By
From the Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Unassembled (free) heavy chains appear during two stages of the class I MHC molecule's existence: immediately after translation but before assembly with peptide and 2-microglobulin,
and later, upon disintegration of the heterotrimeric complex. To characterize the structures of
folding and degradation intermediates of the class I heavy chain, three monoclonal antibodies
have been produced that recognize epitopes along the H-2Kb heavy chain which are obscured
upon proper folding and subsequent assembly with
2-microglobulin (KU1: residues 49-54;
KU2: residues 23-30; KU4: residues 193-198). The Kb heavy chain is inserted into the lumen
of the endoplasmic reticulum in an unfolded state reactive with KU1, KU2, and KU4. Shortly after completion of the polypeptide chain, reactivity with KU1, KU2 and KU4 is lost synchronously, suggesting that folding of the class I heavy chain is a rapid, cooperative process. Perturbation of the folding environment in intact cells with the reducing agent dithiothreitol or the
trimming glucosidase inhibitor N-7-oxadecyl-deoxynojirimycin prolongs the presence of
mAb-reactive Kb heavy chains. At the cell surface, a pool of free Kb heavy chains appears after
60-120 min of chase, whose subsequent degradation, but not their initial appearance, is impaired in the presence of concanamycin B, an inhibitor of vacuolar acidification. Thus, free
heavy chains that arise at the cell surface are destroyed after internalization.
Peptides derived from proteins degraded in the cytosol
are presented to the immune system by class I MHC
molecules (1). Assembly of the class I MHC heavy chain, a
type I membrane glycoprotein, with Antibody reagents that have been prepared against class I
molecules can be categorized as (a) folding or assembly independent, (b) folding or assembly dependent, or (c) specific for the free heavy chain. For example, in the case of
the mouse class I molecules, the antiserum p8 (11), raised
against a peptide derived from the cytoplasmic tail of H-2Kb
molecules, recognizes all forms of the H-2Kb heavy chain,
whereas the mAb Y3 (12) binds only to properly conformed states, and the rabbit anti free-heavy chain serum
binds to nonassembled material exclusively (13). Unfolded
forms of class I heavy chain are likely to appear at two
points during the lifetime of the molecule: early in the
course of folding and assembly in the ER, and later, immediately before degradation. To characterize this population
of class I heavy chains in living cells, we have prepared
three mAbs against denatured Kb molecules that recognize
nonassembled heavy chains exclusively. Folding and assembly studies on other glycoproteins such as the mouse class I
molecule H-2Ld (14, 15), or the influenza hemagglutinin
molecule (5), have utilized conformation-dependent antibodies to characterize the structure of biosynthetic intermediates; this study complements those by utilizing antibodies whose epitopes are exposed only when the protein
is unfolded and has not yet associated with Cell Lines.
The Rauscher virus-transformed mouse lymphoma cell line RMA (16) was grown in RPMI-1640 supplemented with 10% fetal calf serum, L-glutamine (2 mM), penicillin
(1:1000 dilution U/ml), streptomycin (100 µg/ml), sodium
pyruvate (1 mM; GIBCO BRL, Gaithersburg, MD), nonessential
amino acids (0.1 mM; GIBCO BRL), and Antibodies.
The following antisera and mAbs were used: Y3
(IgG2b; recognizes the Western Blotting.
107 freshly isolated splenocytes from C57BL/6
(b haplotype), BALB/c (d haplotype), C3H (k haplotype), or PLJ
(u haplotype) mice were lysed directly in either 2× SDS sample
buffer or 1× IEF sample buffer and run on a 12.5% SDS-PAGE
gel, or 1D-IEF gel (18), respectively. Before blotting, the IEF gel
was washed five times (10 min each) with 300 ml of a solution of
50% (vol/vol) methanol, 5 mm Tris-HCl, pH 8.0, and 1% SDS.
The transfer to nitrocellulose was performed in 25 mM Tris base,
200 mM glycine, and 20% methanol for 2.5 h at 400 mA in a Bio
Rad Trans Blot Cell (Bio Rad, Hercules, CA). After a 2 h incubation in blocking buffer (phosphate-buffered saline with 10%
nonfat powdered milk and 0.05% Tween-20), blots were probed
with either Raf HC (1/3000 dilution) or KU2 (5 µg/ml) overnight, washed in PBS/0.05% Tween-20, and probed for 1 h with horseradish peroxidase-conjugated goat anti-rabbit or goat anti- mouse IgG (Southern Biotechnology Assoc., Birmingham, AL)
before visualization with ECL (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
Pulse Chase Analyses and Immunoprecipitations.
RMA cells or
Con A-stimulated splenocytes were starved in RPMI-1640 medium lacking methionine and cysteine for 45 min before being
pulsed with 500 µCi/ml [35S]methionine/cysteine (80:20) for the
times indicated. When included, DTT (5 mM) was added 5 min
before labeling, and the inhibitors N-7-oxadecyl-dNM (7-0-dec;
2 mM) and concanamycin B (Con B; 20 nM) were added during
the starvation period. Labeling was terminated by adding 1 mM cold
methionine/cysteine to the cell suspension. For the short (1-2
min) pulse-chase analyses (see Figs. 3 and 4), aliquots of cells (2-3 × 106) were removed at each timepoint and directly lysed in ice
cold digitonin lysis buffer (0.5% digitonin [Sigma], 25 mM
Hepes, pH 7.2, 10 mM CaCl2, 1 mM PMSF, 10 mM iodoacetamide) containing mAb or antiserum. After centrifugation of the
cell lysates to remove nuclei and cellular debris, immune complexes were isolated after a 2-h incubation (with agitation) at 4°C
by incubation with 50 µl 10% fixed Staphylococcus aureus for an
additional 45 min. The S. aureus pellets were washed once in ice
cold digitonin lysis buffer and then boiled for 10 min in denaturation/reduction buffer (2% SDS, 5 mM dithiothreitol, 50 mM
Tris/HCl, pH 7.8, 1 mM EDTA). After one preclearing step
with normal rabbit serum, the eluted Kb class I heavy chains were
then reimmunoprecipitated in NP-40 lysis buffer (0.5% NP-40,
50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM PMSF, 10 mM
iodoacetamide) with the anti-p8 antiserum before gel analysis.
Epitope Mapping.
The epitopes recognized by the mAbs
KU1, KU2, and KU4 were determined by immunoprecipitation
from a library of M13 phage displaying a random 10-amino acid
insert (19). The library was constructed from a vector encoding
the fd-tet phage and the tetracycline gene. Before immunoprecipitation, the phage suspension (6 × 1010 phage units/0.8 ml
NP-40 lysis mix) was precleared with normal mouse serum (2 µl)
added with a 1:1 suspension of protein A-Sepharose beads for 1 h
at 4°C. Immunoprecipitations were performed by incubating the
precleared phage suspension with 10 µg/ml mAb for 2 h at 4°C, after which the antibody-phage complexes were adsorbed onto
protein A-Sepharose beads, collected by centrifugation, and
washed five times in NET buffer (0.5% NP-40, 50 mM Tris-
HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA). Phage were eluted
from the beads with 200 µl of glycine HCl, pH 2.2, for 15 min at
4°C, neutralized with Tris-HCl (pH 9) to pH 6-8, and then
grown on K91 (kanamycin-resistant) cells cultured in the presence of 20 µg/ml tetracycline. The mAb-selected phage were
further purified through two additional rounds of immunoprecipitation, elution, and amplification, after which clones were picked
and amplified for sequencing.
With the aim of characterizing partially folded or
assembled forms of the Kb class I heavy chain, we raised
three mAbs against denatured Kb molecules: KU1, KU2,
and KU4. The immunogen was injected as urea-solubilized inclusion bodies obtained from E. coli, and presumably contained little coherent structure. Therefore, we expected that
antibodies from this immunization would recognize unstructured epitopes along the Kb polypeptide chain. Thus,
we employed an M13-based phage display library (19) encoding a random 10 amino acid insert to map the epitopes
for each mAb. Three representative inserts are shown for each
antibody (Fig. 1 A), although we sequenced at least 10 clones
from each pool of selected phage to determine the anchor residues for each epitope. Beneath each consensus sequence
is shown the corresponding Kb sequence and the sequences
of other class I heavy chains in the same region (20). The
location of each epitope could be established without ambiguity, and their projection onto the fully assembled Kb
molecule is shown in Fig. 1 B (21). The epitope recognized by KU1 (residues 49-54) is normally folded into a 310 helix
that immediately precedes the long
The results of the epitope mapping described above and
sequence comparisons for class I heavy chains in that region
suggested that KU1 and KU2 would recognize Kb exclusively, and that KU4 would recognize both Kb and Kk molecules. To probe directly the specificity of the antibodies, Western blots were performed on splenocyte extracts prepared from mice of b, d, k, and u haplotype resolved on
SDS-PAGE and 1D-IEF gels (Fig. 2). As a control, we
blotted with the rabbit anti-free heavy serum (Raf HC), which
recognizes most murine class I heavy chains (13). As anticipated, KU2 recognized Kb heavy chains exclusively (Fig.
2 B), whereas Raf HC decorated all of the class I material
present (Fig. 2 A). Neither KU1 nor KU4 blotted as efficiently as KU2, although the specificity for each was as expected based on the epitope mapping (data not shown). Further confirmation of the predicted epitopes for the antibodies was obtained from immunoprecipitations performed
on Kb COOH-terminal truncation fragments translated in
vitro (KU1, KU2, and KU4 all react with translation products containing Kb residues 1-204; only KU1 and KU2
react with fragments containing residues 1-74; data not
shown).
Having mapped the binding sites
for each monoclonal antibody, we next sought to determine at which points these epitopes would be exposed
during the life of the Kb heavy chain. Little if any detectable material could be immunoprecipitated with the antibodies from RMA cells labeled for 60 min (data not shown), suggesting that, while unfolded forms of the heavy
chain might appear transiently during both biogenesis and
degradation, they were too unstable to accumulate to appreciable levels at steady state. We analyzed reactivity of
KU1, KU2, and KU4 on Kb molecules translated in vitro
under conditions that support folding and assembly (22).
We observed that none of the mAbs recognize any We first chose to examine the early events in the assembly of the Kb molecule, by performing a series of brief pulse
chase experiments on RMA cells with pulse times of 1 min in
order to resolve the rapid early stages of Kb folding and calnexin association. To minimize the lag time between cell
lysis and exposure to antibody, aliquots of cells removed from the chase mixture were added directly to lysis buffer
containing the relevant antibody (1° antibody) and immune
complexes were recovered (8). The crude immunoprecipitates were then denatured completely by exposure to 2%
SDS at 100°C, and reimmunoprecipitated with the p8 antiserum, which recognizes all full-length Kb heavy chains. The
kinetics of assembly of the Kb molecule are evident from the
pulse-chase experiment depicted in Fig. 3 B: properly conformed class I complexes (Y3 immunoprecipitates) form
within 5 min, while, conversely, free heavy chains (Raf HC) are present immediately following completion of the 1-min
pulse, but are largely assembled beyond 5 min of chase. KU2
also binds to newly synthesized heavy chains extracted within
the first 5 min of synthesis, suggesting that the Both KU1 and KU4 could also immunoprecipitate newly
synthesized class I heavy chains within the first few minutes
after completion of the polypeptide chain (Fig. 4 A). To
determine whether disulfide bond formation is required for
the folding of the regions surrounding the epitopes recognized by the antibodies, we performed a short pulse-chase
experiment in the presence of the reducing agent dithiothreitol (DTT; 5 mM), which has been shown to prevent
the formation of disulfide bonds in living cells (Fig. 4 B)
(23). Under these conditions, no properly conformed Kb
molecules are detectable, even after 30 min of chase (Y3).
Unassembled Kb heavy chains persist throughout the chase
(RafHC ), and all three monoclonal antibodies show prolonged reactivity, suggesting that, in the absence of proper
disulfide bond formation, the folding of three distinct regions of the class I heavy chain is impeded.
To investigate the role of calnexin/calreticulin in the
early folding events of the class I heavy chain, we performed a short pulse-chase experiment in the presence of the
glucosidase inhibitor 7-O-dec (Fig. 4 C) (24). By blocking
the trimming of terminal glucose residues on the N-linked
oligosaccharides of the class I heavy chains, 7-O-dec effectively prevents the association of calnexin/calreticulin with
the latter (25). The formation of properly conformed class I
complexes (Y3) still occurs in the absence of calnexin/calreticulin binding, consistent with recent studies performed
on the folding of influenza hemagglutinin (5), although assembly of free heavy chains with To follow the fate of free Kb
heavy chains at the cell surface, we performed a pulse-
chase experiment on Con A-stimulated splenocytes (prepared from C57BL/6 mice) with a chase time of 2 h, and
resolved the Raf HC and KU2-reactive class I material on
SDS (Fig. 5 A) and 1D-IEF (Fig. 5 B) gels. The chase was
performed at 26°C as well as 37°C, because at the former
temperature, the stability of class I molecules has been
shown to be markedly enhanced. IEF allows the visualization of sialylated class I heavy chains. Addition of sialic acids to the class I glycan occurs in the trans-Golgi network,
and immediately precedes surface deposition. For the description of the results, we shall equate sialylation with cell surface expression. At the onset of the chase, free heavy
chains are observed exclusively in the ER (compare SDS
and 1D-IEF 0 min timepoints), but appear at the cell surface by 30 min, most likely from disintegration of unstable
class I complexes. Fewer free heavy chains are detectable
during the 26°C chase, consistent with the increased stability of class I complexes at this temperature.
KU2-reactive heavy chains appear in abundance late in
the chase, following the loss of By what means are class I complexes at the cell surface
degraded? If breakdown occurs in lysosomal compartments,
pH neutralization of these compartments might inhibit the
dissociation of the heavy chain from the class I molecule, or
degradation of the latter, or both. To address this question,
we pulse-labeled RMA cells in the presence or absence of
Con B, a fungal macrolide antibiotic that inhibits vacuolar
H+ ATPases (29), and followed the pools of conformed
(Y3), free (Raf HC ), or unfolded (KU2) Kb molecules over
the 3-h chase period (Fig. 6). Owing to the higher background typically seen in immunoprecipitations from briefly pulse-labeled RMA cells, we utilized the direct immunoprecipitation/reimmunoprecipitation methodology as described for the short pulse-chase experiments in Figs. 3 and 4.
This technique also results in improved recovery of nascent
heavy chains that are only reactive with KU2 immediately following their deposition in the ER. Heavy chains that
dissociate at the cell surface in untreated cells are largely degraded after 3 h of chase (Fig. 6 A). In contrast, when cells
are treated with Con B, the destruction of both Raf HC-
and KU2-reactive heavy chains is inhibited. Quantitation
by phosphoimaging (Fig. 6 C ) reveals that the decay of
properly formed complexes (Y3 panel) is similar in the
presence and absence of the inhibitor, suggesting that degradation, but not disintegration, of the class I complex is dependent on lysosomal function. Neuraminidase digestion of intact cells chased in the presence of Con B demonstrates
that after 3 h, about half of the sialylated KU2-reactive
heavy chains are cell surface disposed, while the remainder
resides within the cell, presumably within lysosomes (data
not shown).
The pool of class I molecules within the cell is comprised
of both partial (i.e., monomeric and heterodimeric) and
complete (i.e., heterotrimeric) complexes. Intermediate forms
of the class I molecule are most evident during the initial
folding and assembly processes that occur in the ER, as
well as during the disintegration of the complex at the cell
surface. The three mAbs introduced in this study have allowed us to characterize in part the structure of non- Newly synthesized class I heavy chains interact with the
ER resident chaperones calnexin and calreticulin rapidly after synthesis (30, 3). We show that three distinct epitopes,
recognized by KU1, KU2, and KU4, persist after synthesis for at least 5 min, during which period the heavy chains
are associated with calnexin (Fig. 3). We have been unable
to observe any differential loss of reactivity over time with
the three antibodies in the course of folding and assembly
of the class I heavy chain, in contrast with recent studies
performed on the much larger glycoprotein, influenza hemagglutinin, which have delineated a number of discrete folding intermediates (5). If the calnexin-calreticulin interaction is abrogated by incubation of cells with the glucosidase inhibitor 7-O-dec, then a pool of mAb-reactive heavy
chains reappears later in the chase, demonstrating that, even
though properly conformed molecules are formed in the
absence of calnexin-calreticulin association, many of the
heavy chains do not fold productively, in agreement with
results from the influenza hemagglutinin system (25) as well
as with a recent study on the murine class I molecules H-2Kb
and Db (4). In the presence of the reducing agent DTT, the
folding of the heavy chains is blocked at an early stage, before the packing of the epitopes recognized by each of the
three mAbs. However, since calnexin contains disulfide
bonds that are essential for its function (31), addition of
DTT most likely perturbs the association between the class
I heavy chain and calnexin as well, and thus heavy chains
synthesized under reducing conditions must attempt to fold
both without disulfide bonds and calnexin.
Once the class I molecule has been properly assembled,
it proceeds to the cell surface via the secretory pathway.
However, soon after deposition at the plasma membrane a
fraction of the class I complexes dissociates, as evidenced by
the appearance of sialylated free heavy chains 60-120 min
after synthesis. In contrast with the pool of intracellular free
heavy chains that persist throughout the chase, the cell surface-disposed free heavy chains are also reactive with the
KU2 mAb, and thus are unfolded to a greater extent. One
explanation for this rapid disintegration of a portion of class I
molecules is that complexes that have bound low affinity
peptides might attain a transport-competent structure, but
then lose peptide once they arrive at the cell surface. This
fraction may be stabilized by suitable peptide ligands added
extracellularly (32) (33), but should no peptides be available, the complex is more likely to resolve into the separate subunits. Treatment of cells with Con B, a vacuolar proton
pump inhibitor, prevents the subsequent destruction of sialylated free heavy chains, implying that degradation occurs
intracellularly in an acidic compartment. While generally
difficult to detect, internalization of class I molecules is unlikely to be restricted to free heavy chains, and thus, this
pathway may provide an opportunity for empty class I molecules that are not degraded to bind peptides derived from
exogenous antigens (34).
2-microglobulin (
2m)1
and peptide occurs rapidly after cotranslational insertion of the subunits into the endoplasmic reticulum (2). En route
to heterotrimer formation, the nascent heavy chain transiently associates with the endoplasmic reticulum (ER) resident proteins calnexin and calreticulin, lectins that bind to
and retain incompletely assembled or misfolded proteins in
the ER (3). Inhibition of the binding of calnexin and/or
calreticulin to the nascent murine class I heavy chain decreases the efficiency of assembly of the latter with
2m (4)
in agreement with studies on influenza hemagglutinin maturation, in which newly synthesized hemagglutinin subunits were more likely to aggregate and/or form aberrant
disulfide bonds in the absence of calnexin/calreticulin association (5). After the heavy chain and
2m associate, the
heterodimer binds to the peptide transporter complex
TAP1-TAP2 (6, 7), an interaction that likely facilitates
peptide loading of the empty class I molecule. Properly assembled class I complexes appear rapidly (<5 min) after initial completion of the polypeptide chains (8), and are subsequently transported to the plasma membrane via the secretory
pathway. Once at the cell surface, class I molecules are
largely stable (9), although exchange of bound
2m for bovine
2m present in serum has been observed for both murine and human class I molecules (10). Little if anything is
known about how class I molecules are destroyed after they
have exceeded their useful lifespan at the cell surface.
2m. We show
that these antibodies can be used to immunoprecipitate both assembly and degradation intermediates from pulselabeled cells. Our results suggest that the folding of the extracellular region is a rapid process, in which all of the
epitopes detected by our antibodies disappear synchronously.
-mercaptoethanol (0.1 mM; American Bioanalytical, Natick, MA). Splenocytes from C57BL/6 mice were stimulated in the above medium supplemented with 2.5 µg/ml ConA for 48 h before labeling.
1/
2 domain of properly conformed Kb
molecules; 12), rabbit anti-mouse free heavy chain (Raf HC; recognizes non-
2m-associated mouse class I heavy chains; 13), rabbit anti-p8 (recognizes the cytoplasmic tail of Kb; prepared in our
lab essentially as described 11) and rabbit anti-calnexin (recognizes the COOH-terminus of calnexin; a gift of Dr. D. Williams,
University of Toronto, Canada). The conformation sensitive rabbit anti-H2 serum was provided by Dr. S. Nathenson. The mAbs
introduced in this study (KU1, KU2, and KU4) were generated by
immunizing mice with inclusion bodies of recombinantly expressed Kb heavy chains (a gift of Dr. S. Nathenson, Albert Einstein University, NY). Production of hybridomas was performed as described (17). All three antibodies were purified from cell culture
supernatants with protein A-Sepharose (Repligen Corp., Cambridge, MA), and used in immunoprecipitations at 10 µg/ml lysate.
Fig. 3.
The monoclonal antibodies KU1, KU2, and KU4 are specific for free heavy chains. (A) mRNAs for H-2Kb and mouse 2m were translated
in vitro for 60 min, following which class I molecules were immunoprecipitated with the anti-H-2 antiserum, or the monoclonal antibodies KU1, KU2,
and KU4, before analysis by SDS-PAGE. (B) RMA cells were pulsed for 1 min and chased for the times indicated. Aliquots of cells were lysed directly in
digitonin lysis mix containing either Y3 (class I complexes), Raf HC (free heavy chains), KU2, or
-calnexin. Before SDS-PAGE analysis, the immunoprecipitated class I material was reimmunoprecipitated with p8.
[View Larger Versions of these Images (68 + 11K GIF file)]
Fig. 4.
Effects of redox potential and calnexin association
on the folding and assembly of
the Kb molecule. RMA cells were
pulsed for 1 min and chased for
the times indicated. Nascent
heavy chains were directly immunoprecipitated from NP-40
detergent lysates as in Fig. 3 with
the antibodies indicated in each
panel. The reducing agent DTT
(B) was added to cells at 5 mM 5 min prior to labeling, while the
glucosidase inhibitor 7-O-dec
(C ) was added to cells at 2 mM
during starvation (45 min prior
to labeling).
[View Larger Version of this Image (30K GIF file)]
Fig. 5.
Appearance of unfolded class I heavy chains at the cell surface. Con A-stimulated splenocytes were pulse-labeled for 5 min and
chased at either 37° or 26°C for the times indicated. Free (Raf HC; recognizes both Kb and Db) or KU2-reactive heavy chains (Kb alone) were immunoprecipitated from precleared lysates and resolved on SDS-PAGE
(A) or 1D-IEF (B) gels.
[View Larger Version of this Image (68K GIF file)]
Fig. 6.
Breakdown of unfolded Kb heavy chains at the cell surface. (A) RMA cells were starved for 45 min in the absence or presence of the vacuolar
H+-ATPase inhibitor Con B, pulse-labeled for 5 min, and then chased for the times indicated. Class I heavy chains were directly immunoprecipitated from NP-40 detergent lysates (no initial preclear) with the antibodies indicated in each panel, and reimmunoprecipitated with the p8 antiserum before
SDS-PAGE. (B) Phosphoimager quantitation of the band densities in A. The data sets for each pulse-chase were normalized around the 60 min chase
point densities.
[View Larger Versions of these Images (20 + 46 + 39 + 34K GIF file)]
Epitope Mapping and Specificity of Free Heavy Chain
mAbs.
helix in the
1 domain which forms one side of the peptide-binding groove.
The involvement of four out of five consecutive residues in
generation of the KU1 epitope argues in favor of recognition of this stretch in nonhelical configuration and, conversely, folding of this region should conceal the KU1
epitope. KU2 binds to an epitope that spans part of the second
strand and connecting loop sequence in the
1 domain (residues 23-30); this region contains residues that directly contact
2m (Y27, E32). The KU4 epitope maps to
the first
strand in the
3 domain (residues 193-198) and
is adjacent to residues that contact
2m (R202, W204) as
well as one of the cysteines (C203) that participates in the
3 disulfide bond. Note that the residues recognized by
each antibody, as identified by phage display, are buried or
rearranged upon folding of the heavy chain and assembly with
2m.
Fig. 1.
Epitope mapping of the monoclonal antibodies KU1, KU2,
and KU4. (A) Three representative phage insert sequences selected by each antibody are shown, with translation, above the Kb sequence that is
predicted to contain the epitope. Residues in the phage sequences that
match the actual Kb sequence are shown in capital letters. For comparison, sequences of other class I heavy chains are shown below each predicted epitope. (B) Ribbon diagrams of the properly conformed Kb heavy
chain, highlighting in grey the locations of the predicted epitopes.
[View Larger Versions of these Images (27 + 76 + 62K GIF file)]
Fig. 2.
Western blot analysis of diverse class I material. Splenocyte
extracts from H-2 b, d, k, and u haplotype mice were resolved on SDS-
PAGE or 1D-IEF gels and blotted with either RafHC (A) or KU2 (B).
[View Larger Versions of these Images (56 + 53K GIF file)]
2m
containing forms of heavy chains, whereas the conformation sensitive rabbit anti-H2 antiserum clearly does (Fig. 3
A). The stronger reactivity of KU2, compared with KU1
and KU4, is demonstrated in this experiment, although the
behavior of KU1, KU2, and KU4, in terms of loss of reactivity with Kb heavy chains, appears indistinguishable (see
also Figs. 3 B and 4). Because of the stronger reactivity of
KU2, much of the remainder of the experiments was performed with this antibody.
1 domain
-strand recognized by the antibody remains accessible until the heavy chain has assembled with
2m. Although the
recovery of calnexin-bound heavy chains was inefficient,
notwithstanding the use of digitonin lysis buffer, the peak of
heavy chains coimmunoprecipitated with calnexin occurs around 2-5 min after the 1-min pulse, slightly later than the appearance of the Raf HC and KU2-reactive material. This
observed lag suggests that calnexin may not be stably bound
to the newly synthesized heavy chains until 1-2 min after
completion of the Kb polypeptide chain. Although folding
clearly is an early and rapid process, and is likely to start on
the nascent chain (5), it is completed posttranslationally.
2 is clearly less complete
over the chase period (Raf HC ). All three mAbs react with
class I heavy chains immediately after their synthesis, and by 15 min, much of the reactivity has been lost. However,
unfolded Kb heavy chains reappear by 30 min, most likely
from the disintegration of unstable class I complexes that
were formed in the absence of the quality control normally
provided by calnexin/calreticulin.
2m from unstable class I molecules at the cell surface (compare KU2 immunoprecipitates, SDS and IEF, 120 min timepoints). At the lower temperature, free heavy chains that accumulate at the cell surface
(see Raf HC, 26° chase, IEF ) maintain a conformation that
is not recognized by the KU2 antibody (compare Raf HC
and KU2, 120 min timepoints, 26°C chase). Thus, the free
heavy chain is capable of retaining the conformation of
properly folded molecules, even after
2m dissociation, consistent with studies on the H-2Ld heavy chain (26), as well
as with our previous observations (13). The KU2 antibody
only recognizes a fraction of the free heavy chains that have
been retained in the ER at either temperature (compare
Raf HC and KU2, IEF gels); clearly, most of these free heavy chains are maintained in a conformation or complex
that obscures the epitope. It is unlikely that calnexin is responsible for this effect, as the noncovalent complex between calnexin and the class I heavy chain does not survive
exposure to NP-40 (27). Because Raf HC-reactive Kb
heavy chains persist at timepoints where little reactivity is observed with the mAb KU2, we infer that the latter is the
more stringent tool for monitoring folding, whereas Raf HC
reports on both folding and assembly: association of Kb heavy
chains with
2m obscures the Raf HC reactivity. We note that intact cells can be stained with the mAbs as revealed by cytofluorimetry, and that such staining is reduced for cells
maintained at 26°C (data not shown), a condition known
to prevent unfolding of otherwise labile Kb molecules (28).
2massociated free heavy chains as they appear in the course of
the lifetime of the class I molecule. These reagents recognize epitopes on the class I heavy chains that are buried
upon folding and assembly with
2m, and thus can be used
to monitor the local conformation of the respective subdomains surrounding each epitope.
Address correspondence to Hidde L. Ploegh, Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, 40 Ames Street, E17-322, Cambridge, MA 02139.
Received for publication 28 June 1996
This work was supported by National Institutes of Health grants R01-AI33456-01 and R01-AI07463-17.We would like to thank Dr. D. Williams for the rabbit anti-calnexin antiserum, and Dr. S. Nathenson for the purified soluble H-2Kb and the rabbit anti-H2 antiserum.
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