This information is current as
of July 31, 2017.
A Single Polymorphic Residue Within the
Peptide-Binding Cleft of MHC Class I
Molecules Determines Spectrum of Tapasin
Dependence
Boyoun Park, Sungwook Lee, Euijae Kim and Kwangseog
Ahn
J Immunol 2003; 170:961-968; ;
doi: 10.4049/jimmunol.170.2.961
http://www.jimmunol.org/content/170/2/961
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References
The Journal of Immunology
A Single Polymorphic Residue Within the Peptide-Binding
Cleft of MHC Class I Molecules Determines Spectrum of
Tapasin Dependence1
Boyoun Park, Sungwook Lee, Euijae Kim, and Kwangseog Ahn2
A
fter targeting to the endoplasmic reticulum (ER),3 nascent MHC class I H chains associate with a multiprotein
complex that assists their assembly with peptide and ␤2
microglobulin (␤2m). This complex includes calnexin, calreticulin,
ERp57, transporter associated with Ag processing (TAP), and tapasin (1). Tapasin is indispensable for proper function of the class
I Ag presentation pathway. In tapasin-mutant mice, the expression
and stability of surface MHC class I molecules are strongly reduced (2). Even though tapasin clearly facilitates Ag presentation
by MHC class I molecules, its precise function in this process has
not been fully elucidated. In addition to the uncertainty regarding
the precise function of tapasin, various MHC class I molecules
differ in their dependency on tapasin for both efficient surface expression and presentation of antigenic determinants to CTL (3– 8).
In tapasin-negative 721.220 cells, tapasin is not required for high
HLA-B2705 allele (B2705) surface expression or for presentation
of viral determinants to CTL (3). In contrast, for the HLA-B4402
allele (B4402), functional Ag presentation and surface expression
are highly dependent on tapasin (3). The factors that determine the
differences in the relative tapasin dependence of various MHC
class I molecules remain elusive. Analysis of the amino acid sequence of some ligands that are loaded into B2705 in the presence
and the absence of tapasin shows no clear correlation between
tapasin dependence and either cellular abundance of the ligands or
their binding affinities for HLA-B2705 molecule (9). Taking these
findings and the relative lack of tapasin polymorphism into account (10), the MHC class I allele-dependent requirement for tapasin is likely a property of the individual MHC class I alleles.
Recent studies indicate that the presence of serine rather than
phenylalanine or tyrosine at position 116 in the HLA-B H chain
correlates with inefficient association with the assembly complex
and correspondingly high surface expression (11, 12). Similarly,
substitution of glutamine to alanine at position 115 of HLA-A2
causes a lack of association with the loading complex but the mutant proteins are expressed on the cell surface at a level similar to
that of wild-type HLA-A2 H chain (13). On the basis of these
findings, positions 115 and 116 are suggested to determine the
tapasin dependence of class I surface expression. However, among
HLA alleles, glutamine at position 115 is highly conserved, and
position 116 alone cannot explain the phenotypic differences in
tapasin dependence between B4402 and B2705 or A0301 alleles
because they are identical at position 116 with aspartic acid. Moreover, the presence of aspartic acid at 116 resulted in either increased (in A68) or decreased (in B7) binding of tapasin to MHC
class I molecules (12, 14). Thus, other positions in the HLA H
chain should act as a major determinant of the tapasin dependence
of MHC class I molecules. In this study, we have determined the
requirements of MHC class I alleles for tapasin dependence.
Graduate School of Biotechnology, Korea University, Seoul, Korea
Received for publication September 9, 2002. Accepted for publication November
8, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a grant from the 21C Frontier for Functional Genome
Analysis of Human Genome.
2
Address correspondence and reprint requests to Dr. Kwangseog Ahn, Graduate
School of Biotechnology, Korea University, 1, 5-Ga, Anam-Dong, Sungbuk-Gu,
Seoul 136-701, Korea. E-mail address: [email protected]
3
Abbreviations used in this paper: ER, endoplasmic reticulum; ␤2m, ␤2 microglobulin; TAP, transporter associated with Ag processing; endo H, endoglycosidase H.
Copyright © 2003 by The American Association of Immunologists, Inc.
Materials and Methods
DNA constructs
The cDNA encoding human tapasin was subcloned into the pcDNA3.1/
hygromycin vector (Invitrogen, Carlsbad, CA). Site-directed mutants of
B2705 with H to D at position 114 (B27H114D), H to N at position 114
(B27H114N), H to R at position 114 (B27H114R), D to Y at position 116
(B27D116Y), and the other substitution mutants of B4402 were made by
changing the codons by PCR with Pfu DNA polymerase (Stratagene, La
Jolla, CA). All HLA cDNAs and their mutagenized derivatives were inserted into the mammalian expression vector pcDNA3.1 (Invitrogen).
0022-1767/03/$02.00
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Different HLA class I alleles display a distinctive dependence on tapasin for surface expression and Ag presentation. In this study,
we show that the tapasin dependence of HLA class I alleles correlates to the nature of the amino acid residues present at the
naturally polymorphic position 114. The tapasin dependence of HLA class I alleles bearing different residues at position 114
decreases in the order of acidity, with high tapasin dependence for acidic amino acids (aspartic acid and glutamic acid), moderate
dependence for neutral amino acids (asparagine and glutamine), and low dependence for basic amino acids (histidine and arginine). A glutamic acid to histidine substitution at position 114 allows the otherwise tapasin-dependent HLA-B4402 alleles to load
high-affinity peptides independently of tapasin and to have surface expression levels comparable to the levels seen in the presence
of tapasin. The opposite substitution, histidine to glutamic acid at position 114, is sufficient to change the HLA-B2705 allele from
the tapasin-independent to the tapasin-dependent phenotype. Furthermore, analysis of point mutants at position 114 reveals that
tapasin plays a principal role in transforming the peptide-binding groove into a high-affinity, peptide-receptive conformation. The
natural polymorphisms in HLA class I H chains that selectively affect tapasin-dependent peptide loading provide insights into the
functional interaction of tapasin with MHC class I molecules. The Journal of Immunology, 2003, 170: 961–968.
962
DETERMINANT FOR TAPASIN DEPENDENCE OF MHC CLASS I MOLECULES
Stable cell lines and Abs
Tapasin expression was restored in 721.220 cells by transfection with the
cDNA-encoding tapasin, and transfectants were selected with 0.2 mg/ml
hygromycin, giving rise to the 721.220.Tpn cell line. The HLA-G-specific
mAb G233 was a gift from Dr. Y. Loke (University of Cambridge, Cambridge, U.K.). Polyclonal rabbit K455 Ab reacts with MHC class I H chains
and ␤2m in both assembled and nonassembled forms (15). The mouse mAb
HLA-B Ab-1 (NeoMarkers, Fremont, CA) recognizes only HLA-B alleles
and mAb W6/32 recognizes only the complex of H chain and ␤2m. The
anti-tapasin Ab (R.gp48N) was a gift from Dr. P. Cresswell (Yale University, New Haven, CT). FITC-conjugated goat anti-mouse IgG Abs and
HRP-conjugated streptavidin were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) and Pierce (Rockford, IL),
respectively.
Pulse-chase and immunoprecipitation
Flow cytometry
The surface expression of MHC class I molecules was determined by flow
cytometry (FACSCalibur; BD Biosciences, Mountain View, CA). Cells
(1 ⫻ 106) were washed twice with cold PBS containing 1% BSA and
incubated for 1 h at 4°C with a saturating concentration of primary Ab.
Normal mouse IgG Ab was used as a negative control for each test. Cells
were washed twice with cold PBS containing 1% BSA and then stained
with FITC-conjugated goat anti-mouse IgG Abs for 30 min. A total of
10,000 gated events were collected by the FACSCalibur cytometer and
analyzed with CellQuest software (BD Biosciences).
Coimmunoprecipitation and Western blot analysis
Cells were lysed in 1% digitonin in buffer containing 25 mM HEPES, 100
mM NaCl, 10 mM CaCl2, and 5 mM MgCl2 (pH 7.6) supplemented with
protease inhibitors. Lysates were precleared with protein G-Sepharose
(Amersham Pharmacia Biotech) for 1 h at 4°C. For immunoprecipitation,
samples were incubated with the appropriate Abs for 2 h at 4°C before
protein G-Sepharose beads were added. Beads were washed four times
with 0.1% digitonin, and bound proteins were eluted by boiling in SDS
sample buffer. Proteins were separated by 12% SDS-PAGE, transferred
onto a nitrocellulose membrane, blocked with 5% skim milk in PBS with
0.1% Tween 20 for 2 h, and probed with the appropriate Abs for 4 h.
Membranes were washed three times with PBS with 0.1% Tween 20 and
incubated with HRP-conjugated streptavidin (Pierce) for 1 h at 4°C. Immunoblots were visualized with ECL detection reagent (Pierce).
Microsomes and peptide-loading assay
Microsomes from 721.220 and 721.220.Tpn cells expressing B2705,
B4402, or their mutant HLA class I H chains, with or without tapasin, were
prepared and purified as previously described (16). Biotinylated peptides
GRIDKPILK (ligand for B2705) and AEIDKVTGY (ligand for B4402)
were conjugated to the photoreactive cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS; Pierce) as described (17). For the peptide-loading assay, reporter peptides, with or without various concentrations of the unlabeled peptides, were mixed with 15 ␮l of microsomes
(concentration of 60 A280/ml) in a total volume of 50 ␮l of RM buffer (250
mM sucrose, 50 mM triethanolamine-HCl, 50 mM potassium acetate, 2
mM magnesium acetate, 1 mM DTT, and 10 mM ATP). The mixture was
incubated for 30 min at 26°C in a flat-bottom 96-well tissue culture plate.
Samples were maintained on ice during a 3-min exposure to shortwave
(365 nm) ultraviolet irradiation. After centrifugation, membranes were
washed once with cold RM buffer and lysed with 1% digitonin, and the
Results
Variable dependence of HLA class I molecules on tapasin for
their cell surface expression
Among different HLA class I alleles, dependence on tapasin for
class I expression is variable. B4402, and to a lesser extent B0801,
are tapasin-dependent for cell surface expression, whereas B2705
is tapasin-independent (3). To evaluate whether these allele-specific differences in tapasin dependence could be further extended to
other MHC class I molecules, we examined the cell surface expression of 10 HLA class I alleles in 721.220 and 721.220.Tpn
cells. In the absence of tapasin, a spectrum of surface expression of
MHC class I alleles was consistently evident in pools of transfected cells (Fig. 1a). High levels of surface expression were observed for B2705, B2702, A0210, A2401, B0801, and B5401 alleles. In contrast to these alleles, ⬎5-fold lower surface expression
was observed for B4402, B3501, A3001, and, interestingly,
HLA-G Ag, a nonclassical MHC class I molecule (Fig. 1a, middle
column). Significantly, expression of tapasin fully restored the surface expression of B4402, B3501, A3001, and HLA-G Ag (Fig.
1a, middle column). Surface levels of B0801 and B5401 were also
increased, albeit to a lesser extent, upon expression of tapasin (Fig. 1a,
right column). However, expression of tapasin did not affect the surface levels of B2705, B2702, A0210, and A2401 (Fig. 1a, left column). These results indicate that the degree to which class I surface
expression is affected by tapasin is indeed allele-dependent.
Residue 114 determines spectrum of tapasin dependence of HLA
class I molecules
As tapasin shows little polymorphism (10), variable dependence
on tapasin should be explained by amino acid differences among
the respective class I alleles. These differences are mainly located
in the ␣1␣2 domain of MHC class I molecules. To define the
amino acids potentially involved in determining tapasin dependence, we, therefore, aligned the sequences of the ␣1␣2 domain of
HLA class I molecules (Fig. 1b). Interestingly, tapasin-independent HLA class I alleles contain histidine or arginine at position
114, whereas tapasin-dependent alleles contain acidic amino acid
residues, such as aspartic acid or glutamic acid. Alleles that are
weakly dependent on tapasin contain neutrally charged asparagine
at this position. Database analysis reveals that position 114 of all
HLA class I molecules is represented by one of the following six
amino acids: histidine, arginine (basic), aspartic acid, glutamic
acid (acidic), and asparagine, glutamine (neutral).
To test whether the side chain of the amino acid at position 114
governs the extent of tapasin dependence of HLA class I alleles,
we constructed substitution mutants and examined their dependence on tapasin for surface expression. Surprisingly, B44D114H
was no longer tapasin-dependent and instead displayed a tapasinindependent phenotype for their surface expression (Fig. 2a). The
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Cells (5 ⫻ 106) were transfected by electroporation (Gene Therapy Systems, San Diego, CA), starved for 40 min in medium lacking methionine,
labeled with 0.1 mCi/ml [35S]methionine (TranS-label; NEN, Boston, MA)
for 20 min, and chased in normal medium for the indicated times. Cells
were lysed by using 1% Nonidet P-40 (Sigma-Aldrich, St. Louis, MO) in
PBS with a protease inhibitor mixture for 30 min at 4°C. After preclearing
cell lysates with protein G-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ), primary Abs and protein G-Sepharose were added to the
supernatant and incubated at 4°C with rotation for 2 h. The beads were
washed three times with 0.1% Nonidet P-40 in PBS. Proteins were eluted
from the beads by boiling in SDS sample buffer and separated by 12%
SDS-PAGE. The gels were dried, exposed to BAS film for 14 h, and then
analyzed with Phosphor Imaging System BAS-2500 (Fuji Film, Tokyo,
Japan). For endoglycosidase H (endo-H) treatment, immunoprecipitates
were digested with 3 ␮ of endo H (Roche, Indianapolis, IN) at 37°C overnight in 50 mM sodium acetate (pH 5.6), 0.3% SDS, and 150 mM 2-ME
(Sigma-Aldrich).
cross-linked proteins were immunoprecipitated with mAb HLA-B Ab-1.
Precipitates were separated by 12% SDS-PAGE and transferred to an immobilon-P membrane (Millipore, Bedford, MA). The membrane was incubated with HRP-conjugated streptavidin for 1 h 4°C, and biotinylated
proteins were visualized by using ECL Western blotting reagent (Pierce).
Peptide translocation was determined after incubating microsomes with
biotin-conjugated reporter peptides in the absence of competitor for 30 min
at 26°C with or without 1 mM ATP. Microsomal membranes were recovered by centrifugation at 75,000 ⫻ g for 10 min through a 0.5 M sucrose
cushion in cold RM buffer. After washing with cold RM buffer twice, the
membrane pellet was directly dissolved in sample buffer. The samples were
analyzed on tricine/SDS-PAGE, appropriate for resolution of low mass
polypeptides as described (18), and probed with HRP-conjugated streptavidin. The relative densities of the peptide bands were determined by use
of an imaging densitometer (GS-700; Bio-Rad, Richmond, CA) and MultiAnalyst densitometer software (Bio-Rad).
The Journal of Immunology
963
B44D114N and B27H114N, in which residue 114 was replaced by
the neutral-charged asparagine, fell between the B4402 and B2705
in the spectrum of tapasin dependence. The reciprocal mutation,
B27H114D, rendered the otherwise tapasin-independent B2705 allele dependent on tapasin for surface expression. As arginine, a
basic amino acid, is also found in some MHC class I alleles, such
as A0101, A0301, A1101, or A2902, we substituted arginine for
histidine in B2705 (B27H114R) and examined its dependence on
tapasin for surface expression. In the absence of tapasin, the surface expression of B27H114R was comparable to the level observed for wild-type B2705 and was not affected by expression of
tapasin (Fig. 2b), indicating that arginine can functionally replace
histidine at position 114 in respect to conferring tapasin independence on MHC class I alleles. These results indicate that the tapasin dependency of HLA alleles is determined by the nature of
the amino acid at position 114.
Differential effect of tapasin on maturation of HLA class I
alleles is exerted by residue 114
To investigate the mechanism by which the amino acid at position
114 influences dependence of MHC class I molecules on tapasin
for surface expression, we compared the intracellular maturation
and transport of wild-type HLA class I alleles with their respective
point mutants in the absence or presence of tapasin. We used
pulse-chase analysis to estimate the extent of intracellular transport
of MHC class I molecules by examining their acquisition of
endo-H resistance. In the absence of tapasin, the majority of B4402
H chains remained sensitive to endo-H digestion, even after 90 min
(Fig. 3a, upper panel), reflecting complete retention of these molecules in the ER over this period. This finding suggests that the
impaired intracellular transport of B4402 molecules in the absence
of tapasin accounts for their lower levels of surface expression.
Conversely, in the presence of tapasin, B4402 molecules had become endo H-resistant by this time, consistent with their efficient
expression at the cell surface (Fig. 3a, lower panel). Pulse-chase
experiments from tapasin-negative and -positive cells revealed
comparable acquisition of endo-H resistance by B44D114H (Fig.
3b), indicating its tapasin independence for normal intracellular
transport. Without tapasin, the maturation kinetics of B2705 and
B27H114D were essentially the opposite of the maturation kinetics
of B4402 and B44D114H, respectively. After a 90-min chase,
B2705 had become resistant to endo H irrespective of tapasin,
indicating its transport out of the ER (Fig. 3c). At the 90-min
chase, B27H114D remained sensitive to endo H in the absence of
human tapasin but had become resistant to endo H in its presence
(Fig. 3d). In the absence of tapasin, B44D114N, in which aspartic
acid is replaced with the neutrally charged asparagine at position
114 of B4402, was efficiently transported out of the ER (Fig. 3e,
upper panel) at a rate that was similar, albeit lower, to the rate
observed for B44D114H. Tapasin slightly promoted the transport
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FIGURE 1. Determinant for tapasin dependence of
HLA class I molecules. 721.220 and 721.220.Tpn
cells were transfected with DNA encoding the indicated HLA class I H chains. a, Surface expression
level of MHC class I molecules was measured by
FACS analysis. FACS histograms are shown for cells
without tapasin (thin lines) and with tapasin (thick
lines). Staining of mock-transfected cells is shown in
the filled histograms (control). b, Amino acid comparison of tapasin-independent and -dependent HLA
class I alleles.
964
DETERMINANT FOR TAPASIN DEPENDENCE OF MHC CLASS I MOLECULES
FIGURE 2. Residue 114 governs a spectrum of tapasin dependence for efficient surface expression.
721.220 and 721.220.Tpn cells were transfected with
DNA encoding either B4402, B44D114H, or
B44D114N (a), and B2705, B27H114D, B27H114N,
or B27H114R H chains (b). Cells were stained 48 h
posttransfection with the mAb HLA-B Ab-1 followed
by FACS analysis. Tapasin-positive (thick lines), tapasin-negative (thin lines), and mock-transfected
721.220 cells (dotted lines) are shown.
reports (12, 14), these data confirm the importance of position 116
for chaperone association and indicate that position 116 indeed
affects the association with tapasin. Taken together, our findings
indicate that position 114 influences tapasin association and that
the residue 114-mediated tapasin dependence of HLA class I alleles correlates with the strength of tapasin interaction.
Residue 114 influences the association of HLA class I alleles
with tapasin
Position 114 determines the peptide-loading characteristics of
HLA class I molecules
To further understand the molecular basis for the critical role of
residue 114 in defining the tapasin dependence of HLA class I
molecules, we examined by coimmunoprecipitation the interaction
of wild-type HLA and its mutant derivatives with tapasin in the loading complex. Remarkably, B44D114H, in comparison with wild-type
B4402, showed an 8-fold lower affinity for tapasin (Fig. 4a, lanes 1
and 2), suggesting that introduction of a basic amino acid residue at
position 114 disrupts its interaction with tapasin. B44D114N exhibited weaker association with tapasin than did B4402 but exhibited
stronger association with tapasin than did B44D114H (Fig. 4a,
lane 3). In contrast, B27H114D had an affinity for tapasin that was
even higher than the tapasin affinity of the wild-type B2705 in this
and repeating experiments (Fig. 4b, lanes 1 and 2); B27H114N fell
between B27H114D and B2705 in tapasin affinity. To confirm that
the observed differential binding of HLA class I molecules to tapasin was not due to differences in sample loading, we reacted the
same blots with the K455 Ab and verified that each lane contained
a comparable amount of MHC class I molecules (Fig. 4, a and b,
lower panels). Several studies have shown that positions 115 and
116 affect association of HLA alleles with chaperones and that the
interaction of chaperones with HLA class I H chains is cooperative
(11–14). We wanted to confirm the effect of residue 116 on the
association of HLA class I H chains with chaperones and thereby
validate our coprecipitation system. Substitution of aspartic acid to
tyrosine at position 116 caused disparate binding of tapasin by
B2705 and B4402. In the case of 2705, the substitution
(B27D116Y) significantly decreased tapasin (Fig. 4b, lane 4),
whereas the B44D116Y mutant had a stronger association with
tapasin (Fig. 4a, lane 4). In general agreement with the previous
In the absence of tapasin, the impaired intracellular transport and,
consequently, the low surface expression of B4402 and
B27H114D might be due to their inability to load peptides. To
determine the influence of residue 114 on peptide loading as a
function of tapasin, we examined peptide loading into MHC class
I molecules by using the reporter peptide. We incubated intact
microsomes derived from B4402 and B44D114H transfectants
with or without tapasin and AEIDKVTGY peptide, a high-affinity
ligand for B4402 (19), in the presence of the competitors at different concentrations. The amount of tapasin-dependent peptide
loading was determined by measuring the level of incorporation of
labeled peptide into W6/32-reactive class I complexes. We were
surprised at the marked differences in the ability of B4402 and
B44D114H to load peptide in the absence of tapasin. When no
competitor was added, the level of binding of the reporter peptide
by B4402 was only 25% of the binding observed in B44D114H
(Fig. 5a, lane 0). Furthermore, the reporter peptides loaded into
B4402 were completely out-competed by a lower concentration
(1.6 ␮M) of unlabeled peptide, whereas as much as 12.8 ␮M of
unlabeled peptide was not even sufficient for completely out-competing reporter peptide binding to B44D114H. However, in the
presence of tapasin, no discernible difference in the peptide-binding ability of B4402 and B44D114H was seen (Fig. 5b). Analysis
of B2705 and B27H114D as to tapasin dependence for peptide
loading revealed that wild-type B2705 resembles B44D114H,
whereas B27H114D behaves like wild-type B4402. In the absence
of tapasin, B2705 efficiently binds the reporter peptide, GRIDK
PILK, a high-affinity ligand for B2705 (20). A high concentration
(12.8 ␮M) of unlabeled peptide was not sufficient to out-compete
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of B44D114N (Fig. 3e, lower panel). The intracellular maturation
and transport results correlate with the cell surface expression of
MHC class I molecules. From these experiments, we have established that the amino acid residue at position 114 governs a spectrum of dependence on tapasin for biochemical maturation and
surface expression.
The Journal of Immunology
965
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FIGURE 3. Tapasin-dependent effect of residue 114 on intracellular transport of MHC class I molecules. 721.220 (tapasin-negative) and 721.220.Tpn
(tapasin-positive) cells were transfected with B4402 (a), B44D114H (b), B2705 (c), B27H114D (d), or B44D114N cDNAs (e). Cells were labeled and
chased for the indicated times. Cell lysates were immunoprecipitated with mAb HLA-B Ab-1, and then digested with endo H (⫹) or mock digested (⫺)
for 16 h. Endo-H-resistant (r) and endo-H-sensitive (s) proteins bands are indicated.
the reporter peptide binding (Fig. 5c, upper panel). In contrast,
substitution of aspartic acid for histidine at position 114 of B2705
dramatically abolished its ability to load peptides (Fig. 5c, lower
panel). However, expression of tapasin greatly enhanced the ability of B27H114D to load peptides to the same level as observed for
wild-type B2705 (Fig. 5d, lower and upper panels, respectively).
These results indicate that B4402 molecules are highly dependent
on tapasin for efficient peptide loading, but the aspartic acid-tohistidine substitution at position 114 renders the molecules independent of tapasin for peptide loading. Accordingly, the impaired
intracellular transport and reduced surface expression of B4402
and B27H114D in the absence of tapasin are likely the result of
their inability for peptide loading.
To further investigate whether the differential peptide loading
observed in the absence of tapasin might be due to differences in
the luminal availability of the peptide, we quantitated the amount
of reporter peptides that were translocated into the ER lumen by
UV irradiation time point. Comparable amounts of peptides were
recovered between B2705 and B27H114D (Fig. 5e, lanes 1 and 2)
and between B4402 and B44D114H (Fig. 5e, lanes 3 and 4).
Therefore, we exclude the notion that luminal availability of the
peptide plays an important role in dictating class I loading in
966
DETERMINANT FOR TAPASIN DEPENDENCE OF MHC CLASS I MOLECULES
tapasin-deficient cells. In control experiments in which ATP was
omitted, little peptide was recovered (Fig. 5e, lanes 5 and 6). Because peptide translocation via TAP requires ATP (21), we conclude that the modified reporter peptides entered the ER lumen in
a TAP-dependent manner. Overall, our data clearly show that
functional polymorphism in tapasin dependence for efficient peptide loading of MHC class I molecules is defined by the presence
of different amino acids at position 114.
Discussion
Based on the available class I crystal structures, some considerations on the peculiar behavior of position 114, an integral part of
the D pocket of the peptide-binding groove, may be discussed. The
general structure of different MHC class I molecules is similar,
diverging only at the polymorphic residues, which are relatively
few and located mostly in the peptide-binding groove (22). A
slight structural variation affecting this region can dramatically
change the requirements for binding peptides and might result in a
different set of bound peptides. Our findings reveal that tapasin
dependence and the nature of the side chain of amino acid residue
114 are clearly correlated (the “tapasin-rule”). HLA class I alleles
with acidic amino acids at position 114 associate strongly with
tapasin and are dependent on tapasin for high-affinity peptide loading and surface expression. The negatively charged side chain of
aspartic acid or glutamic acid might pose steric hindrances in the
peptide-binding groove that would interfere with the strong binding of peptides. Upon interaction with tapasin, the peptide-binding
cleft transits to the open, peptide-receptive form. In contrast to
tapasin-dependent HLA class I alleles, the peptide-binding groove
of MHC class I molecules containing basic residues at position 114
seemingly forms the open state to fit the appropriate peptides, regardless of tapasin. Accordingly, these HLA class I alleles are
capable of binding a broad spectrum of the peptide repertoire without the assistance of tapasin and are subsequently transported to
the cell surface. In corroboration with this concept, HLA class I
alleles with a neutral-charged amino acid display an intermediate
level of tapasin dependence for surface expression (3). These findings intimate that the dependence upon tapasin for loading a repertoire of peptides of sufficient stability to allow egress from the
ER might reside in the ability of the D pocket to facilitate the
loading of peptides or perhaps to initiate a peptide-induced conformational change in the class I molecule that signals release from
the loading complex. We predict that the tapasin rule might also be
imposed upon other nonclassical MHC class I molecules, such as
HLA-E, HLA-F, as well as the unexamined other classical MHC
class I molecules.
Recently, Williams et al. (23) observed that unlike tapasin-dependent B4402, B4405 (tyrosine at position 116), which was assumed to be identical to B4402 (aspartic acid at position 116) apart
from the polymorphism at position 116, is tapasin-independent for
the cell surface expression. This observation led them to conclude
that amino acid residue 116 determines the tapasin dependence of
cell surface expression. This result contrasts with our observations
with B44D116Y. We found that the single amino acid change of
tyrosine for aspartic acid at position 116 does not change the phenotype of B4402 for tapasin dependence (data not shown) and that
B4402 and B2705, which share the same amino acid residue at
position 116, exhibit different phenotypes for tapasin dependence
in their surface expression (Fig. 1). Likewise, HLA-G and A2401
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FIGURE 4. Residue 114 and the association
of HLA class I molecules with tapasin.
721.220.Tpn cells were transfected with DNA
encoding the indicated HLA class I H chains.
Cells were lysed by 1% digitonin, and lysates
were immunoprecipitated with mAb HLA-B
Ab-1. The immunoprecipitated proteins were
separated by SDS-PAGE and blotted with antitapasin Ab. The intensities of precipitated bands
were quantified. The results of several independent experiments are shown as mean values and
SD. The relative intensities of coprecipitated tapasin with mutants B4402 and B2705 were
compared with those with wild-type B4402 and
B2705 (100% control), respectively.
The Journal of Immunology
have tyrosine at position 116 but they display the opposite phenotype for tapasin dependence (Fig. 1). In fact, the structures of
B4405 and B4402 are quite different. In comparison with B4402,
B4405 is devoid of the N-terminal 48 amino acids and the entire
␣3 region, including the transmembrane and cytoplasmic domains
(24). It is intriguing how Williams et al. (23) could detect the
putative “soluble” B4405 proteins at the cell surface by FACS
analysis. On the basis of our findings and the findings of others, we
propose that residue 114 acts as a major determinant of tapasin
dependence for efficient peptide loading, maturation, and surface
expression of HLA class I alleles while residue 116 can also affect
association of certain MHC class I molecules with the components
of the loading complex, albeit, in unpredictable manner.
Several studies have shown that TAP association of MHC class
I molecules requires tapasin (2, 25–27), as expected if tapasin
bridges MHC class I molecules to TAP. Therefore, a spectrum of
residue 114-determined dependence of HLA class I alleles on tapasin might be an indirect effect of the residue on TAP-related
function. However, our data argue against this possibility. First,
residue 114 appears to have no effect on the function of TAP in
translocating peptides into the ER lumen (Fig. 5e). Second, as
loading of peptides onto B44D114H and B2705 proceeds normally
in the absence of tapasin (Fig. 5, a and c), bridging of MHC class
I molecules to TAP, which is one of the proposed functions of
tapasin, appears to be unnecessary for enhanced peptide loading,
which is consistent with a previous report (3).
What is the clinical relevance of our findings on tapasin dependence of MHC class I alleles? HLA alleles that encode tapasinindependent molecules might have evolved in response to evolutionary pressures exerted by microbial pathogens. Adenovirus
protein E19 targets tapasin to prevent Ag presentation and in turn
to evade CTL recognition (28). The flexibility offered by tapasin
independence might permit certain MHC class I molecules to continue presenting viral Ags even when tapasin function is disrupted.
Peptide loading that is independent of tapasin, although beneficial
during infection with such microorganisms, might also result in the
up-regulated presentation of poorly tolerized self-peptides in an
inflammatory context. Such up-regulation might be a novel mechanism for triggering autoimmune responses. This hypothesis might
be particularly relevant to the development of inflammatory spondyloarthritis, known to be strongly associated with the “tapasinindependent” HLA-B27 alleles (29). Differences in the residues at
positions 114 and 116 are likely to contribute to allele-specific
recognition by different CTL clonotypes, changing the requirements for binding peptides and modifying the set of bound peptides. This might provide a possible molecular mechanism by
which a molecular mismatch involving positions 114 and 116 of
MHC class I H chain influences the outcome of unrelated bone
marrow transplantation in donor-recipient pairs, otherwise
matched for HLA-DRB and -DQ loci; patients with substitution at
positions 114 and 116 are at increased risk for transplant-related
mortality (30). The description of the natural polymorphisms in
HLA class I H chains that selectively affect tapasin-dependent peptide loading provides insights into the functional interaction of
tapasin with MHC class I molecules and indicates that manipulation of class I sequences can affect MHC class I maturation and
cell surface expression.
Acknowledgments
We thank Drs. P. Cresswell and Y. Loke for providing materials.
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CORRECTIONS
Boyoun Park, Sungwook Lee, Euijae Kim, and Kwangseog Ahn. A Single Polymorphic Residue Within the PeptideBinding Cleft of MHC Class I Molecules Determines Spectrum of Tapasin Dependence. The Journal of Immunology
2003;170:961–968.
In the original article, both the concepts and language in the last paragraph of the Discussion were taken without
attribution from the previous publications (References 3 and 9). We deeply regret and apologize for this lack of appropriate attribution.
Sang-Heon Lee, Dong Kyung Chang, Ajay Goel, C. Richard Boland, William Bugbee, David L. Boyle, and Gary S.
Firestein. Microsatallite Instability and Suppressed DNA Repair Enzyme Expression in Rheumatoid Arthritis. The Journal of Immunology 2003;170:2214 –2220.
In Figure 2B, the mismatch repair enzyme expression for osteoarthritis (OA) and rheumatiod arthritis (RA) was
compared in a bar graph. The designation for RA and OA were reversed in the figure. The corrected figure is shown below.
Copyright © 2003 by The American Association of Immunologists, Inc.
0022-1767/03/$02.00