(Received for publication, September 27, 1994; and in revised form, November 22, 1994)
From the
Molecular chaperones are believed to retain misfolded and
incompletely assembled oligomeric proteins in the endoplasmic reticulum
(ER). Here, we have further analyzed the association of one such
chaperone, calnexin, with human major histocompatibility complex class
II and
subunits and the invariant chain. Calnexin
associates with transport-competent invariant chain trimers (p33 or
p41), as well as ER-retained trimers (p35/33 or p43/41), suggesting
that ER retention of the latter is not because of calnexin association.
Neither the replacement of the transmembrane segment of the DR
subunit with a glycosyl phosphatidylinositol anchor nor deglycosylation
of any of these proteins with tunicamycin or endoglycosidase H
treatment abolished calnexin association. Using a cell-permeabilization
system, we were unable to observe association of newly synthesized
glycopeptides with calnexin, arguing that calnexin may not act like a
simple lectin for association with glycoproteins. The results indicate
that neither transmembrane regions nor N-linked glycans are
exclusively responsible for calnexin association. Based on our data and
the observations of others, we suggest that these features may have
varying significance for different glycoproteins in determining their
interaction with calnexin.
Proteins synthesized within the endoplasmic reticulum (ER) ()undergo folding, assembly, and a variety of modifications
before they are secreted or transported to various compartments within
cells. Proper conformational maturation of such proteins is vital for
their function (1, 2, 3) and is facilitated
by an oxidative environment, favoring disulfide bond formation, and the
presence in the ER of molecular
chaperones(4, 5, 6) . Misfolded proteins and
incompletely assembled oligomeric proteins are often retained and
degraded in the ER(4) . Several monomeric and multimeric
proteins in the ER have been shown to transiently associate with
resident ER proteins, such as BiP/GRP78 and GRP94, collectively known
as chaperones (reviewed in (6) and (7) ). Association
of different proteins with such chaperones is transient, and their
dissociation coincides with the folding, assembly, and transport of
proteins(8) . The chaperones are believed to exert a control on
the interactions within and between polypeptides, thus limiting the
formation of incorrect structures. Moreover, they are thought to retain
improperly folded and assembled proteins in the ER. One of the newly
emerging chaperones, calnexin (also known as IP90 or p88), has been
found in association with unassembled Ig, T cell receptor, and major
histocompatibility complex (MHC) class I and class II
subunits(9, 10, 11, 12, 13, 14) ,
newly synthesized viral glycoproteins(15) , and monomeric
secretory glycoproteins(16) . Calnexin is a nonglycosylated
type I integral membrane protein with an apparent molecular mass of 90
kDa by SDS-PAGE(10, 11, 12, 17) .
Recently, we reported the association of calnexin with MHC class II
and
chains, the associated invariant chain, and partially
assembled
-invariant chain complexes(14) . There are
four forms of invariant chain (p33, p35, p41, and p43) in humans
generated by the alternative initiation of translation (p33 versus p35 and p41 versus p43) and alternative splicing (p33 versus p41 and p35 versus p43)(18, 19) . In the absence of class II, p33
and p41 forms, which lack the N-terminal 16 residues, exit the ER,
whereas the longer forms (p35 and p43) are
retained(20, 21, 22) . Similarly, class II
and
chains expressed alone remain in the ER. In normal
circumstances, cells expressing all four isoforms of invariant chain
generate mixed trimers that are retained in the
ER(22, 23) . Each invariant chain subunit is thought
to bind to one MHC class II
dimer, forming a
transport-efficient nonamer complex through a series of
intermediates(24, 25) . Addition of the
and
chains is believed to mask the invariant chain ER retention
signal, resulting in transport of the complex through the Golgi
apparatus into the endocytic pathway. Calnexin associates with these
chains individually and remains associated until the complete nonamer
is formed(14) .
Chaperones associate with a variety of newly
synthesized proteins perhaps by recognizing some common feature(s).
Recently, BiP has been shown to preferentially bind peptides containing
a subset of aromatic and hydrophobic amino acids in alternating
positions(26) . Attempts have been made to find the feature
that is recognized by calnexin. Using various truncated mutants of MHC
class I heavy chain, Margolese et al.(27) showed that
replacement of the transmembrane segment and cytoplasmic domains of
class I heavy chain with a glycosyl phosphatidylinositol anchor
resulted in loss of calnexin association. However, the same group
observed that a soluble, truncated mutant of T-cell antigen receptor
(TCR) chain lacking a transmembrane region retained a trace level
of association with calnexin. Ou et al.(16) reported
that preventing the glycosylation of secretory glycoproteins by
tunicamycin treatment of cells abolished most calnexin association.
Recently, Hammond et al.(15) reported that altering
the glycosylation state of influenza hemagglutinin and vesicular
stomatitis virus G protein, either by tunicamycin treatment or by
inhibiting the trimming of N-linked glycans by glucosidase
inhibitors, resulted in complete loss of calnexin association,
suggesting that calnexin may interact with the N-linked
carbohydrates of newly synthesized glycoproteins. Here, we have
analyzed this question using MHC class II
and
subunits and
the associated invariant chain and report that neither the
transmembrane region nor N-linked glycosylation of these
proteins is exclusively responsible for their association with
calnexin. However, both play a significant role. Based on our data and
existing information in the literature, we propose that various
features on glycoproteins are recognized by the calnexin. The
transmembrane region, glycosylation state, or unidentified feature(s)
may have varying significance for different proteins in establishing a
detectable interaction, explaining the conflicting observations with
different proteins.
Figure 1:
Association of invariant chain isoforms
with calnexin. HeLa cells transiently expressing invariant chain
isoforms (p33, p35/33, p41, p43/41) were pulsed with
[S]methionine for 30 min. The cells were
extracted in 1% digitonin/TS and immunoprecipitated with anti-calnexin (lanes1 and 3) or anti-invariant chain (lane2) antibody. Proteins were eluted from the
protein G-Sepharose beads with TS, 2% SDS, and 2 mM DTT,
diluted with TS, 1% Triton X-100, and 10 mM IAA, and
reprecipitated with control (lane1) or
conformation-independent anti-invariant chain antibodies (lanes2 and 3). Precipitates were subjected to
SDS-PAGE (10.5%) under reducing conditions. The position of molecular
mass markers (kDa) are indicated on the left. Discrete bands
representing p43 and p41 are not seen due to lack of resolution in
10.5% gels.
Figure 2:
Association of calnexin with invariant
chain trimers. T2 cells were pulsed with
[S]methionine for 15 min, and the cells were
extracted in 1% C
E
/BS with 200 µg/ml DSP.
The reaction was quenched with glycine, and proteins were
immunoprecipitated with anti-calnexin (lanes1 and 3) or anti-invariant chain (lanes2 and 4) antibodies. The proteins were stripped from the protein
G-Sepharose beads using 2% SDS and reprecipitated with control (lane1) or anti-invariant chain (lanes2 and 3) or anti-calnexin (lane4) antibodies. Precipitates were subjected to
non-reducing (6%, upperpanel) or reducing (10.5%, lowerpanel) SDS-PAGE. The lower and upperarrow heads indicate positions of
I
and I
-calnexin, respectively. Molecular mass
markers (kDa) are indicated on the left.
We followed
different strategies to determine whether the 180-kDa band is solely
the result of an I-calnexin complex or if it contains some
genuine invariant chain hexamers. Initially, we attempted to preclear
calnexin from cross-linked extracts by sequential immunoprecipitation
using anti-calnexin antibodies. However, we were unable to completely
deplete the sample of the 180-kDa band, perhaps because the antibody
did not recognize all of the calnexin-containing complex (data not
shown). Another approach was based on the observation that
calnexin-invariant chain association is retained in digitonin and not
in C
E
or Triton X-100(14) . Here, we
labeled invariant chain-positive, class II-negative T2 cells for 15 min
and extracted them in 1% digitonin/TS. Proteins were precipitated with
anti-calnexin antibody (Fig. 3, lanes1 and 2) and dissociated from calnexin using
C
E
. They were then cross-linked with DSP.
Dissociated materials were reprecipitated with control (Fig. 3, lane1) or anti-invariant chain (Fig. 3, lane2) antibodies. Samples were analyzed in 6 and
10.5% gels under non-reducing and reducing conditions, respectively.
This procedure yields less material than stripping with SDS. However,
unlike in Fig. 2, only the 90-kDa trimer band and no 180-kDa
band was observed (Fig. 3, lane2) under
non-reducing conditions. This suggests that no hexameric invariant
chain associates with calnexin. In addition, preliminary experiments in
which invariant chain multimers were studied in a calnexin-negative
mutant cell line showed only the 90-kDa trimer band (data not shown).
We therefore believe that the invariant chain does not form genuine
hexamers.
Figure 3:
Dissociation of calnexin from I results in loss of I
-like complex. T2 cells were
pulsed with [
S]methionine for 15 min, and the
cells were extracted in 1% digitonin/TS. Proteins were precipitated
with anti-calnexin antibody; the proteins in their native conformation
dissociated from calnexin using 1% C
E
/BS and
then cross-linked with DSP. Dissociated proteins were reprecipitated
with control (lane1) or anti-invariant chain (lane2) antibodies. Precipitates were subjected to
non-reducing (6%, upperpanel) or reducing (10.5%, lowerpanel) SDS-PAGE. The arrowhead indicates position of I
. The position of molecular
mass markers (kDa) are indicated on the left.
Figure 4:
Association of glycosylated and
deglycosylated proteins with calnexin. HeLa cells transiently
expressing p33 form of invariant chain, chicken hepatic lectin, or
DR or DR
1 chain were untreated or treated with tunicamycin
(10-20 µg/ml) for 2 h before and during labeling (30 min)
with [
S]methionine. Cells were extracted in 1%
digitonin/TS and precipitated with anti-calnexin (lanes1 and 3) antibodies or antibodies specific for the proteins
expressed (lane2). Proteins were eluted from protein
G-Sepharose beads as shown for Fig. 1and reprecipitated with
control antibodies (lane1) or antibodies specific
for the proteins expressed (lanes2 and 3).
Precipitates were subjected to reducing (10.5%) SDS-PAGE. The position
of molecular mass markers (kDa) are indicated on the left.
The experiments shown in Fig. 4showed that
unglycosylated class II MHC proteins do associate with calnexin. We
also performed experiments to show that removal of carbohydrate from
glycoproteins already associated with calnexin does not dissociate the
complex. The results are shown in Fig. 5. Calnexin and
associated glycoproteins, bound to anti-calnexin antibody on Protein
G-Sepharose beads, were treated or mock-treated with the enzyme
endoglycosidase H (leftpanel). Proteins remaining
associated with calnexin after endoglycosidase H treatment or mock
treatment were eluted from the complex and re-precipitated with
specific antibodies (leftpanel). Similarly, we
looked for the specific proteins that might have dissociated from
calnexin in the supernatants of endoglycosidase H-treated samples.
Invariant chain, DR, DR
1, and GPI-DR
1 were found in the
endoglycosidase H-treated pellets (leftpanel). No
released proteins were found in the supernatants (data not shown). The
reduction in apparent molecular weight of the associated proteins in
the endoglycosidase H-treated samples compared with the untreated
samples demonstrates the complete removal of carbohydrate from the
proteins examined. In parallel, proteins in the calnexin-depleted
extracts were precipitated with specific antibodies and treated or
mock-treated with endoglycosidase H. We again looked for expressed
proteins both in the pellet (rightpanel) and
supernatant (data not shown). The majority of the proteins were found
in the pellets. These experiments clearly show that despite removal of
the N-linked glycans, the proteins remain associated with
calnexin. Similar observations have been made by others studying MHC
class I-calnexin association. (
)This is consistent with the
observations shown in Fig. 4and argues that the N-linked glycans of the proteins tested here are not essential
in the association with calnexin. There is a small amount of
endoglycosidase H-undigested GPI-DR
1 both in anti-calnexin (leftpanel) and in specific antibody precipitates (rightpanel), suggesting that in some of the
molecules the N-linked glycans are inaccessible.
Conventionally, endoglycosidase H treatment is performed in the
presence of SDS to ensure complete denaturation and accessibility.
Digestion in a non-denaturing buffer may explain the lack of complete
removal in these particular cases. The reduction in apparent molecular
weight after endoglycosidase H treatment in these experiments and in
experiments conducted under denaturing conditions is comparable (data
not shown). The retention of the calnexin-protein interaction in these
experiments also shows that the presence of digitonin, CHAPS, or
cholate, normally used to retain the association of glycoproteins with
calnexin upon solubilization, is not required to maintain the
association.
Figure 5:
Endoglycosidase H (Endo H)
treatment of calnexin-glycoprotein complexes does not cause their
dissociation. HeLa cells transiently expressing invariant chain
(p43/41) or DR, DR
1, or GPI-DR
1 chain were labeled with
[
S]methionine and extracted in 1% digitonin/TS.
Calnexin and the associated glycoproteins were precipitated with
anti-calnexin antibodies (leftpanel). Expressed
proteins in the calnexin-depleted extracts were precipitated with
specific antibodies (rightpanel). Both sets of
immunoprecipitates on protein G-Sepharose beads were suspended in 20
mM sodium phosphate buffer, pH 6.5, and treated or mock
treated with endoglycosidase H enzyme for 16 h at 37 °C. Presence
of expressed proteins both in the pellet and supernatant of processed
materials were analyzed as described under ``Materials and
Methods.'' The position of molecular mass markers (kDa) are
indicated on the left.
To further strengthen the argument that glycosylation
of proteins is not critical for their association with calnexin, we
followed a different strategy using a permeabilized cell system that we
and others recently reported(39, 40) . A I-labeled nonamer peptide that has an N-linked
glycosylation acceptor site was introduced in the cytoplasm of
streptolysin O-permeabilized Swei cells. The labeled peptides were
translocated into the ER through the transporters associated with
antigen processing (TAP1 and TAP2). Glycosylation of this peptide
allowed its binding with concanavalin A-Sepharose beads (Fig. 6). While concanavalin A-Sepharose beads clearly bound the
radioactive glycopeptide, precipitation of calnexin did not bring down
any detectable associated radioactive glycopeptide (Fig. 6).
This argues against the view that calnexin acts like a simple lectin
for association with glycoproteins. Other groups have made similar
arguments and speculated that the glycosylation machinery, e.g. the oligosaccharyl transferase or the processing glucosidases, may
mediate the interaction between calnexin and
glycoproteins(46) . However, in the above experiments, we
cannot rule out the possibility that trimming of core saccharide unit
on glycopeptides inefficiently generates peptides with monoglucosylated
oligosaccharides, which are suggested to be substrates for calnexin (15) .
Figure 6:
Lack of association of glycopeptides with
calnexin. Swei cells (10) were permeabilized with
streptolysin O and incubated with labeled peptide (50 nM) for
10 min at 37 °C. Cells were washed and extracted in 1% digitonin/TS
and precipitated with concanavalin A (ConA)-Sepharose beads, control (PIN.1), or
anti-calnexin (AF8) antibodies. Radioactivity of the
precipitate was counted in a
counter, and the cpm is shown on the y axis.
Recently, Rajagopalan et al.(42) demonstrated that CD3 chain associates with
full-length or truncated forms of calnexin when co-expressed in COS
cells. Its ER-retention or transport to the Golgi, lysosomes, and cell
surface was shown to be determined by the form of associated calnexin.
Interestingly, CD3
chain is naturally unglycosylated. Similarly,
viral proteins whose glycosylation acceptor sites were mutationally
eliminated have also been found in association with calnexin. (
)Recently, Kearse et al.(45) observed
association of many proteins with calnexin in glucoside II-deficient
mutant cell line and in castanospermine-treated wild-type cells, even
though association of TCR
and
with calnexin was
considerably reduced. Taken together, our results from tunicamycin,
endoglycosidase H, and cell-permeabilization experiments and the
observations of others suggest that the carbohydrate moieties of many
glycoproteins are not essential for calnexin association.
Figure 7:
Association of GPI-DR1 with calnexin.
HeLa cells transiently expressing GPI-DR
1 were treated with
tunicamycin (10 µg/ml) or under control conditions for 2 h before
and during labeling (30 min) with [
S]methionine.
The cells were extracted in 1% digitonin/TS and immunoprecipitated with
anti-calnexin (lanes1 and 3) or anti-class
II
chain (lane2) antibody. Proteins were
eluted from the protein G-Sepharose beads with TS, 2% SDS, and 2 mM DTT, diluted with TS, 1% Triton X-100, and 10 mM IAA, and
reprecipitated with control (lane1) or
conformation-independent anti-class II
antibodies (lanes2 and 3). Precipitates were subjected to
reducing (10.5%) SDS-PAGE. The position of molecular mass markers (kDa)
are indicated on the left.
Deglycosylation of GPI-DR1 by
tunicamycin (Fig. 7, rightpanel) or by
endoglycosidase H digestion (Fig. 5, leftpanel) did not abolish the calnexin association. This
rules out the possibility that the calnexin must recognize both the N-linked glycans and transmembrane segments of glycoproteins
for its association. Removal of either of them individually does not
abolish the association. It is well documented that for many
glycoproteins, N-linked glycans are not critical for achieving
a functional conformation (reviewed in Refs. 47 and 48). Soluble MHC
class II molecules (49) and soluble invariant chain (
)synthesized without glycosylation in a prokaryotic
expression system were found to form heterodimers and homotrimers,
respectively. The MHC class II heterodimers were also able to bind and
present peptides to specific T cells. This suggests that neither their
transmembrane regions nor glycosylation are essential for these
molecules to achieve a functional conformation. Based on our results
and information available from other laboratories, we suggest that
neither N-linked glycans nor the transmembrane segment of
glycoproteins are critical for calnexin association. There are two
possibilities. 1) Calnexin may mediate lectin-like as well as
protein-protein interactions with glycoproteins, and the degree of
dependence on these interactions for detectable association may vary
between proteins. In a similar way, the cell surface receptor CD23 has
been shown to recognize both a sugar moiety and the protein backbone
structure of its ligand, CD21, during binding(50) . 2)
Calnexin, like other chaperones such as GroEL, and members of the Hsp
60 family (51, 52) may recognize an intermediate
structure in the protein-folding process that exposes certain features.
The degree of dependence on glycosylation and on the transmembrane
region for attaining a calnexin-recognizable conformation may vary
between proteins, explaining the conflicting observations with
different glycoproteins.