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Department of Pathology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Calnexin binding to many proteins is dependent on the presence of monoglucosylated N-oligosaccharides generated in the ER by trimming of glucose residues from the core glycan by glucosidases I and II (17, 18, 19, 20). Ware et al. (7) and more recent work by Zapun et al. (21) demonstrated by direct binding studies that calnexin can associate with N-oligosaccharides, confirming that calnexin contains a lectin-like binding site. We had previously shown that mutagenesis of the carbohydrate attachment site of A*0201 abolished calnexin binding, suggesting that class I heavy chain binding to calnexin is carbohydrate mediated (8).
In the present study, we asked whether the structural basis for the observed difference in calnexin binding between mouse and human class I molecules is due to differing number and location of N-oligosaccharides. Human class I heavy chains contain a single glycosylation site at position 86, whereas mouse heavy chains contain either two or three sites, at positions 86 and 176, and in the latter subset, also at position 256 (reviewed in 22 . We hypothesized that additional N-glycans may allow for simultaneous binding of multiple calnexin molecules or alternatively could strengthen binding of a single calnexin molecule by providing additional attachment sites for lectin binding. To test these possibilities, we introduced a N-glycan acceptor site at position 176 of HLA-A2 by substitution of Asn for Arg. In addition, a mutant molecule bearing a single glycosylation site at position 176 was generated. The resulting class I heavy chain mutants were then characterized for intracellular stability, assembly with ß2m, and association with calnexin and calreticulin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The B cell lines CIR (HLA-A negative, HLA-B*3503 low, HLA-C*0401 normal) (23) and Daudi (ß2m negative) (24) were grown in RPMI 1640 (Irvine Scientific, Irvine, CA) containing 10% transferrin-supplemented bovine calf serum (HyClone, Logan, UT). Castanospermine was purchased from Genzyme (Cambridge, MA). Protein A-Sepharose beads, formalin-fixed Staphylococcus aureus Cowan I strain (10% suspension), and fluorescein isothiocyanate-conjugated goat anti-mouse Ab were purchased from Sigma Chemical Co. (St. Louis, MO). Trans35S-label ([35S]methionine and [35S]cysteine) was from ICN (Costa Mesa, CA). Methionine-free RPMI 1640 medium was obtained from Life Technologies (Gaithersburg, MD). Endoglycosidase H (Endo H) was purchased from New England Biolabs (Beverly, MA).
Antibodies
mAb W6/32 (American Type Culture Collection, Rockville, MD) recognizes an epitope on all HLA-A, -B, and -C heavy chains dependent on the presence of ß2m, which is not present on free class I heavy chains (25). mAb BB7.2, which binds to HLA-A2 and -A69 complexes (26), and anti-invariant chain Ab PIN1.1 (27) were obtained from Dr. Peter Cresswell (Yale University School of Medicine). Monoclonal Ab AF8 against calnexin (28) was obtained from Dr. Michael Brenner (Harvard Medical School). Antiserum UCSF#2 reacts with the cytoplasmic tail of class I HLA heavy chains and was provided by Drs. Bruce Koppelman and Frances Brodsky (both at University of California, San Francisco). Antisera against calreticulin was purchased from Affinity Bioreagents (Golden, CO). Antisera reactive with human ß2m and human IgG were purchased from Sigma Chemical Co.
Site-directed mutagenesis and transfections
A cDNA clone encoding HLA-A*0201 was subcloned into the HindIII and SalI sites of M13 mp18 (29). A mutant with substitution of Ser to Ala at position 88 (S88A) was constructed previously (8). Mutant A*0201 with a second N-glycosylation site at position 176 (176dg) was generated by substituting Arg with Asn at position 176, using the site-directed mutagenesis method of Kunkel (30). A mutant with a single N-glycosylation site at position 176 (176g) was generated by a second round of mutagenesis using the S88A mutant. Sequences of the mutants were confirmed by dideoxy sequencing. SalI- and HindIII-purified inserts were then subcloned into the vector pREP10 (Invitrogen, San Diego, CA). Stable transfectants were generated by electroporation followed by selection of cultures in 300 µg/ml hygromycin (Sigma Chemical Co.).
Metabolic labeling, immunoprecipitation, and gel electrophoresis
Cells were washed in deficient medium (methionine-free RPMI
1640) and then incubated at 37°C for 1 h in the presence or
absence of castanospermine. Cells were labeled with 150 µCi of
Trans35S-label for the times indicated. For pulse
chase experiments, 10 vol of nonradioactive RPMI 1640 containing 10%
bovine serum were added at the end of labeling, and aliquots were
removed at times indicated and stored on ice. After one wash in
Dulbecco’s PBS (0.2 g/ml KCl, 0.2 g/ml KH2PO4,
0.047 g/ml anhydrous MgCl2, 8 g/ml NaCl2, 1.15
g/ml Na2HPO4, pH 7.4), cells were lysed at
4°C in Tris-buffered saline (TBS) containing 2% (w/v)
(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 0.1
mM sodium-p-tosyl-L-lysine chloromethyl ketone
(CHAPS), and 1 mM PMSF for 20 min on ice. Lysates were centrifuged at
13,000 x g for 5 min, and postnuclear lysate was
precleared at 4°C with 1 µl of rabbit 
-human IgG and 30 µl of
10% suspension of formalin-fixed Staph A for 90 min. Aliquots were
incubated with 1 µl of antiserum or 50 µl of hybridoma supernatant
for 60 min at 4°C. Ag-Ab complexes were isolated with 20 µl of 50%
suspension of protein A-Sepharose beads. After 4 washes in 0.5%
CHAPS-TBS, beads were boiled in reducing gel buffer for 10 min at
95°C to elute proteins. In some experiments (noted in text), proteins
were eluted from beads with 2% Triton-TBS for 2 h to disrupt
interactions between calnexin and associated proteins. Released
proteins were then isolated using specific Abs as before. Samples were
separated on 12% slab gels and fluorography and scanning densitometry
performed as described.
Endoglycosidase H treatment
Proteins were eluted from beads by boiling in 1% SDS for 10 min. Samples were diluted in PBS that had been adjusted to pH 5.75 with citric acid, divided into aliquots, and either mock treated or treated with Endo H for 16 h at 37°C. Samples were then precipitated in 70% ethanol and resuspended in reducing gel buffer and separated by SDS-PAGE.
In vitro assembly of class I heavy chains with ß2m
Radiolabeled cells were lysed in CHAPS-TBS containing 10 µg/ml human ß2m (Sigma Chemical Co.). Formalin-fixed S. aureus were added to postnuclear lysates, and samples were incubated at 4°C for 16 h. Abs were then used to isolate specific proteins as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Following mutagenesis, mutant and wild-type class I cDNA were
transfected into CIR cells and surface expression characterized with
Abs specific for HLA-A2 by flow cytometry. The mutant bearing two
glycans (176dg) was consistently expressed at between 70 and 80% of
wild-type levels in multiple experiments (n>5) using independently
transfected cell lines (data not shown). To examine the ability of the
introduced site at position 176 to accept N-glycan, cells
were radiolabeled with [35S]methionine, class I proteins
isolated with class I-specific Ab UCSF#2 and subjected to Endo H
digestion, followed by SDS-PAGE. Figure 1
shows that glycosylated 176dg migrates more slowly than the wild-type
protein, consistent with the presence of two glycans. Following
digestion with 10 mU Endo H, wild-type and mutant proteins migrate at
an identical position, as expected following removal of glycans.
Digestion of the mutant with a lower concentration of Endo H (6 mU)
generated a band that comigrates with undigested wild-type heavy chains
and presumably corresponds to the mutant heavy chain with one or both
of the glycans removed.
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To test whether the presence of a second glycan strengthens
binding of class I heavy chains to calnexin, calnexin-associated
proteins were isolated with anti-calnexin Ab (AF8) from
radiolabeled cells expressing either mutant or wild-type A*0201
proteins. Cells were lysed in CHAPS detergent for this experiment to
maintain the association between calnexin and ligands, as previously
described. Following immunoprecipitation with AF8, calnexin-associated
material was eluted from protein A beads with 2% Triton X-100 and
individual proteins then reisolated with specific Abs reactive with
class I heavy chains (UCSF#2), invariant chain (PIN1), and
ß2m (anti-ß2m). Figure 2 shows that wild-type and mutant heavy
chains can be isolated from lysates with Abs reactive with either
UCSF#2 or anti-ß2m. Following elution from
AF8-protein A beads, both wild-type and mutant class I heavy chains
were isolated with UCSF#2, whereas only the mutant could be isolated
with anti-ß2m. The amount of mutant heavy chain bound
by UCSF#2 was greater than for wild-type in most experiments, when
binding of the invariant chain molecule to calnexin was used as an
internal standard for scanning densitometry (Table I). This suggests that the presence of
the second glycan strengthens binding to calnexin. This was not due to
differences in radiolabeling efficiency between cell lines (data not
shown). In addition, the amount of invariant chain associated with
calnexin was similar amounts in these samples (Fig. 2A).
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We considered whether the presence of a glycan at position 176
could by itself increase calnexin binding, or whether both glycans
needed to be present. To distinguish between these possibilities, a
mutant was generated with a single glycan acceptor site at position
176. This mutant was glycosylated and migrated at the same position as
wild-type class I on SDS-PAGE (data not shown). Figure 2
B
and Table I show that following AF8 immunoprecipitation, mutant heavy
chains could be readily isolated with UCSF#2, but only in small amounts
with anti-ß2m. Thus, the mutant with a single glycan
at position 176 appears to bind calnexin rather weakly, similar to
wild-type A*0201, and in contrast to the doubly glycosylated mutant
does not include ß2m in the heavy chain-calnexin
complex.
Binding of calnexin to 176dg depends on trimming of glucose residues from N-glycans
It seemed possible that the presence of two glycans attached to 176dg could cause it to misfold, perhaps resulting in strong calnexin binding through determinants exposed in protein portions of the heavy chain. There is a relatively small group of nonglycosylated proteins that associate with calnexin, and it has been shown that calnexin binding to some class I MHC proteins does not require glycan (31). It was thus important to determine whether calnexin binding to 176dg is dependent on glycan, as is the wild-type A*0201 molecule. This was tested using castanospermine, an inhibitor of glucosidases I and II, which blocks trimming of the two outermost glucose residues from the core N-glycan and prevents generation of the monoglucosylated carbohydrate ligand recognized by calnexin.
Figure 3A demonstrates that
binding of 176dg to calnexin is blocked by concentrations of
castanospermine >100 µg/ml, confirming the involvement of
N-glycan. At a concentration of 50 µg/ml, however, a
calnexin-associated band of intermediate mobility is seen which
represents a population of heavy chains bearing one trimmed and one
untrimmed glycan. Relatively strong binding of calnexin to invariant
chain following treatment is also seen, consistent with other reports
(15, 32, 33). In Figure 3B, calnexin binding to the mutant
bearing a single glycan at position 176 is shown also to be dependent
on glucose trimming, as previously demonstrated for wild-type class I
HLA molecules. Together, these results suggest that calnexin binding
can be directly mediated by glycans attached to either position 86 or
position 176 and are inconsistent with misfolding of either the 176dg
or the 176g proteins promoting calnexin association.
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Pulse-chase analyses were conducted to determine the efficiency of
176dg assembly with ß2m and subsequent transport
through the Golgi (Fig. 4). Following a
short period of radiolabeling, cells were incubated for the indicated
times in nonradioactive medium before lysis and with use of specific
Abs to isolate total class I heavy chains (UCSF#2) or
ß2m-associated class I heavy chains (w6/32). In Figure 4A, it is apparent that a proportion of the 176dg has
decreased mobility and is heterogeneous, characteristic of addition of
sialic acid residues to N-glycans. Molecules within this
subpopulation are evident in increasing amounts 30 min after synthesis
(lane 3, Fig. 4A), and are Endo H
resistant (lanes 7–10 and
15-20, Fig. 4B), indicating
that they have passed through the Golgi. UCSF#2 and w6/32, but not AF8,
react with these molecules, suggesting that they are associated with
ß2m, and not calnexin. A second subpopulation, which
remained sensitive to Endo H for at least 2 h (Fig. 4B), was also observed and represented a major
proportion of the total heavy chain even at later time points (Fig. 4A). Failure of these molecules to become resistant
to Endo H suggests that they are not transported through the Golgi,
which is typically due to a failure in assembly with ß2m.
It was unexpected that this latter subpopulation reacted with w6/32 and
thus appeared to be associated with ß2m (Fig. 4B). In contrast, wild-type A*0201 molecules in CIR
cells pass through the Golgi within 15 to 30 min after assembly with
ß2m and do not remain sensitive to Endo H for long
periods. Together, these experiments demonstrate that 176dg molecules
are heterogeneous in phenotype, with a proportion assembling normally
and passing rapidly through the Golgi, while the rest remain in the ER
in a transport-incompetent state.
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One potential explanation for the apparent uncoupling of
ß2m assembly and transport competence in a
subpopulation of class I molecules described above is that the presence
of the second glycan on 176dg imposes a conformation that allows
binding of Ab w6/32 independently of ß2m interaction.
This might be a direct consequence of mutation at position 176,
resulting in structural alteration of the protein, or may be more
indirectly due to strengthened interaction of 176dg with calnexin. To
test whether w6/32 binding to the mutant occurs in the absence of
ß2m, the 176dg construct was transfected into Daudi
cells. Following radiolabeling, cells were lysed using CHAPS and
incubated for 16 h with or without exogenously added
ß2m; then class I proteins were isolated with w6/32 or
UCSF#2, and SDS-PAGE analysis was performed (Fig. 5). In samples to which no
ß2m has been added, there is significant reactivity of
w6/32 with 176dg, which contrasts with the endogenous class I heavy
chains found in Daudi. Addition of ß2m during the
incubation period results in increased binding of w6/32 to the
endogenous Daudi heavy chains, but there is no additional increase in
binding observed for 176dg. Pretreatment of cells with castanospermine
prevented the ß2m-induced increase in w6/32 binding,
suggesting that calnexin is required to maintain a conformation
receptive to ß2m (data not shown). These results
demonstrate that the epitope recognized by w6/32 is not absolutely
dependent on ß2m, but can be achieved by 176dg following
association with calnexin.
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Calreticulin binding to 176dg is reduced relative to wild-type A*0201 protein
After assembly with ß2m, class I HLA heavy
chains associate with calreticulin, a chaperone with structural and
functional similarities to calnexin (11, 12, 34). Like calnexin,
binding of calreticulin to many ligands requires glucose trimming to
generate the appropriate carbohydrate structure. We tested whether the
presence of two N-glycans on 176dg affected binding to
calreticulin. Figure 6 shows that 176dg
associates with calreticulin, but to a lesser extent than the wild-type
A*0201 molecule. Quantitation of data from a separate experiment is
shown in Table II, demonstrating a marked
reduction in the percentage of ß2m-associated 176dg heavy
chains bound to calreticulin. In contrast to 176dg, A*0201 molecules
with a single N-glycan at position 86 (wild-type) or 176
(176g mutant) bound more strongly to calreticulin and, as shown
earlier, interacted weakly with calnexin. Furthermore, interaction of
176dg with TAP was reduced, consistent with previous suggestions that
calreticulin is required to mediate binding of TAP and tapasin (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Calnexin associates with human class
I heavy chains via the single N-glycan attached at position
86 and is then displaced when ß2m binds to a sterically
overlapping site. In mouse class I molecules, the presence of two or
three N-oligosaccharides allows binding of multiple calnexin
molecules, possibly one to each glycan. ß2m displaces
only calnexin bound to the glycan at position 86, while leaving
intact calnexin attached at positions 176 or 256, which are relatively
distant from position 86. After binding of ß2m, class I
heavy chains bearing one or more N-oligosaccharides would
associate with calreticulin, possibly via the position 86 glycan. This
model would predict that the binding stoichiometry of calnexin to class
I heavy chains bearing more than one glycan could change following
interaction with ß2m and that calreticulin and calnexin
might jointly associate with such a class I molecule. We are currently
testing these possibilities.
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Structural requirements for recognition of ligands by calnexin and calreticulin are presently unclear, with widely disparate results reported in the literature. Several groups have reported that human class I heavy chains dissociate from calnexin either before or during binding to ß2m (8, 14, 15, 16), while others have detected association of calnexin with human class I heavy chain-ß2m dimers (9, 13). These differences may be due to the type of assay or Abs used to detect this association, as previously suggested. The use of mAb AF8 to isolate calnexin-class I complexes has been criticized as potentially disrupting a pre-existing complex of calnexin, class I HLA heavy chain, and ß2m, causing the release of ß2m from the complex. This would presumably occur only with human class I molecules, since we and others have shown that complexes including human calnexin, human ß2m, and mouse class I heavy chains can be readily detected using this assay (16) (our unpublished data). The present results argue against this possibility, since complexes containing human 176dg heavy chains in association with ß2m and calnexin were detected using Ab AF8.
In addition, we have demonstrated a requirement for N-glycans present on class I heavy chains to mediate calnexin association (8), while others have seen no requirement for glycosylation (32). It is not clear how to explain these differences. The results in this study clearly show that N-glycans can influence interactions of class I molecules with calnexin and calreticulin and are consistent with our previous demonstration that N-glycan of A*0201 is required for calnexin binding. We have also shown that calreticulin does not bind to aglycosylated A*0201, suggesting that the lectin-like components of the two chaperones function similarly (our unpublished data).
Data obtained by comparison of ß2m-deficient and positive
cell lines suggests that calnexin and calreticulin bind mutually
exclusive subsets of class I HLA proteins (11, 34), and other studies
suggest that both chaperones require the presence of monoglucosylated
N-oligosaccharide (7, 8, 9, 10, 17, 18, 19, 20, 21). This can be interpreted
as evidence that distinct peptide determinants are recognized by the
two chaperones in addition to the shared carbohydrate ligand.
Alternatively, it may be that accessibility of
N-oligosaccharides change as proteins fold and assemble with
subunits and that following ß2m binding only calreticulin
has access to N-oligosaccharides on class I heavy chains, as
suggested for other proteins (35). The model for class I MHC
biosynthesis proposed in Figure 7 suggesting that ß2m
sterically hinders binding of calnexin, but not calreticulin, to
position 86 glycan is consistent with either of these
possibilities.
The observation that Ab w6/32 can recognize non-ß2m-associated 176dg molecules in Daudi cells demonstrates that the presence of two N-oligosaccharides has a significant effect on the conformation of the class I heavy chain. We know of no report of w6/32 binding to class I heavy chains without ß2m, which was previously interpreted as evidence that the epitope is formed only after assembly of class I dimers (25). Our data suggest that stronger binding of calnexin to class I heavy chains bearing a second glycan results in a conformation recognized by w6/32, which is usually obtained only after association with ß2m. Further maturation of these class I molecules is not seen, however, probably due to an absolute requirement for ß2m in providing longer term stability in folding or in facilitating binding to TAP. In support of the latter possibility, ß2m has been shown to bind directly to TAP and perhaps to directly mediate association of unassembled class I heavy chains with TAP (13).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Russell D. Salter, W957 Biomedical Science Tower, 200 Lothrop Street, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213. E-mail address:
3 Abbreviations used in this paper: ER, endoplasmic reticulum; ß2m, ß2-microglobulin; TAP, transporter of antigenic peptides; CHAPS, (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), 0.1 mM sodium-p-tosyl-L-lysine chloromethyl ketone; TBS, Tris-buffered saline (0.01 M Tris, 0.15 M NaCl, pH 7.4); Endo H, endoglycosidase H; 176dg, mutant with a second N-glycosylation site at position 176; 176g, mutant with a single N-glycosylation site at position 176.
Received for publication June 10, 1997. Accepted for publication September 26, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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