 pocket, thereby stabilizing the pMHCII complex (data not
shown). Remarkably, occupation of the P1 position by the Wanchor, in combination with the DRB1-N82A mutation, generated a DR1-peptide molecule with high affinity for DM (Fig. 4E–
H). Even at 10-fold lower MHC/peptide concentrations, DRB1N82A/CM107W showed a significantly increased response compared with wild-type DR1/CM107W (Fig. 4I). Examination of
the association rates (ka) for DR1 containing DRB1-N82A displayed an ∼3-fold higher ka for DMA*0101 R184H relative to
DMA*0101 (1.06 3 104 6 0.03 3 104 M21s21 versus 3.7 3 103 6
0.1 3 103 M21s21), whereas only a minor reduction of the association rate was observed for DMA*0101 G155A. DMA*0103
with both substitutions shows a ka value that is ∼1.8-fold higher
than that of DMA*0101 (6.91 3 103 6 1.4 3 103 M21s21 versus
3.7 3 103 6 0.1 3 103 M21s21; Fig. 4J). DMA*0101 G155A,
which showed the lowest association rate, displayed the highest
DM-DR1 dissociation rate. The kd of DMA*0103 and DMA*0101
R184H displays values of ∼9 3 1024 s21, which is 1.4-fold higher
than that of DMA*0101 (Fig. 4K). This compensatory effect of
G155A and R184H was also reflected in the KD (Fig. 4L). However, when combined, these two mutations did not result in a considerable change in affinity between DMA*0101 and DMA*0103
(Fig. 4L). We further analyzed the peptide-induced release of DR
from the sensor surface, but could not observe a difference (not
shown). In conclusion, using the DRB1-N82A/CM107W complex,
we observed a contrary effect on the kinetic rates ka and kd of both
substitutions present in DMA*0103.
We then decided to investigate the DM-induced dissociation of
the DR-bound peptide, as this is considered to be a kinetically
critical step of the catalytic mechanism. The additional effect of
DM catalysis on CLIP exchange against MBP is difficult to detect,
as this high-affinity peptide is efficiently exchanged against DR2bound CLIP in the absence of DM. We thus loaded DR2 molecules
Vmax (mA*min21)
Vmax/Km
6 0.3
6 0.13
6 0.3
6 0.013
6 0.05
6 0.1
6 0.06
6 0.084
6 0.2
6 0.4
6 0.17
ND
15.4 6 0.1
5 6 0.8
0.6 6 0.07
0.03 6 0.03
7.2 6 0.4
4.9 6 0.2
1.7 6 0.1
0.5 6 0.04
10 6 0.3
6.3 6 0.2
2.4 6 0.08
1.0 6 0.07
6.12E-03
2.93E-03
9.20E-04
1.60E-04
0.0155
4.69E-03
8.24E-04
1.79E-04
0.0113
3.43E-03
1.02E-04
ND
0.0605
6.99E-03
1.01E-03
4.87E-05
0.011
0.01
2.86E-03
5.00E-04
0.015
0.014
4.30E-03
7.16E-04
3.5
1.7
0.63
0.11
3.1
2
0.9
0.25
10.4
3.5
0.15
with FITC-labeled, high-affinity MBP83–101 peptide, to determine
the dissociation rates at different pH values, in the presence of DM
and an excess of unlabeled MBP83–101 to prevent rebinding of the
labeled peptide (Fig. 5A for pH 5.2, Supplemental Fig. 4A for pH
4.6–6.4). In this way we intended to see if the kinetics of the first
catalytic step, the dissociation of the bound peptide, is affected. As
expected, only negligible peptide release was observed in the
absence of DM for this DR2/MBP83–101 complex (Fig. 5A). As
a further test, we performed a DM concentration-dependent release rate experiment at a fixed pH (5.2). Varying the DM concentration had little effect on the difference between DMA*0101
and DMA*0103, with an ∼2-fold smaller rate for the latter in
the range of 0–300 nM enzyme concentration (Fig. 5B). The
DM-catalyzed peptide release was highly influenced by pH, and
DMA*0103 showed a reduced ability for enzymatic peptide dissociation relative to DMA*0101, at all tested pH values (Fig. 5C).
Interestingly, the DMA*0101 G155A mutant showed properties
nearly identical to those of DMA*0103, whereas the R184H
substitution alone did not significantly change the initial velocity
of peptide dissociation compared with DMA*0101 (Fig. 5A, 5B).
For DMA*0101 R184H this effect at high pH was more pronounced, with a smaller decrease of the dissociation rate with
increasing pH (Supplemental Fig. 4A, 4B). Thus, on average the
half-life of DR2/MBP-FITC was increased 2-fold in the presence
of DMA*0103 compared with DMA*0101 (data not shown). With
the same complex and DM concentration used in the peptidedissociation experiments of MBP-FITC and DR2, we were only
able to observe a significant rate of HA peptide release from DR4/
HA-FITC complexes over a period of 300 h (Supplemental Fig.
4C, 4D). In this case, at pH 5.2 DMA*0101 shows faster rate
values than those of DMA*0101 G155A, even though in this study
the overall effect of decreased efficiency of DM-catalyzed peptide
dissociation in the presence of DMA*0103 is less pronounced.
Finally, we evaluated peptide dissociation of the DM-susceptible,
HA-FITC–loaded DRB1-N82A mutant. As seen with DR2/MBP,
we detected a lower peptide dissociation rate in the presence of
DMA*0103 when compared with DMA*0101 (Fig. 5D). Similar
to the peptide loading experiments and the release experiments
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Protein Variant
The Journal of Immunology
811
with DR2/MBP, we observed a sustained reduction of DMA*0103
activity in the pH range from 4.6 to 6.4 relative to DMA*0101
(Fig. 5E, 5F).
In summary, DMA*0103 and DMA*0101 G155A showed
moderately lowered affinity (to DR2/C) and decreased DMcatalyzed peptide dissociation. In contrast, DMA*0101 and
DMA*0101 R184H bound DR2/C with similar affinities and released MBP-FITC from DR2 with similar kinetics. This behavior,
especially with regard to the immune dominant epitope of MBP,
raised the question of whether the release of autoimmune-relevant
self-peptides is generally compromised in DMA*0103.
Decreased DMA*0103 efficiency leads to increased half-life
times of autoimmune relevant peptide–MHCII
Our results indicate that DMA*0103 shows decreased activity for
peptide exchange, more precisely for peptide release, which could
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FIGURE 4. SPR analysis shows a compensational effect of both substitutions present in DMA*0103 for thermodynamics and kinetics with moderate
affinity decrease of DR2/C-DMA*0103 interaction. (A–D) Titration of DR2/C to the following HLA DM variants: (A) DMA*0101 (n = 3), (B) DMA*0103
(n = 2), (C) DMA*0101 G155A (n = 2), and (D) DMA*0101 R184H (n = 2). Differently concentrated (0–2 and 0–4 mM, respectively) DR2/C complexes
were injected with a flow rate of 15 ml/min for 300 s into flow cells with HLA-DM–coupled variants, followed by 300 s buffer injection and 20 mM MBP
until the baseline was reached. Dissociation and association rates were obtained from separate fits, and the KD was determined by fitting a steady state
model to obtained Req values (E–H) Titration of the DM-susceptible DRB1-N82A bound to CLIP M107W (DRB1-N82A/CM107W) to DMA*0101 (E),
DMA*0103 (F), DMA*0101 G155A (G), and DMA*0101 R184H (H). Protein was injected for 600 s with a flow rate of 15 ml/min followed by 300 s buffer
injection and 50 mM high-affinity HA306–318 peptide for 450 s to finish the peptide exchange reaction on the chip surface. (I) Effect of disruption of two
conserved hydrogen bonds on DM susceptibility, as shown by SPR. A total of 5 mM DR1/CM107W and 0.5 mM DRB1-N82A/CM107W were applied as in (E)–
(H) to a DMA*0101 coupled SPR-chip surface exhibiting the pronounced effect of the P2 hydrogen bond for DM susceptibility. (J) Comparison of association rates obtained from the simultaneous fitting of ka and kd for all the DM variants to DRB1-N82A/CM107W. (K) Comparison of dissociation rates for
all the DM variants to DRB1-N82A/CM107W. (L) Dissociation constants (16) for binding of DMA*0101, DMA*0103, DMA*0101 G155A, and DMA*0101
R184H to DRB1-N82A/CM107W.
812
HLA-DMA POLYMORPHISMS
Table III. Thermodynamic and kinetic constants as determined by SPR for the DM/DR interaction
DM Variant
KD (nM)
ka (M21s21)
kd (s21)
350
640
490
110
6
6
6
6
100
10
10
20
ND
ND
ND
ND
ND
ND
ND
ND
180
155
315
85
6
6
6
6
20
10
5
5
3.70 E3 6 0.05 E3
6.91 E3 6 1.44 E3
3.56 E3 6 0.01 E3
1.06 E4 6 0.28 E3
6.72 E24 6 8.6 E25
9.71 E24 6 1.3 E24
1.13 E24 6 1.4 E25
9.02 E24 6 1.5 E25
DR Variant
DR2/C
DMA*0101
DMA*0103
DMA*0101 G155A
DMA*0101 R184H
DRb1-N82A/CM107W
DMA*0101
DMA*0103
DMA*0101 G155A
DMA*0101 R184H
MHCII molecules, after incubation with an excess of highaffinity peptides in the presence and in the absence of DM
(DMA*0101 and DMA*0103), was quantified over a period of
24 h. The signal of a control containing MHCII–peptide complexes alone was used in subsequent measurements as the basal
fluorescence value. As shown in Fig. 6, in the absence of DM,
most of the peptide–MHCII complexes remained stable in the
time period assayed, with the exception of the low-affinity DR2/
mbp and DR4/mbp complexes, which spontaneously dissociated
after 3–6 h in the absence of DM. Of interest, upon addition of
either DMA*0101 or DMA*0103, we were able to observe
significant differences in peptide occupancies of DR molecules.
To quantify the observed difference, we plotted the amount of
biotinylated peptide relative to the initial biotinylated peptide
bound at each time point and obtained the half-life in the presence of the two DMA variants (Table IV). The half-life of DR
complexes in the presence of DMA*0103 was consistently increased for all MHCII–peptide complexes. This 1.6- to 2.2-fold
increase relative to the half-life of complexes incubated with
DMA*0101 is consistent with our previous observations for DR
FIGURE 5. Decreased peptide-dissociation catalysis of DMA*0103 is mostly dominated by the DMA-G155A mutation. (A) Release of MBP-FITC
loaded onto DR2 (150 nM) in the presence of different DM variants and 50 mM unlabeled MBP determined by FP. (B) MBP-dissociation rates of DR2
catalyzed by DMA*0101, DMA*0103, DMA*0101 G155A, and DMA*0101 R184H plotted against the enzyme concentration at pH 5.2. (C) DM-catalyzed
(150 nM) release of MBP-FITC from DR2 (150 nM) in the presence of 50 mM unlabeled MBP in dependence of the pH. (D) Release of HA-FITC loaded
onto DRB1-N82A (150 nM) in the presence of DMA*0101 and DMA*0103 plus 20 mM unlabeled HA. (E) DM-catalyzed (150 nM) release of HA-FITC
from DRB1-N82A (150 nM) in the presence of 20 mM unlabeled HA evaluated at different pH values (4.6 to 6.4). (F) HA-dissociation rates of DRB1N82A catalyzed by DMA*0101 or DMA*0103 plotted versus the enzyme concentration at pH 5.2.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
conceivably affect antigenic peptide presentation. The given
peptide repertoire displayed by a specific MHCII allele is determined by many factors, including Ag availability, processing and
loading conditions, and kinetic stability of the loaded peptides
(40). Moreover, the kinetic stability of a pMHCII complex has
a large impact on whether a peptide is potentially immunogenic
(44). Such kinetic stability, normally measured as half-life, is
defined not only by the pMHCII affinity at a given pH but also by
its susceptibility to DM activity (38).
We therefore investigated how widely the residence time of
DR-bound, autoimmunity-related self-antigens would differ in
DMA*0101- and DMA*0103-catalyzed reactions. We investigated collagen peptides (DR1/CII and DR4/CII, both of which
have been linked to RA), two variants of MS-associated MBP
peptides (DR1/MBP, DR2/MBP and DR2/mbp: lower affinity
nonimmunodominant epitope; mbp114–126), as well as DR4/MBP
and DR4/mbp, and finally an insulin-derived peptide (DR4/Ins,
which has been found to be immunodominant in T1D) (45–48).
The aforementioned self-peptide–loaded MHCII complexes
were generated in vitro. Subsequently, the peptide bound to
The Journal of Immunology
813
peptide release measured by FP (Fig. 5). The half-life dissociation values obtained ranged from 20 min in the case of the mbp
peptide in complex with DR2 and DR4 in the presence of
DMA*0101 to 26 h in the case of MBP83–101 in complex with
DR4 and in the presence of DMA*0103. It is worth noting that
the collagen 2 peptide CII in complex with DR1 (with a half-life
of 7.8 for DMA*0101 and 14.1 h for DMA*0103) exhibits
slower dissociation rates in the presence of DM than when
complexed with DR4 under the same conditions (2.7 and 4.8 h
when catalyzed by DMA*0101 and DMA*0103). In contrast,
MBP83–101 forms peptide complexes kinetically more stable with
DR4 than with DR2: 11.8 h versus 2.25 h in the presence of
DMA*0101 and 26 h versus 4.2 h in the presence of DMA*0103.
In summary, we observed slower peptide dissociation in the
DMA*0103-catalyzed reactions than in the DMA*0101-catalyzed reactions for all autoimmune-relevant MHCII–peptide
complexes we investigated.
Discussion
Our results clearly show that the two substitutions (G155A and
R184H) of the DMA*0103 allelic variant affect the biochemical properties of HLA-DM heterodimers when compared with
DMA*0101. Protein stability is negatively affected by the G155A
substitution, which dominates the properties of DMA*0103.
Nevertheless, proper folding of DM heterodimers bearing either
DMA*0101 or DMA*0103 was demonstrated by the formation of
SDS-stable dimers upon cotransfection of either of the two DMA
variants with, HLA-DR and Li in HeLa cells. Although the two
substitutions found in DMA*0103 are located in surface-exposed
loops, there are some relevant differences with respect to structural changes accompanying MHC class II binding. The DMAG155 position, which seems to be more conserved than other
polymorphic residues in DMA, is directly located at the DM–DR
interface and has almost the same conformation in the free and the
bound form (21). However, residue D154, neighboring the poly-
Table IV. Dissociation rates and t1/2 for different peptide–MHCII complexes catalyzed by DMA*0101 and DMA*0103
t1/2 (h)
Soluble DR
DR1
DR2
DR4
Peptide
Sequence
CII276–294
MBP83–101
MBP83–101
mbp114–126
CII276–294
Ins73–90
MBP83–101
mbp114–126
EPGIAGFKGEQGPKGEPGP
DENPVVHFFKNIVTPRTPP
DENPVVHFFKNIVTPRTPP
FSWGAEGQRPGFG
EPGIAGFKGEQGPKGEPGP
GAGSLQPLALEGSLQKRG
DENPVVHFFKNIVTPRTPP
FSWGAEGQRPGFG
DMA*0101
6.4
2.4
2.3
0.35
2.8
3.0
9.5
0.3
6
6
6
6
6
6
6
6
1
0.7
0.6
0.5
0.5
0.5
0.9
0.01
DMA*0103
DMA*0101: DMA*0103
6
6
6
6
6
6
6
6
1.7
1.5
1.5
1.4
1.7
1.8
1.8
2.3
10.7
3.5
3.4
0.51
4.73
5.6
17.3
0.65
0.7
0.5
0.8
0.07
0.9
0.9
1.8
0.07
Complexes of the DR alleles indicated in the first column with the peptides in the second column were prepared in vitro and t1/2 were determined in the presence of
DMA*0101 or DMA*0103. Peptide sequence is indicated in the third column, and the known or expected binding motif to the different DR alleles is underlined. The t1/2
dissociation for each complex in the presence of one or the other DMA allele is shown in h and compared in the last column.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Dissociation rates of autoimmune relevant peptide–MHCII complexes in the presence of different DMA variants. Experiments on autoimmune relevant DR–peptide complexes were performed in vitro with biotinylated peptides. Dissociation experiments were performed in the absence or in
the presence of DMA*0101 or DMA*0103 with an excess of high-affinity peptides (100 mM of HA306–318 for DR1 and DR4 complexes or MBP83–101 for
DR2 complexes) at pH 5.2. In addition, the spontaneous peptide release was measured in the same time frame. Remaining biotinylated peptide was
measured by a capture ELISA, as described in the Materials and Methods section. The peptide–DR complexes used for these experiments were as follows:
(A) DR1/collagen II276–294 and DR1/MBP83–101, (B) DR2/MBP83–101 and DR2/mbp114–126, (C) DR4/collagen II276–294, DR4/MBP83–101, DR4/insulin73–90
and DR4/mbp114–126. Remaining fluorescence relative to the fluorescence measured at t = 0 is shown as bar plots at the indicated times. Empty bars
represent the noncatalyzed reactions, black bars represent the DMA*0101, and gray bars represent DMA*0103-catalyzed reactions.
814
with the different CLIP affinities of these molecules (40, 41).
Vmax/Km coefficients are more indicative of the enzymatic properties of HLA-DM than the release rates at fixed protein concentrations. Whereas release rates are measured at a single concentration
of DR–peptide complex in the presence of competitor peptide
excess, Vmax/Km rates were obtained at substoichiometric concentrations of reporter peptide over a wide range of DR/C concentrations. For receptive MHCII proteins, peptides with very
different affinities have similar rates that depend on peptide concentration (51). However, in the case of DM-catalyzed exchange
reactions, the situation is partly different, as peptides with different affinities vary in their ability to induce HLA-DM dissociation from the DR–DM complex. Hence binding of peptides
to DR in complex with DM depends on the peptide affinity to
DR (16).
It has been long discussed and assumed that DM recognition of
a particular MHCII–peptide complex is not the limiting step of
peptide exchange catalysis, but rather peptide dissociation (40).
However, under conditions in which peptide dissociation is favored, a long-lived interaction of DM/MHCII is required until an
incoming peptide dissociates the complex. Presumably, it is the
relative stability of a receptive, empty state of a particular MHCII
allele that governs the DM/MHCII interaction under these conditions (17). Under cellular conditions, however, DM is more
likely to encounter a preloaded MHCII molecule, and the stability
of the DM–MHCII complex is not critical per se.
To delineate individual steps of DM-mediated catalysis, we
used SPR to determine peptide-dependent dissociation constants
for two different DM–DR complexes. In the case of DR2/C, the overall KD values align very well with the observed enzymatic parameters for Km of this complex (see Tables II and III). Nevertheless, the
23 decreased peptide dissociation rates could not be explained
solely by the KD or Km values. To obtain kinetic information for the
respective DM/DR interaction, we used the mutant DRB1-N82A
with a CLIP peptide variant containing a tryptophan as a P1 anchor. This N82A mutant interacts faster and more tightly with
DM. KD values are smaller compared with DR2/C, showing again
a pattern similar to what we observed in the analysis of enzyme
kinetics (see Table III). The on/off rates for DM-DRB1-N82A/CW
revealed that DMA*0101 R184H associated significantly faster
than DMA*0101 and DMA*0101 G155A, whereas DMA*0101
G155A displayed the highest kd. In addition, these experiments
revealed a compensatory effect of both substitutions (G155A and
R184H) on DMA*0103 affinity. To further understand how peptide editing is affected and to confirm the lower efficiency of
DMA*0103, we used DR–peptide complexes with different DM
susceptibilities (34, 52). Dissociation experiments with both natural DMA*0101 and DMA*0103 confirm that DMA*0103 was
∼2-fold less efficient for DR–peptide complexes of different intrinsic affinities relative to DMA*0101.
Adaptive immune responses and the normal development of
CD4+ T cells require DM-mediated peptide exchange. Loading of
internalized Ags is known to be DM dependent (38), and the
expression of the particular DMA allele in an individual would be
expected to have direct consequences on the peptide repertoire.
Moreover, in the absence of HLA-DM, CLIP-MHCII molecules
accumulate on the cell surface, as observed in human (53, 54) and
murine B cell lines (55, 56). However, conclusions of the in vivo
relevance of HLA-DM expression were first drawn from studies
with H2-DM (HLA-DM homolog) knockout mice (57–59). The
main findings of these studies revealed that T cell development
and hence the composition of CD4+ T cell populations were altered in the absence of H2-DM. Positive selection in the thymus in
the three models seemed to be differentially affected with respect
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
morphic G155, makes a charge interaction with R50 of the DR-a–
chain in the complex, and this arginine undergoes a large sidechain repositioning when compared with the free form of DR1. It
is quite likely that substituting glycine with alanine will affect the
exact conformation of the loop containing D154 and G155 and
thereby might influence the charge–charge interaction–dependent
encounter of the two proteins. It is also noteworthy that the DMAG155A mutation introduces a reduction in the protein thermal
stability of ∼3˚C, indicating that the replacement by the less
mobile and more hydrophobic alanine comes at a thermodynamic
cost of several kilojoules per mole in free energy. In the case of
R184, the side-chain conformation is changed upon interaction
with DR1 (17) or DO (20). In the free HLA-DM structure (26) the
R184 side chain is within the region where HLA-DR1 or HLADO encounters DM. Upon complex formation, however, as part of
a loop repositioning, this residue flips away and forms an intramolecular contact with DMA-E181. It is clear that the substitution
of arginine by histidine would allow maintaining this polar contact
to DMA-E181, especially at acidic pH values.
In a previous study aimed at understanding the HLA-DM interaction with DR, the authors described how the substitutions
DMB-G17V, DMB-Q100P, or DMA-N195S negatively affect the
assembly and/or expression of functional HLA-DM heterodimers
(49). Many other positions were also addressed by Pashine et al.
(49), and at least seven substitutions resulted in an impaired cellular activity of DM. It was then shown in vitro that DMB-L51D
narrows the pH range for optimal HLA-DM function, whereas
DMB-D31N and DMB-E47Q have no effect on peptide exchange
as individual mutations but do increase DM peptide exchange
activity 2-fold at low pH and 9 times at high pH when combined
(26). In addition to these critical positions in DMB, DMA-F100A
and DMA-I173N in DMA were also shown to influence activity.
Although the latter substitution introduces a glycosylation site
interfering directly with the DM/DR interaction, the effect of
DMA-F100A in reducing DM activity was less clear. Of note,
DMA-F100 is in close spatial proximity to R184 in free HLA-DM,
but its side-chain aromatic ring is occupying a different rotamer
upon complex formation. We conclude that both polymorphic
substitutions within DMA*0103 are placed in conformationally
sensitive regions of the protein that affect exchange catalysis in
distinct ways.
HLA-DM–mediated peptide exchange is required for selection
of kinetically stable MHCII–peptide complexes (40). The reaction
has been proposed to display a bimolecular mechanism whereby
empty MHC complexes can undergo inactivation in a fast and
DM-reversible manner (50). To circumvent the issue of empty
receptive versus nonreceptive states and to estimate DM catalytic
properties, we made use of preloaded proteins that were always
freshly prepared and purified before measurements. Our results
clearly indicate that DM heterodimers containing the DMAG155A mutation are less efficient in peptide exchange catalysis
by directly affecting the release of peptide from the MHCII
complexes. Peptide dissociation rates measured in the presence of
G155A containing heterodimers were ∼2 times slower in all cases
investigated in this study. Of interest, using the Vmax/Km coefficient to compare the catalytic efficiency of DMA*0103 with that
of DMA*0101, we observed a 2-fold reduction over the pH range
tested in the case of DR1/C as substrate, whereas a very moderate
but statistically significant reduction for the catalysis on DR2/C
and DR4/C was observed (1.3- and 1.2-fold, respectively). The
Vmax/Km values reported in this article for DMA*0101 are in
a similar range to those obtained by Nicholson et al. (26) using the
same system (DR2/C and MBP). Interestingly, the difference in
the Vmax/Km coefficients for the individual DR alleles correlates
HLA-DMA POLYMORPHISMS
The Journal of Immunology
Acknowledgments
We thank Eliot Morrison and Larry Stern for critically reading the manuscript; Kai W. Wucherpfennig for providing the original cDNA constructs
encoding HLA-DR4 and HLA-DM for baculovirus expression; and Adam
Benham for the cDNA constructs encoding full-length HLA-DMA, HLADMB, and CD74.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
Disclosures
The authors have no financial conflicts of interest.
28.
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HLA-DMA POLYMORPHISMS