Maturation of Qa-1b Class I Molecules Requires
β2-Microglobulin But Is TAP Independent
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J Immunol 1998; 160:3217-3224; ;
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Copyright © 1998 by The American Association of
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References
Peter J. Robinson, Paul J. Travers, Arthur Stackpoole, Lorraine
Flaherty and Hakim Djaballah
Maturation of Qa-1b Class I Molecules Requires
b2-Microglobulin But Is TAP Independent1
Peter J. Robinson,2* Paul J. Travers,† Arthur Stackpoole,* Lorraine Flaherty,‡ and
Hakim Djaballah*
C
lass I histocompatibility Ags alert the immune system to
viruses by displaying peptides derived from virally encoded proteins on the surface of infected cells. Cells expressing class I molecules bearing viral epitopes are efficiently
destroyed by host CTL. Newly synthesized class I heavy chains
(Hcs) first associate with calnexin, an endoplasmic reticulum
(ER)3-resident chaperone, and then bind b2-microglobulin (b2m)
(1, 2). Stabilization of class I/b2m heterodimers is usually dependent upon acquisition of a suitable peptide, delivered by the TAP
peptide transporter located in the ER (3, 4). In TAP-deficient cells
where the peptide supply to class I molecules is severely reduced,
many Hcs are unable to form stable structures and are targeted for
degradation (5).
Peptides that enter the ER independently of TAPs, such as
cleaved leader sequences, can induce assembly of class I molecules in TAP-deficient cells (6). It has also been shown that the
expression of some class I molecules is intrinsically TAP independent (7, 8). The class I-like molecules CD1 and TL can
both be expressed on the surface of cells lacking a functional
TAP transporter, and there is increasing evidence that they may
acquire antigenic epitopes in intracellular compartments other
than the ER (9, 10).
Mouse Qa-1 is one of a group of class I molecules designated as
nonclassical or class Ib histocompatibility Ags, and that elicits
*MRC-CSC, Hammersmith Hospital, and †Department of Crystallography, Birbeck
College, London, United Kingdom; and ‡Wadsworth Center for Laboratories and
Research, Albany, NY 12201
Received for publication September 29, 1997. Accepted for publication December
5, 1997.
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
H.D. is supported by the Leukaemia Research Fund.
2
Address correspondence and reprint requests to Dr. Peter J. Robinson, Transplantation Biology Group, MRC-CSC, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, U.K. E-mail address:
[email protected]
Abbreviations used in this paper: ER, endoplasmic reticulum; Hc, heavy chain; b2m,
b2-microglobulin; Endo, endoglycosidase.
3
Copyright © 1998 by The American Association of Immunologists
strong CTL responses (11). At least two other members of this
group, CD1 and H-2 M3, are involved in the presentation of bacterial and mycobacterial Ags to T cells (12, 13). Qa-1b is expressed
in many mouse tissues, associates with b2m, and can present peptide Ags to CTL (11). Previous studies have shown that some CTL
can recognize Qa-1b on cells defective in TAP function (14).
Maturation of class I molecules has usually been studied using
alloantibodies that recognize correctly folded heterotrimers of Hcs,
b2m, and peptide. More recently, alternative conformations of
class I molecules, including precursor forms as well as cell surface
molecules, have been described using other Hc-specific Abs (15).
We have produced rabbit antisera against unique peptide sequences present in the cytoplasmic domain of Qa-1b. These antisera do not cross-react with other mouse class I molecules, and
recognize both correctly folded and conformationally altered
Qa-1b molecules. By comparing the reactivity of alloantiserum
with peptide-specific Abs, we have identified two forms of Qa-1b
that differ in their association with b2m and with other components
of the maturation pathway.
Materials and Methods
Antibodies
Alloantiserum specific for Qa-1b was produced by immunization of
B6.Tlaa 3 A strain mice with cells from A.Tlab. Anti-KSFQ (anti-Qa-1b
polyclonal) was produced by immunizing New Zealand White rabbits with
the peptide KSFQKDAMLMF. Hybridoma cells producing monoclonal
anti-Kb (34.2.12) and anti-Kk (16.3.1) were obtained from the American
Type Culture Collection (Rockville, MD).
Cloning and expression of Qa-1b/Ld
A genomic clone of H-2Ld carrying the regular H-2 promoter region was
kindly provided by Dr. A. Mellor. The Qa-1b/Ld construct was made by
introducing the a1 and a2 domains of Qa-1b into the Ld gene as described
previously for the Qa-1b/Dd construct (16). The construct was cloned into
pBR327 and the linearized DNA was used to transfect L cells. Cells were
grown in RPMI 1640 (Life Technologies, Grand Island, NY) containing
10% FCS, 50 IU/ml penicillin, 50 IU/ml streptomycin, and 2 mM glutamine (complete medium) at 37°C in 5% CO2.
0022-1767/98/$02.00
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Two conformationally distinct and stable forms of Qa-1b, one strongly associated with b2-microglobulin (b2m) and the other
associated with a novel molecule, gp44, were observed during immunochemical studies on the expression of Qa-1b molecules in
mouse spleen cells. Both forms are efficiently processed and expressed at the cell surface. However, a large proportion of Qa-1b
was found to be disulfide linked to gp44 without any detectable b2m. In TAP1-deficient mice, both forms undergo carbohydrate
processing and are expressed on the cell surface, suggesting that they may traffic using a pathway not requiring a TAP association
step. Consistent with this, size exclusion chromatography of newly synthesized class I molecules shows that high molecular mass
complexes containing H-2Kk do not contain Qa-1b. Although Qa-1b can be stably expressed without b2m, there was no maturation
of either form in cells from b2m-deficient mice where heavy chains were rapidly degraded. These results suggest that Qa-1b, like
most other class I molecules, requires b2m for an initial folding step. However, b2m is not essential for subsequent processing of
Qa-1b molecules. The Journal of Immunology, 1998, 160: 3217–3224.
MATURATION OF Qa-1b REQUIRES b2m BUT NOT TAPs
3218
Biosynthetic labeling and immunoprecipitation
CBA mouse spleen cells were cultured for 40 to 50 h with 2 mg/ml of Con
A in complete medium containing 1 mM 2-ME. For labeling, 2 3 107 cells
were washed three times at room temperature in RPMI medium lacking
methionine and cysteine and containing no additives. A total of 200 mCi of
Pro-Mix, [35S](methionine 1 cysteine) (Amersham, Little Chalfont, U.K.)
was added and cells were incubated for 10 min at 37°C. After labeling,
cells were either placed on ice, or chased in complete medium for appropriate times.
Labeled cells were washed once in ice-cold isotonic TBS to remove
radioisotopes, and lysed in 1 ml of PBS containing 1% Triton X-100
(Pierce, Chester, U.K.), proteinase inhibitors, and 10 mM iodoacetamide.
After 10 min on ice, lysates were centrifuged at 20,000 3 g in a Sigma (St.
Louis, MO) refrigerated bench microfuge. Cleared lysates were then
treated with protein A beads and unimmunized serum (mouse or rabbit) at
4°C overnight. Protein A beads preloaded with specific antiserum were
rotated at 4°C with precleared lysates for 2 h, and then harvested at 100 3
g. After extensive washing, immunoprecipitates were treated with SDS
containing sample buffer at 95°C with or without 10 mM DTT. Samples
were then subjected to electrophoresis on 12% polyacrylamide gels, treated
with autoradiographic enhancers (Amplify, Amersham, U.K.), dried, and
autoradiographed on Kodak BioMax MR film.
125
I
Cells, 2 3 10 , were washed thoroughly in cold PBS and suspended in 200
ml of PBS on ice. A total of 5 ml of 1 mg/ml of lactoperoxidase solution
was added followed by 5 ml of a dilute hydrogen peroxide solution (30 vol
diluted 1:104). Labeling was started by adding 10 ml (1 mCi) of [125I]Na
solution (Amersham) and removing the tube from the ice. The reaction was
continued for 30 min, adding 5 ml of dilute hydrogen peroxide solution at
10-min intervals. Cells were then washed in PBS containing 1 mM sodium
iodide and lysed. 125I-labeled samples were visualized using Kodak BioMax MS film using an appropriate intensifying screen.
7
Pulse-chase analysis
Cells labeled with [35S]methionine for 5 min were diluted to 7 ml with
warm complete RPMI medium in a 10-ml polypropylene tube, and 1 ml
was removed and placed immediately in ice. This chase suspension was
maintained at 37°C with occasional mixing, removing samples at 30-min
intervals. Lysis and immunoprecipitation were performed as described
above, and then immunoprecipitates were treated with Endo (endoglycosidase) H (Boehringer Mannheim, Mannheim, Germany) according to the
manufacturer’s instructions before gel electrophoresis.
Two-stage immunoprecipitation
First, immunoprecipitates were treated with 20 ml of 1.0% SDS at 95°C for
5 min with or without 1 mM DTT. Then, 1 ml of PBS containing 1% Triton
X-100 (Pierce) was added and the beads removed. The supernatant containing the released Ag was then subjected to a second round of immunoprecipitation using protein A beads preloaded with Ab. Samples were then
subjected to gel electrophoresis under reducing conditions.
Fractionation of metabolically labeled lysates by size exclusion
chromatography
CBA spleen cell blasts were metabolically labeled for 20 min as described
above and lysed in PBS containing proteinase inhibitors and 1% digitonin.
Lysates were centrifuged at 20,000 3 g for 15 min and precleared with
unimmunized serum. Precleared lysates were loaded onto a Superose 6 size
exclusion gel filtration column (Pharmacia, St. Albans, U.K.) equilibrated
with PBS containing 1% digitonin and run at a flow rate of 0.25 ml/min.
The column was calibrated using gel filtration protein standards (Bio-Rad,
Hemel Hempstead, U.K.), thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), and chicken OVA (44 kDa). One-milliliter fractions were
collected and each fraction subjected to immunoprecipitation with protein
A beads preloaded with specific antiserum/Ab at 4°C for 4 h. After three
washes in digitonin-containing PBS buffer, samples were analyzed by gel
electrophoresis under reducing conditions.
Results
Two conformations of Qa-1b
To visualize Qa-1b molecules, Con A-activated CBA mouse
spleen cells (H-2k, Qa-1b) were either metabolically labeled
with [35S]methionine or surface labeled with 125I. Cell lysates
were treated either with an anti-Qa-1b alloantiserum or with a
FIGURE 1. Alloantiserum and anti-KSFQ define different forms of Qa1b. A, [35S]methionine-labeled CBA lysates immunoprecipitated using alloantiserum or anti-KSFQ; B, 125I surface-labeled lysates treated similarly;
and C, [35S]methionine-labeled lysates precleared three times sequentially
with alloantiserum, then treated with anti-KSFQ. Hc denotes class I heavy
chain.
rabbit antiserum prepared against a Qa-1b cytoplasmic peptide
motif (anti-KSFQ) and protein A beads. Immunoprecipitates
were subjected to gel electrophoresis under reducing conditions
and the results shown in Figure 1, A and B. Both reagents detect
Qa-1b Hcs, but in very different amounts. This was not due to
differences in Ab titer, since Ab was always present in excess.
Interestingly, both reagents coprecipitated equal amounts of
b2m. Similar results were obtained with metabolically labeled
(Fig. 1A) and surface-labeled (Fig. 1B) cells. When lysates
were extensively precleared with the alloantiserum, all of the
b2m-associated Hcs were removed (Fig. 1C). However, Hcs
devoid of b2m were still detectable using the anti-KSFQ Ab
(Fig. 1C). To investigate whether anti-KSFQ Ab might crossreact with proteins other than Qa-1b, it was tested on lysates of
[35S]methionine-labeled B10.BR (H-2k, Qa-1a) spleen cells. No
40- to 50-kDa material was immunoprecipitated from these lysates (data not shown), indicating that all of the labeled 40- to
50-kDa material detected with anti-KSFQ was associated with
Qa-1b. These results suggested that CBA lysates contain two
serologically distinguishable forms of Qa-1b Hcs. One is a stable Qa-1b/b2m heterodimer recognized both by the alloantiserum and by anti-KSFQ. There are also Qa-1b Hcs present
that contain little or no b2m and are recognized only by
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Surface labeling with
The Journal of Immunology
FIGURE 3. Coisolation of Qa-1b with gp44 using conformation-independent Abs. A, Immunoprecipitates of 125I-labeled Qa-1b molecules
treated with Endo F. B, CBA cells were [35S]methionine labeled for 1 h
(lane 2) or 5 h (lanes 1 and 3) and immunoprecipitated with anti-KSFQ.
Immunoprecipitates in lanes 2 and 3 were treated with Endo F. C, Immunoprecipitates of 125I-labeled Qa-1b/Ld molecules not treated with Endo F.
Hc denotes class I heavy chain. Hc(EF) denotes Endo F-treated Hc.
gp44(EF) denotes Endo F-treated gp44.
anti-KSFQ. It also shows that a significant proportion of the
Qa-1b does not form a stable association with b2m.
tion of the samples, high m.w. forms of Qa-1b were found immediately after the labeling pulse, and were increased in quantity
during the chase period (Fig. 2C). Potential artifacts due to oxidation occurring in the lysate were minimized by including iodoacetamide in the lysis buffer. No high m.w. forms of H-2Kk
molecules were found in the same lysate (data not shown). These
results show that some Qa-1b molecules form disulfide-linked
complexes early in the maturation pathway.
Trafficking of Qa-1b molecules
Cell surface Qa-1b is associated with additional molecules
To examine the intracellular trafficking of Qa-1b molecules, CBA
spleen cell blasts were labeled for 10 min with [35S]methionine
and chased for various times in medium containing excess unlabeled methionine. Precleared lysates were treated with anti-KSFQ
and maturation of Qa-1b monitored by treatment with the glycosidase Endo H to monitor the conversion of immature, high-mannose, carbohydrates to complex forms. As shown in Figure 2A,
Endo H-sensitive Qa-1b molecules have a half-life of about 60 min
before becoming resistant. The half-life of immature carbohydrates
on H-2Kk molecules in the same cells was less than 30 min (Fig.
2B). Samples from the pulse-chase analysis were also investigated
by electrophoresis under nonreducing conditions. Without reduc-
CBA spleen cells were surface-labeled and lysates were treated
with either the alloantiserum or with anti-KSFQ. To reduce the
complexity of the gel patterns, N-linked carbohydrate side
chains were removed by treatment of immunoprecipitates with
endoglycosidase F (Endo F). Endo F treatment of labeled material isolated using the alloantiserum reduced the size of Qa-1b
Hcs to a single band of 35 kDa, consistent with removal of the
two N-linked carbohydrate chains. However, Endo F treatment
of anti-KSFQ immunoprecipitates revealed the 35-kDa Hc band
and an additional component of 37 kDa (Fig. 3A). The 37-kDa
component was not eliminated by extended treatment with excess Endo F and is not, therefore, a consequence of incomplete
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FIGURE 2. Pulse-chase analysis of Qa-1b and H-2Kk molecules in
CBA cells. A, Cells were labeled for 10 min with [35S]methionine, chased
for the times indicated, and immunoprecipitated with anti-KSFQ. B, Lysates from A were immunoprecipitated with anti-H-2Kk. In A and B, samples were treated with Endo H before SDS-PAGE. Hc denotes class I heavy
chain. EHR Hc denotes running position of Endo H-resistant Hcs. C, Samples from A were run unreduced. **, Denotes high molecular mass
aggregates.
3219
3220
MATURATION OF Qa-1b REQUIRES b2m BUT NOT TAPs
carbohydrate removal. To determine whether the 37-kDa
polypeptide was synthesized internally, cells were labeled with
[35S]methionine for 1 h and a similar immunoprecipitation conducted. Very little 37-kDa material was detectable after a 1-h
label, but if cells were labeled continuously for 5 h with
[35S]methionine, 37-kDa material was clearly detectable using
anti-KSFQ (Fig. 3B). This indicates that the 37-kDa Qa-1bassociated polypeptide is indeed synthesized by the cells but
either has a slow turnover rate or a large intracellular pool size.
Since the fully glycosylated cell surface form of the 37-kDa
polypeptide is approximately 44 kDa, it has been called gp44.
A hybrid class I molecule consisting of the a1 and a2 domains
of Qa-1b linked to the a3 domain of H-2Ld was also examined.
Transfected L cells expressing the hybrid molecule were surface
labeled with 125I and lysates treated with either anti-Qa-1b alloantiserum or with the 28.14.8 Ab, which is specific for the a3 domain
of H-2Ld. The hybrid molecule showed a weak b2m association
similar to native Qa-1b. However, since the hybrid Qa-1b/Ld Hc
carries an additional N-linked carbohydrate chain, it is 2 to 3 kDa
larger than Qa-1b. As shown in Figure 3C, the a3-specific Ab
28.14.8 reveals the hybrid class I Hc and gp44, which are clearly
distinguishable without Endo F treatment. Consistent with previous data, the anti-Qa-1b alloantiserum detect only the hybrid Hc,
indicating that Hcs associated with gp44 are not recognized by
alloantibodies. This result shows that gp44 is detectable using Abs
other than anti-KSFQ.
To investigate further the nature of the association between
Qa-1b and gp44, a two-stage immunoprecipitation experiment was
performed. Lysates of surface-labeled cells were treated with antiKSFQ and immunoprecipitates washed as before. Labeled
polypeptides were then released from the immunosorbent with a
hot SDS solution, fresh lysis buffer added, and the eluted material
treated a second time with anti-KSFQ on protein A beads. Finally,
the samples were treated with Endo F and subjected to gel electrophoresis. As shown in Figure 4, when immunosorbents from the
first immunoprecipitation were treated with SDS alone, Qa-1b and
gp44 remain associated in the second round (Fig. 4, lane 1). If,
however, the reducing agent DTT is included in the first SDS
retrieval step, gp44 does not reappear in the second immunoprecipitate (Fig. 4, lane 2). This indicates that Qa-1b and gp44 are
disulfide linked. It also shows that anti-KSFQ does not recognize
FIGURE 5. Expression of Qa-1b in TAP1-deficient mice. A, Surface
expression of Qa-1b and H-2Kb in 125I-labeled B10 and TAP1-deficient
mice. B, Pulse-chase analysis of Qa-1b in TAP1-deficient mice. Lysates
were immunoprecipitated with anti-KSFQ and samples were Endo H
treated. Hc denotes class I heavy chain. EHR Hc denotes running position
of Endo H-resistant Hcs.
gp44 directly but does so only by virtue of its association with
Qa-1b.
Expression of Qa-1b in TAP1-deficient cells
It has been previously reported that T cells can recognize Qa-1b in
a TAP-dependent or a TAP-independent way (14). This implies
that Qa-1b molecules can reach the cell surface in the absence of
TAP molecules. To test this, activated spleen cells from TAP1deficient mice (H-2b, Qa-1b) were surface labeled and lysates prepared as before. Using anti-KSFQ, Qa-1b was easily detectable by
immunoprecipitation (Fig. 5A, lane 2). Surface forms were also
detected using the alloantiserum (data not shown). In contrast, only
low levels of H-2Kb were found in the same lysates (Fig. 5A, lane
4). Pulse-chase analysis showed that a significant proportion of
Qa-1b molecules are processed to Endo H-resistant forms in
TAP1-deficient cells, indicating that intracellular transport of
Qa-1b occurs in the absence of TAP function (Fig. 5B).
Gel filtration analysis of Qa-1b- and H-2Kk-containing
complexes
It has been shown recently that class I molecules associate transiently in the ER with a protein complex consisting of the molecular chaperons calnexin and calreticulin, another molecule called
tapasin, and the TAP1/TAP2 heterodimer, as a prelude to peptide
loading (17). To investigate a possible physical association between Qa-1b and the TAP machinery, lysates of metabolically labeled CBA blasts were fractionated by size-exclusion chromatography in digitonin-containing buffer to preserve weak molecular
associations. Fractions were then treated sequentially with antiKSFQ and 16.3.1 to detect Qa-1b and H-2Kk, respectively. The
results are shown in Figure 6. Using this methodology, we were
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FIGURE 4. Qa-1b and gp44 are covalently linked. 125I-labeled CBA
lysates were immunoprecipitated with alloantiserum (lane 3) or anti-KSFQ
(lane 4). Alternatively, anti-KSFQ immunoprecipitates were treated with
either SDS alone (lane 1) or SDS and DTT (lane 2) and subjected to a
second anti-KSFQ immunoprecipitation step as described in Materials and
Methods. All samples were Endo F treated. Hc denotes class I heavy chain.
The Journal of Immunology
3221
Requirement for b2m for Qa-1b expression
FIGURE 6. Gel filtration analysis of Qa-1b- and H-2Kk-containing
complexes. Fractions 7 to 18 were immunoprecipitated using anti-H-2Kk
(A) or anti-KSFQ (B) as described in Materials and Methods. Running
positions of size markers are shown above each panel. Hc denotes class I
heavy chain.
Our results show that b2m is not associated with a large proportion
of Qa-1b Hcs in CBA cells. This suggested that Qa-1b expression
might be independent of b2m. We therefore examined the maturation and cell surface expression of Qa-1b in b2m-deficient mice
by pulse-chase analysis. We were unable to detect any Endo Hresistant forms of Qa-1b in b2m-deficient mice, and Endo H-sensitive Hcs disappear during the chase period (Fig. 7). Furthermore,
no cell surface Qa-1b was detectable by surface labeling (data not
shown). These results show that, without b2m, Qa-1b is rapidly
degraded. They also show that, once the Hcs have undergone their
initial folding step, b2m is no longer essential for subsequent maturation and stable cell surface expression.
Discussion
able to detect class I molecules associated with different molecular
mass ranges. Some H-2Kk was found in complexes with molecular
masses above the 670-kDa marker, possibly representing molecules associated with the TAP machinery (Fig. 6A). H-2Kk molecules were also found in complexes with molecular masses below
the 158-kDa standard marker, probably representing assembled
heterotrimeric complexes of Hc, b2m, and peptide. In contrast,
Qa-1b was found in complexes with molecular masses in the range
of 100- to 300-kDa only, and not in the high molecular mass complexes that contain H-2Kk (Fig. 6B). The presence of material in
the 300- to 400-kDa range in Figure 6B indicates some variability
in the composition of Qa-1b-containing complexes. In a separate
experiment, anti-TAP1 immunoprecipitates, in digitonin-containing buffer, of metabolically labeled CBA lysates were eluted and
reprecipitated with anti-KSFQ. No Qa-1b molecules were found
(data not shown), consistent with their failure to strongly associate
with TAPs.
The stability of cell surface heterotrimeric complexes of class I
Hcs, b2m, and peptide is critically dependent upon the affinity of
the peptide bound. Release of peptide either during intracellular
transport or at the cell surface results in loss of b2m and alteration
of the Hc conformation. Most class I molecules bind a range of
peptides with a spectrum of affinities. Inevitably, therefore, some
cell surface Hcs will acquire an aberrant conformation. These
forms are not readily detectable, since the majority of mAbs recognize only stable heterotrimeric forms. The availability of conformation-independent Abs, which recognize class I Hcs irrespective of their association with b2m and peptide, has allowed detailed
studies on the trafficking and cell surface presentation of several
class I molecules, including H-2Kb (15), H-2Db (18), H-2Ld (19),
HLA-B27 (20), HLA-Cw1 (21), and Qa-1b (this study). The
emerging picture is that conformational variants of class I molecules do exist and are stably expressed at the cell surface. The
function of these variants remains unclear.
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FIGURE 7. Pulse-chase analysis of Qa-1b in cells from b2m-deficient
mice. [35S]methionine-labeled cells were chased for the indicated times
and immunoprecipitated with anti-KSFQ. Samples were Endo H treated.
Hc denotes class I heavy chain. EHR Hc denotes running position of Endo
H-resistant Hcs.
3222
FIGURE 8. Molecular images of the peptide-combining site of Qa-1b
highlighting the position of Cys114. The main part of the figure shows a
cartoon view of the a2 domain of the model of Qa-1b (this region is indicated by the gray shading in the inset, where the molecule is seen from the
top). The positions of the serine residues at positions 143 and 147, which
are well conserved as threonine and tryptophan in other class I molecules,
are shown, as is the conserved disulfide bond between Cys101 and Cys164.
A free cysteine residue is present at position 114, on the second b strand
of the a2 domain and points up into the peptide binding groove. The figure
was produced using MOLSCRIPT (32).
another polypeptide, gp44, which associates with Qa-1b in H-2k
and H-2b strains. Using biosynthetic labeling, gp44 labels poorly,
and is therefore difficult to detect. Also, if carbohydrates are not
removed, gp44 was difficult to distinguish from Qa-1b on SDSPAGE due to the closeness of their polypeptide chain sizes. It was,
in contrast, easy to distinguish Qa-1b/Ld Hc from gp44 since the
extra carbohydrate on the chimeric class I Hc reduces substantially
its SDS-PAGE mobility (Fig. 3C). This experiment also demonstrates that the a3 domain of Qa-1b is unimportant for gp44
association.
Evidence that gp44 is a novel molecule comes from the following observations. First, gp44 associates with Qa-1b in mice deficient in the class II-associated invariant chain (data not shown).
Second, other known accessory molecules in the TAP pathway,
e.g., calreticulin (28), ectocalreticulin (28), and tapasin (29, 30)
have higher molecular masses: 52 kDa, 62 kDa, and 47.5 kDa,
respectively. And third, preliminary N-terminal sequence analysis
of gp44 appears to exclude other known class I Hcs (H. Djaballah
and P. J. Robinson, unpublished observations). It is not yet clear
whether gp44 is membrane bound; further characterization is
under way.
All class I molecules have four conserved cysteine residues
that form two intrachain disulfide bonds, Cys101-Cys164 and
Cys203-Cys259. The additional cysteine residue at position 114
of Qa-1b is located on one of the b strands at the base of the
peptide-binding cleft, making it solvent accessible and able to
form a disulfide bridge with free cysteine residues on gp44 (Fig.
8). A cysteine residue at this position of class I Hcs is unusual.
Although some mouse class I molecules have a free cysteine
residue at position 121, this is less likely to form disulfide
bridges because of poor solvent accessibility. However, this
may account for the formation of disulfide-linked homodimers
observed previously (31). Cysteine residues are also present in
the cytoplasmic domains of several class I Hcs, including
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An interesting and special feature of Qa-1b is its ability to bind
the nonamer peptide, AMAPRTLLL, derived from the leader sequences of H-2D and H-2L class I molecules, in preference to
other peptides (11). Recently, peptide elution studies show AMAPRTLLL indeed to be the major peptide bound, with very few
others detected (16, 22). In this study, we have shown that, as
expected, association of Qa-1b with b2m is poor in CBA (H-2k)
mice that lack AMAPRTLLL. We were, however, surprised to find
that, despite poor b2m association, Qa-1b molecules do not undergo rapid proteolysis, but rather traffic normally and are stably
expressed on the cell surface. These cell surface Qa-1b molecules,
which lack b2m and did not react with our conformation-dependent alloantiserum, were detected with the conformation-independent peptide-specific antiserum (anti-KSFQ). This suggests that, if
AMAPRTLLL is not present, Qa-1b adopts an alternative stable
conformation.
It is widely accepted that trafficking and maturation of class I
Hcs involves the recruitment of several accessory molecules. One
of these is calnexin, an ER resident lectin-like chaperone, to which
nascent Hcs bind cotranslationally and remain bound while they
undergo correct folding upon acquisition of b2m. On subsequent
binding to the TAP machinery and on binding peptides they are
then released and bound for the cell surface. It should be noted that
recent studies have shown that, in calnexin-deficient cells, rates of
class I Hc maturation were not affected, suggesting that other ER
molecular chaperons can perform the same job (23, 24). One possible explanation for the weak association between Qa-1b and b2m
observed here is that Hcs are capable of folding correctly independently of b2m. This is not clearly the case, since Qa-1b Hcs are
rapidly degraded in b2m-deficient mice, and cell surface labeling
experiments showed them to be absent. Therefore, Qa-1b molecules appear to share the same b2m-dependent initial folding steps
as other class I molecules, but subsequent b2m association appears
not to be essential for their trafficking and maturation. An exception to the b2m-dependence rule is H-2Db, which is expressed on
the surface of cells from b2m-deficient cells, although at low
levels (18).
Previous studies have shown that, in cells defective in TAP expression and function, the rate of intracellular transport of class I
molecules is reduced, possibly because calnexin remains associated for longer with Hc-b2m heterodimers (25). Our pulse-chase
experiments show that Qa-1b becomes Endo H resistant 60 to 90
min after synthesis, much slower than H-2Kk molecules, which
become resistant within 30 min. Similar rates of maturation and
cell surface expression of Qa-1b were observed in both normal and
TAP1-deficient mice. Cell surface expression of most class I molecules in TAP-deficient cells is generally low, since Hc-b2m
dimers without peptide are unstable. However, Qa-1b molecules
are abundant and stable on the cell surface in TAP1-deficient mice.
In this respect, Qa-1b resembles the class Ib molecules TL and
CD1 (7, 8). Earlier work has shown that CTL recognition of Qa-1b
is not dependent on TAPs (14, 26). Paradoxically, many Qa-1specific CTL recognize their targets in a TAP-dependent manner,
possibly reflecting a requirement for the transporter in delivering
peptide epitopes into the lumen of the ER. Our results, therefore,
clearly distinguish the dual roles of TAP molecules in the assembly of class I molecules and in the delivery of peptides.
It is not unusual for free class I Hcs to form either homodimers
or associate with additional proteins. Indeed, this may be a way to
stabilize free Hcs that would otherwise be targeted for degradation.
Wolf and Cook reported that Qa-1b Hcs associate with free H-2Ld
Hc and with another unidentified molecule, Qsm, and in doing so
exclude b2m (27). In their experiments, no additional molecules
were detected in H-2k strains. In contrast, our results demonstrate
MATURATION OF Qa-1b REQUIRES b2m BUT NOT TAPs
The Journal of Immunology
3223
order of events, and establish whether other class Ib molecules
may follow a similar maturation pathway.
The results described above, obtained using a variety of immunochemical and biochemical techniques, shed some light on the
trafficking and maturation of Qa-1b and suggest that stable cell
surface expression of Qa-1b Hcs lacking b2m can be accounted for
by covalent association with a novel molecule gp44.
Acknowledgments
We thank Drs. O. Smithies and M. Merkenschlager for b2m-deficient mice;
Drs. D. Kioussis, O. Williams, and S. Tonegawa for TAP1-deficient mice;
Drs. C. Benoist and D. Mathis for Ii-deficient mice; and John Trowsdale for
anti-TAP1 Abs. We also thank Drs. Jim Kaufman and Danny Altman for
critically reading the manuscript.
References
Qa-1b. However, the reducing environment of the cytoplasm
effectively rules out formation of disulfide bridges in vivo involving cytoplasmic cysteine residues. In addition, we find no
evidence for dimers of Qa-1b, but detect larger complexes that
form within minutes of Hc synthesis and contain gp44. One
might predict that formation of such complexes may not allow
a good contact between the Hcs and the TAP molecular complex. Our gel filtration studies showing that Qa-1b and H-2Kk
are present in different molecular mass fractions is consistent
with this hypothesis.
The scheme we propose for the maturation of Qa-1b molecules
is shown in Figure 9. It is based upon a recent maturation scheme
for class I molecules proposed by Sadasivan et al. (17). Hcs associate cotranslationally with calnexin in the ER, where they bind
b2m and commence folding. Next, calnexin is replaced by calreticulin, as is the case for most class Ia molecules, forming a quasistable complex with the chaperone. Here, similarity with the class
Ia maturation pathway ends. Rather than associating with the TAP
machinery, a decision is now made as to whether Hcs bind peptide
or gp44. If peptide binds, b2m association is stabilized and the
heterotrimeric complex is detectable by alloantiserum. If, instead,
gp44 forms a disulfide bridge with Qa-1b, possibly via cysteine
114, b2m is released and the complex is no longer detectable using
our alloantiserum. In either case, calreticulin is recycled while the
Hcs undergo further maturation and transport to the cell surface.
Additional experiments will be required to determine the precise
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FIGURE 9. Proposed scheme for Qa-1b maturation and possible origins
of the two cell surface forms.
1. York, I., and K. L. Rock. 1996. Antigen processing and presentation by the class
I major histocompatibility complex. Annu. Rev. Immunol. 14:369.
2. Jackson, M. R., and P. A. Peterson. 1993. Assembly and intracellular transport of
MHC class I molecules. Annu. Rev. Cell Biol. 9:207.
3. Suh, W. K., E. K. Mitchell, Y. Yang, P. A. Peterson, G. L. Waneck, and
D. B. Williams. 1996. MHC class I molecules form ternary complexes with
calnexin and TAP and undergo peptide regulated interaction with TAP via their
extracellular domains. J. Exp. Med. 184:337.
4. Ortmann, B., M. J. Androlewicz, and P. Cresswell. 1994. MHC class I/b2-microglobulin complexes associate with TAP transporters before peptide binding.
Nature 368:864.
5. Van Kaer, L., P. G. Ashton Rickardt, H. L. Ploegh, and S. Tonegawa. 1992.
TAP1 mutant mice are deficient in antigen presentation, surface class I molecules,
and CD4281 T cells. Cell 71:1205.
6. Wei, M. L., and P. Cresswell. 1992. HLA-A2 molecules in an antigen processing
mutant cell contain signal sequence derived peptides. Nature 356:443.
7. Holcombe, H. R., A. R. Castaño, H. Cheroutre, M. Teitell, J. K. Maher,
P. A. Peterson, and M. Kronenberg. 1995. Nonclassical behavior of the thymus
leukemia antigen: peptide transporter independent expression of a nonclassical
class I molecule. J. Exp. Med. 181:1433.
8. Brutkiewicz, R. R., J. R. Bennink, J. W. Yewdell, and A. Bendelac. 1995. TAP
independent, b2-microglobulin dependent surface expression of functional mouse
CD1.1. J. Exp. Med. 182:1913.
9. Prigozy, T. I., P. A. Sieling, D. Clemens, P. L. Stewart, S. M. Behar,
S. A. Porcelli, M. B. Brenner, R. L. Modlin, and M. Kronenberg. 1997. The
mannose receptor delivers lipoglycan antigens to endosomes for presentation to
T cells by CD1b molecules. Immunity 6:187.
10. Sugita, M., R. M. Jackman, E. Van Donselaar, S. M. Behar, R. A. Rogers,
P. J. Peters, M. B. Brenner, and S. A. Porcelli. 1996. Cytoplasmic tail dependent localization of CD1b antigen presenting molecules to MIICs. Science
273:349.
11. Soloski, M. J., A. DeCloux, C. J. Aldrich, and J. Forman. 1995. Structural and
functional characteristics of the class Ib molecule, Qa-1. Immunol. Rev. 147:
67.
12. Moody, D. B., M. Sugita, P. J. Peters, M. B. Brenner, and S. A. Porcelli. 1996.
The CD1 restricted T-cell response to mycobacteria. Res. Immunol. 147:550.
13. Bouwer, H. G., K. F. Lindahl, J. R. Baldridge, C. R. Wagner, R. A. Barry, and
D. J. Hinrichs. 1994. An H2-T MHC class Ib molecule presents Listeria monocytogenes-derived antigen to immune CD81 cytotoxic T cells. J. Immunol. 152:
5352.
14. Aldrich, C. J., R. Waltrip, E. Hermel, M. Attaya, K. F. Lindahl, J. J. Monaco, and
J. Forman. 1992. T cell recognition of Qa-1b antigens on cells lacking a functional TAP2 transporter. J. Immunol. 149:3773.
15. Machold, R. P., and H. L. Ploegh. 1996. Intermediates in the assembly and
degradation of class I major histocompatibility complex (MHC) molecules
probed with free heavy chain specific monoclonal antibodies. J. Exp. Med.
184:2251.
16. Cotterill, L. A., H. J. Stauss, M. M. Millrain, D. J. Pappin, D. Rahman, B. Canas,
P. Chandler, A. Stackpoole, E. Simpson, P. J. Robinson, and P. J. Dyson. 1997.
Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide.
Eur. J. Immunol. 27:2123.
17. Sadasivan, B., P. J. Lehner, B. Ortmann, T. Spies, and P. Cresswell. 1996. Roles
for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class
I molecules with TAP. Immunity 5:103.
18. Allen, H., J. Fraser, D. Flyer, S. Calvin, and R. A. Flavell. 1986. b2-microglobulin is not required for cell surface expression of the murine class I histocompatibility antigen H-2Db or of a truncated H-2Db. Proc. Natl. Acad. Sci. USA 83:
7447.
19. Smith, J. D., N. B. Myers, J. Gorka, and T. H. Hansen. 1993. Model for the in
vivo assembly of nascent Ld class I molecules and for the expression of unfolded
Ld molecules at the cell surface. J. Exp. Med. 178:2035.
20. Carreno, B. M., and T. H. Hansen. 1994. Exogenous peptide ligand influences the
expression and half-life of free HLA class I heavy chains ubiquitously detected
at the cell surface. Eur. J. Immunol. 24:1285.
3224
21. Setini, A., A. Beretta, C. De-Santis, R. Meneveri, A. Martayan, M. C. Mazzilli,
C. Appella, A. G. Siccardi, P. G. Natali, and P. Giacomini. 1996. Distinctive
features of the alpha 1-domain alpha helix of HLA-C heavy chains free of b2microglobulin. Hum. Immunol. 46:69.
22. DeCloux, A., A. S. Woods, R. J. Cotter, M. J. Soloski, and J. Forman. 1997.
Dominance of a single peptide bound to the class Ib molecule, Qa-1b. J. Immunol.
158:2183.
23. Sadasivan, B. K., A. Cariappa, G. L. Waneck, and P. Cresswell. 1995. Assembly,
peptide loading, and transport of MHC class I molecules in a calnexin negative
cell line. Cold Spring Harbor Symp. Quant. Biol. 60:267.
24. Scott, J. E., and J. R. Dawson. 1995. MHC class I expression and transport in a
calnexin-deficient cell line. J. Immunol. 155:143.
25. Degen, E., M. F. Cohen Doyle, and D. B. Williams. 1992. Efficient dissociation
of the p88 chaperone from major histocompatibility complex class I molecules
requires both b2-microglobulin and peptide. J. Exp. Med. 175:1653.
26. Hermel, E., E. Grigorenko, and K. F. Lindahl. 1991. Expression of medial class
I histocompatibility antigens on RMA-S mutant cells. Int. Immunol. 3:407.
MATURATION OF Qa-1b REQUIRES b2m BUT NOT TAPs
27. Wolf, P. R., and R. G. Cook. 1995. The class Ib molecule Qa-1b forms heterodimers with H-2Ld and a novel 50 kDa glycoprotein encoded centromeric to
I-E beta. J. Exp. Med. 181:657.
28. Zhu, Q., P. Zelinka, T. White, and M. L. Tanzer. 1997. Calreticulin-integrin
bidirectional signaling complex. Biochem. Biophys. Res. Commun. 232:354.
29. Li, S., H-O. Sjogren, U. Hellman, R. F. Pettersson, and P. Wang. 1997. Cloning
and functional characterization of a subunit of the transporter associated with
antigen processing. Proc. Natl. Acad. Sci. USA 94:8708.
30. Ortmann, B., J. Coperman, P. J. Lehner, B. Sadasivan, J. A. Herberg,
A. G. Grandea, S. R. Ridell, R. Tampe, T. Spies, J. Trowsdale, and P. Cresswell.
1997. A critical role for tapasin in the assembly and function of multimeric MHC
class I-TAP complexes. Science 277:1306.
31. Capps, G. G., B. E. Robinson, K. D. Lewis, and M. C. Zuniga. 1993. In vivo
dimeric association of class I MHC heavy chains: possible relevance to class
I-b2-microglobulin dissociation. J. Immunol. 151:159.
32. Kraulis, P. J. 1991. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystal. 24:946.
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