From the Departments of Immunology and
¶ Biochemistry, University of Toronto,
Toronto, Ontario M5S 1A8, Canada
Received for publication, January 11, 2001, and in revised form, April 27, 2001
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ABSTRACT |
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Calnexin and calreticulin are molecular
chaperones of the endoplasmic reticulum that bind to newly synthesized
glycoproteins in part through a lectin site specific for
monoglucosylated
(Glc1Man7-9GlcNAc2) oligosaccharides. In addition to this lectin-oligosaccharide
interaction, in vitro studies have demonstrated that
calnexin and calreticulin can bind to polypeptide segments of both
glycosylated and nonglycosylated proteins. However, the in
vivo relevance of this latter interaction has been questioned. We
examined whether polypeptide-based interactions occur between calnexin
and its substrates in vivo using the glucosidase inhibitor
castanospermine or glucosidase-deficient cells to prevent the formation
of monoglucosylated oligosaccharides. We show that if care is taken to
preserve weak interactions, the block in lectin-oligosaccharide binding
leads to the loss of some calnexin-substrate complexes, but many others
remain readily detectable. Furthermore, we demonstrate that calnexin is
capable of associating in vivo with a substrate that
completely lacks Asn-linked oligosaccharides. The binding of calnexin
to proteins that lack monoglucosylated oligosaccharides could not be
attributed to nonspecific adsorption nor to its inclusion in protein
aggregates. We conclude that both lectin-oligosaccharide and
polypeptide-based interactions occur between calnexin and diverse
proteins in vivo and that the strength of the latter
interaction varies substantially between protein substrates.
Glycoprotein folding within the endoplasmic reticulum
(ER)1 is facilitated in part
by the membrane-bound chaperone calnexin (CNX) and its soluble
homolog calreticulin (CRT) (1). These proteins are unique among
molecular chaperones in that they utilize a lectin site as a means to
associate with unfolded glycoproteins (2-4). The lectin site is
specific for Glc1Man7-9GlcNAc2 oligosaccharides, which exist transiently as intermediates during the
processing of Asn-linked glycoproteins. It is a widely held view that
the removal and re-addition of the terminal glucose residue of these
oligosaccharides, catalyzed, respectively, by the ER enzymes
glucosidase II and UDP-glucose:glycoprotein glucosyltransferase, regulate cycles of CNX and CRT binding to glycoproteins (5, 6). In this
model, CNX and CRT do not function as classical molecular chaperones
that prevent aggregation by binding to hydrophobic polypeptide
segments. Rather, they are thought to promote folding by recruiting
other chaperones and folding enzymes, such as the thiol oxidoreductase
ERp57 (7, 8), to the glycoprotein substrate.
The concept that CNX and CRT associate with glycoproteins solely
through lectin-oligosaccharide interactions is based primarily on
experiments wherein cultured cells were treated with tunicamycin to block Asn-linked oligosaccharide addition or with the glucosidase I
and II inhibitors castanospermine or deoxynojirimycin to prevent the
conversion of the Glc3Man9GlcNAc2
precursor to the monoglucosylated Glc1Man9GlcNAc2 species. Subsequent
immunoprecipitation with anti-CNX or anti-CRT antibodies frequently
revealed a dramatic reduction in the amounts of various glycoproteins
co-isolating as complexes with these chaperones (2, 9-14). Similar
results were obtained with mutant cell lines that lack the glucosidases
involved in producing the monoglucosylated oligosaccharide (15, 16). In addition, glucosidase inhibitors added after complexes with CNX or CRT
were formed prevented or slowed glycoprotein dissociation, thus
implicating glucosidase activity in the dissociation process (5, 6,
17).
In contrast with the preceding results, many lines of evidence have
suggested that CNX and CRT can also associate with non-native proteins
via protein-protein interactions. First, several studies show that
complexes between CNX and either membrane-bound or soluble glycoproteins cannot be dissociated by enzymatic removal of
oligosaccharides (3, 18, 19). Second, there are several examples of CNX interacting with proteins that either lack Asn-linked oligosaccharides naturally (20) or have lost them through mutagenesis or
under-glycosylation (21-23). Third, after treatment of cells with
castanospermine to block the formation of monoglucosylated
oligosaccharides, CNX or CRT have been detected in association with
specific glycoproteins such as invariant chain (24), CD3 Despite this accumulated information, the concept that CNX and CRT are
capable of associating in vivo with unfolded proteins via
polypeptide-based interactions in addition to lectin-oligosaccharide binding has been largely discounted. It has been speculated that the
lack of dissociation of CNX-substrate complexes after complete deglycosylation may be due to the trapping of the two species within
the same detergent micelle (1, 35) or that the substrate, being
nonnative, might become insoluble upon dissociation (1, 35, 36).
Similarly, it has been suggested that the association of CNX with
nonglycosylated proteins in vivo may arise through nonspecific inclusion of CNX within misfolded protein aggregates (1,
36, 37). However, apart from a single instance in which CNX was
detected in association with aggregates of nonglycosylated vesicular
stomatitis virus G protein (37), there has been no direct evidence to
support such speculations. Finally, the in vitro studies
demonstrating direct binding of nonglycosylated peptides to CRT or the
molecular chaperone functions of CNX and CRT with non-glycoproteins
have been questioned in terms of their relevance to the in
vivo situation (1).
In an effort to address the question of the existence of
polypeptide-based interactions between CNX and its diverse substrates in vivo, we chose to utilize the same methodology used most
commonly in previous studies to demonstrate the apparent exclusivity of lectin-oligosaccharide interactions, i.e. block the
formation of monoglucosylated oligosaccharides and assess by
co-immuno-isolation if complexes between diverse substrates and CNX can
be detected. We reasoned that upon loss of the lectin-oligosaccharide
interaction, any remaining polypeptide-based association might be too
weak to survive rigorous immuno-isolation conditions. Consequently, care was taken to employ mild, yet highly specific isolation
procedures. Using either pharmacologic or genetic methods to block the
formation of the Glc1Man9GlcNAc2
oligosaccharide in diverse cell types, we show that although many
complexes were lost, a large number of CNX-substrate complexes remained
readily detectable. Complementary results were also obtained using a
substrate that lacked oligosaccharides through mutation of its
Asn-X-(Ser/Thr) sequence. Interactions with CNX (and
CRT) were maintained in the absence of any detectable aggregation. We
conclude that in addition to the well established lectin-oligosaccharide interaction, polypeptide-based association does
indeed exist in vivo between CNX or CRT and a diverse array of protein substrates.
Cell Lines and Antibodies--
Murine BW5147 thymoma cells,
their glucosidase II-deficient variant PhaR2.7 (38) (both
provided by Dr. R. Kornfeld, Washington University), and L cells were
grown in Dulbecco's modified Eagle's minimum essential medium. Murine
EL-4 thymoma cells and the human C1R cell line, that stably expresses
the HLA-B27 molecule (39) (provided by Dr. P. Cresswell, Yale
University), were cultured in RPMI 1640. CHO-K1 and its glucosidase
I-deficient variant CHO-Lec23 (40) were obtained from Dr. A. Helenius,
Swiss Federal Institute of Technology, and were grown in
A rabbit antiserum (anti-8) directed against the C terminus of the
H-2Kb H chain, which reacts with all conformational states
of Kb, was provided by Dr. Brian Barber, University of
Toronto. Antiserum UCSF#2 reacts with the cytoplasmic tail of class I
HLA H chains and was provided by Dr. Frances Brodsky, Stanford
University. mAb PIN1.1, which reacts with invariant chain, was obtained
from Dr. Tania Watts, University of Toronto. Two rabbit antisera were used to isolate CNX. One was directed against the C-terminal 14 amino
acids (anti-C-CNX), and the second was raised against the entire
462-residue ER luminal domain (anti-N-CNX). mAb 12CA5, which reacts
with the influenza hemagglutinin (HA) epitope tag on CNX(HA) and the
CNX 1-387(HA), mutant was provided by Dr. Paul Hamel, University of Toronto.
Construction of N-Glycosylation Mutants and Expression in L
Cells--
The N-glycosylation mutants of the
H-2Kb H chain were generated by mutating the consensus
glycosylation sequence, Asn-X-(Ser/Thr), using the
QuikChangeTM site-directed mutagenesis kit (Stratagene) and
full-length H-2Kb cDNA in pcDNA3 (Invitrogen) as
template. To remove the glycosylation site at residue 176, Asn-176 was changed to Lys using the mutagenic oligonucleotide (mutated
base in lowercase) 5'-GC AGA TAT CTG AAG AAC GGG AAg GCG ACG CTG CTG
CGC-3'. The glycosylation site at residue 86 was removed by
substituting Asn-86 with Lys using 5'-C CTG CTC GGC TAC TAC AAg CAG AGC
AAG GGC GGC-3' as the mutagenic oligonucleotide. To add a glycosylation
site at position 256, Tyr-256 was changed to Asn with the mutagenic
oligonucleotide 5'-GGG AAG GAG CAG aAT TAC ACA TGC CAT GTG TAC C-3'.
Recombinant plasmids were introduced into L cells using the
SuperFectTM transfection reagent (Qiagen), and stably
transfected cells were established by G418 selection. Transient
expression of HA-tagged calnexin (CNX(HA) and a truncated ER luminal
segment of calnexin (CNX-(1-387(HA))) in L cells was conducted
as described previously (41).
Metabolic Radiolabeling and Immuno-isolation--
BW5147,
PhaR2.7, CHO-K1, CHO-Lec23, or L cells at a density of
1 × 107 cells/100-mm dish were incubated in Met-free
medium for 60 min at 23 °C to deplete intracellular Met pools. They
were then radiolabeled at 23 °C for the times indicated in the
various figure legends by the addition of 400 µCi/ml
[35S]Met. Castanospermine (CAS), when added, was present
throughout the prelabeling and labeling periods. Cells were lysed for
30 min at 4 °C in 1 ml of lysis buffer containing either 1%
digitonin or 1% CHAPS in PBS, pH 7.4, 10 mM iodoacetamide,
60 µg/ml PefablocR (Roche Molecular Biochemicals), and 10 µg/ml each of leupeptin, antipain, and pepstatin. For isolation of
CNX and associated molecules, lysates were incubated with preimmune or
anti-CNX antibodies for 2 h. Immune complexes were collected for
1 h using protein A-agarose beads and analyzed by SDS-PAGE as
described previously (41). For detection of calnexin-associated
Kb, Db, HLA-B27, and invariant chain, digitonin
lysates were subjected to sequential immunoprecipitation (42). Briefly,
CNX-substrate complexes were recovered with anti-CNX antiserum,
dissociated by heating at 42 °C for 1 h in 0.2% SDS, adjusted
to 2% Nonidet P-40, 5% skim milk, and incubated with anti-class I H
chain or anti-invariant chain antibodies. Immune complexes were
collected and analyzed as above.
Radiolabeling of transfected Drosophila cells with
[35S]Met, lysis, and immuno-isolation was carried out as
described previously (14). Briefly, after induction of the
metallothionein promoter with 1 mM CuSO4 for
16 h, Drosophila cells were incubated for 1 h in
Met-free Schneider's medium in the presence or absence of 1 mM CAS. Cells were then radiolabeled with 0.5 mCi/ml
[35S]Met for 5 min in the presence or absence of CAS and
lysed in digitonin lysis buffer. Lysates were incubated with anti-class I H chain or anti-C-CNX antibodies, and immune complexes were collected
on protein A-agarose followed by SDS-PAGE analysis using 10% gels
(43). Radioactive proteins were visualized by fluorography.
Glycerol Density Gradient Centrifugation--
L cells (1 × 107) expressing wild type or nonglycosylated
H-2Kb were radiolabeled with [35S]Met for 30 min, lysed in 1 ml of 1% digitonin buffer, and centrifuged briefly at
top speed in an Eppendorf microcentrifuge to remove insoluble
material. A 0.5-ml aliquot of lysate was loaded onto a 12-ml, 10-40%
(w/v) linear glycerol gradient prepared in digitonin buffer. The
gradients were centrifuged at 4 °C for 15 h at 35,000 rpm using
a Beckman SW 41 rotor. Fractions (0.75 ml) were collected from the top
of the gradients, and Kb H chains were immuno-isolated from
each fraction using anti-8 antiserum. As a control for the total amount
of Kb molecules loaded onto the gradient, an additional
0.5-ml sample of lysate was also immuno-isolated with anti-8 antiserum.
Immunoblotting--
For detection of H-2Kb-CNX
complexes by immunoblotting, 1 × 107 L cells
expressing wild type Kb or various Kb
glycosylation mutants were lysed in digitonin lysis buffer and immuno-isolated with anti-8 antiserum and protein A-agarose. After SDS-PAGE analysis, proteins were transferred to nitrocellulose membrane
(44), and the membrane was incubated with rabbit anti-N-CNX antiserum
at 1:5,000 dilution followed by donkey anti-rabbit IgG horseradish
peroxidase conjugate at 1:10,000 dilution (Jackson Laboratories).
Immune complexes were visualized using enhanced chemiluminescence
(Amersham Pharmacia Biotech).
Calnexin Associates with Many Substrate Proteins when the Formation
of Monoglucosylated Oligosaccharides Is Blocked--
To establish
whether CNX associates with its substrates only via its lectin site or
whether protein-protein interactions also contribute to this
association, we examined the formation of CNX-substrate complexes in
glucosidase I- or glucosidase II-deficient cell lines and in the
presence of the glucosidase inhibitor, CAS. In an effort to minimize
protein aggregation and to preserve potentially weak protein-protein
interactions, metabolic radiolabeling of cells was conducted at
25 °C, and lysis was performed using the mild detergents digitonin
and CHAPS. Furthermore, immune complexes were washed for the minimum
number of times (typically three) required to preserve CNX-substrate
interactions while minimizing recovery of nonspecifically associated proteins.
Initially, the BW5147 mouse lymphoma cell line and its glucosidase
II-deficient mutant, PhaR2.7, were radiolabeled with
[35S]Met, and digitonin lysates were subjected to
immuno-isolation with two separate anti-CNX antisera. The anti-C-CNX
antibody recognizes the last 14 residues of the cytoplasmic tail of
CNX, and the anti-N-CNX antibody is directed against the entire ER
luminal domain (residues 1-462). As reported previously (15, 16), in
addition to CNX, which appeared as a major band of 90 kDa, a large
number of newly synthesized proteins co-isolated as complexes with CNX
from the parental BW5147 cells (Fig.
1A). A similar pattern of
proteins was observed with the two independent anti-CNX antisera (Fig. 1A, lanes 2 and 3). A substantial
number of these proteins were lost or reduced in intensity in the
glucosidase II-deficient PhaR2.7 cells, reflecting their
apparent requirement for monoglucosylated oligosaccharides for stable
association with CNX. However, it is noteworthy that many other
proteins remained firmly associated with CNX (Fig. 1A,
lanes 5 and 6). A similar result was obtained when parental BW5147 cells were treated with CAS to block glucosidase activity (Fig. 1A, compare lanes 2 and
3 with lane 7). Indeed, the patterns of
CNX-associated proteins were remarkably similar in the
PhaR2.7 and CAS-treated BW5147 cells. It is conceivable
that these proteins arise as a result of nonspecific interactions
either with the precipitating immunoglobulins or with protein A-agarose beads. However, we consider this possibility unlikely since a similar
spectrum of proteins was obtained with the two independently generated
anti-CNX antibodies (containing different arrays of immune globulins),
and they were absent from control isolations performed with preimmune
serum and protein A-agarose beads (Fig. 1A, lanes
1 and 4). To confirm these findings, we also compared glucosidase I-deficient Lec23 cells to their parental CHO cell line
(Fig. 1B). Lec23 cells have been shown to possess little or
no glucosidase I activity, and no monoglucosylated oligosaccharides could be detected on glycoproteins (40). Remarkably, despite the block
in formation of monoglucosylated oligosaccharides, there was no obvious
reduction in the number of CNX-associated proteins recovered with each
antiserum, although some differences in the patterns of recovered
proteins were apparent (Fig. 1B, compare lane 2 with lane 5 and lane 3 with lane 6).
Again, these proteins were absent in control isolations performed with
preimmune antiserum.
To further exclude the possibility that the proteins remaining
associated with CNX in glucosidase-deficient cells or after CAS
treatment were due to nonspecific associations with immune complexes,
HA-epitope tagged CNX (CNX(HA)) and a soluble variant (CNX-(1-387(HA))) were prepared and transfected into mouse L cells. We
showed previously that the CNX-(1-387(HA)) variant fails to form
complexes with newly synthesized proteins (41). The transfectants were
radiolabeled with [35S]Met, lysed in 1% CHAPS, and
subjected to immuno-isolation with anti-HA mAb. As shown in Fig.
1C, lane 1, a large number of newly synthesized
proteins were recovered with the anti-HA mAb from cells expressing
full-length CNX(HA). Consistent with the experiments presented in Fig.
1, panels A and B, many proteins were also
recovered in association with CNX(HA) after treatment of cells with CAS (Fig. 1C, lane 3). In contrast, only trace levels
of proteins were recovered in association with the binding-impaired
CNX-(1-387(HA)) variant (Fig. 1C, lane 2). This
was also the case when the CNX-(1-387(HA)) variant was isolated from
CAS-treated cells (data not shown). That the CNX-(1-387(HA)) variant
was recovered under identical conditions of immune isolation as the
CNX(HA) construct establishes that the CNX-associated proteins
remaining after CAS treatment are indeed bona fide complexes
and not merely proteins nonspecifically adsorbed to anti-HA immune precipitates.
Our finding that CNX remains capable of specific association with many
proteins under conditions where the formation of monoglucosylated oligosaccharides is blocked contrasts with a number of previous studies. For example, Ora and Helenius (16) compared CNX-associated proteins in CHO versus glucosidase I-deficient Lec23 cells
and showed that many fewer proteins associated with CNX in parental CHO
cells than we observed and that none of these could be detected in the
Lec23 mutant cells. To determine if the disparity in results arises
from differences in the radiolabeling and immune isolation methods, we
directly compared CNX-associated proteins in CHO and Lec23 cells using
our mild conditions and those of Ora and Helenius (16). The
major differences in methodology included our radiolabeling of cells at
23 °C versus 37 °C, our use of digitonin
versus CHAPS during cell lysis and washing of immune
complexes, our elimination of a pre-clearance step with fixed
Staphylococcus aureus cells, and our recovery of immune
complexes over a 3-h period versus the overnight isolation
employed by Ora and Helenius (16). The results of this
comparison are depicted in Fig. 1D. Consistent with the
results presented in Fig. 1B, we observed few differences in
the patterns of CNX-associated proteins recovered from CHO cells
relative to glucosidase I-deficient Lec23 cells (compare lanes
2 and 4). In contrast, the procedure of Ora and
Helenius (16) recovered far fewer CNX-associated proteins from
parental CHO cells, and most of these were not detectable in isolates
from Lec23 cells (Fig. 1D, compare lanes 6 and 8). These results demonstrate that the ability to detect
CNX-associated proteins either from wild type cells or from cells
deficient in the ability to produce monoglucosylated oligosaccharides
is highly dependent on the particular conditions chosen for
radiolabeling, cell lysis, and immune isolation.
Differential Effects of Castanospermine on Defined CNX-Glycoprotein
Complexes--
To obtain further insight into the nature of proteins
that remain associated with CNX under conditions that block the
formation of monoglucosylated oligosaccharides, we examined the
interactions of CNX with several defined glycoproteins. Initially,
complexes between CNX and the mouse class I histocompatibility
molecules, H-2Kb and H-2Db, were studied using
the EL4 thymoma cell line. Murine class I molecules consist of three
subunits that assemble within the ER, a ~46-kDa transmembrane heavy
(H) chain that possesses 2 or 3 Asn-linked glycans, the soluble 12-kDa
A different situation was observed when we expressed the Kb
H chain,
Since the murine Kb and Db H chains possess two
or three glycans, respectively, there was the formal possibility that
CAS treatment may not fully block the formation of monoglucosylated
oligosaccharides at all sites, thereby permitting some degree of
interaction with CNX. Consequently, we examined CNX association with
the human class I molecule, HLA-B27, which has only a single glycan at
position 86. C1R cells stably expressing HLA-B27 were treated for
1 h with 1 mM CAS and lysed, and CNX-associated
proteins were isolated with anti-CNX Ab. As shown in Fig.
3A, CAS treatment resulted in
the loss of many but not all complexes of newly synthesized proteins
with CNX. To assess the fate of CNX-HLA-B27 complexes, immune complexes
containing CNX and associated proteins were dissociated, and the
released proteins were subjected to a second round of immune isolation
with anti-HLA antibody. As shown in Fig. 3B, left
panel, equivalent amounts of HLA-B27 H chains were recovered from
CNX complexes in the absence or presence of 0.25 or 1 mM CAS. Furthermore, the CNX-associated H chains from CAS-treated cells
exhibited the reduced mobility indicative of blocked glucose trimming.
This was most apparent at 1 mM CAS; at the lower
concentration of 0.25 mM, the mobility shift was less
pronounced, suggesting an incomplete block in glucose trimming (Fig.
3B, left and center panels). We also
assessed the tendency for HLA-B27 to form insoluble aggregates after
CAS treatment. However, no H chains could be sedimented after
centrifugation at 100,000 × g (Fig. 3B,
right panel). Therefore, for a singly glycosylated
glycoprotein that clearly lacked monoglucosylated oligosaccharide,
interactions with CNX were fully maintained.
We extended these experiments to include an endogenous glycoprotein of
C1R cells, the invariant chain. Invariant chain is the major
substrate of CNX in these cells, and it can be readily observed as an
intense ~35-kDa band in anti-CNX immune isolates (Fig. 3A,
lanes 2 and 3). Invariant chain also appeared to
be present at reduced intensity in anti-CNX immune complexes after 1 mM CAS treatment (Fig. 3A, lanes 5 and 6). To confirm this, immune complexes containing CNX and
associated proteins were isolated from cells treated with 0-1
mM CAS, dissociated, and subjected to re-immuno-isolation
with anti-invariant chain mAb. In this experiment, the bulk of
invariant chain molecules remained associated with CNX after CAS
treatment (Fig. 3C, left panel), and they
exhibited the reduced electrophoretic mobility that accompanies a block in glucose trimming (Fig. 3C, left and
center panels). Invariant chain also did not form insoluble
aggregates after CAS treatment (Fig. 3C, right
panel).
We conclude that for three different glycoproteins, the presence of
monoglucosylated oligosaccharides is not required for association with
CNX. Furthermore, the associations detected in CAS-treated cells are
unlikely to be a consequence of the nonspecific inclusion of CNX in
large glycoprotein aggregates.
Calnexin Associates with Nonglycosylated Class I Molecules
in Vivo--
Although CNX fails to bind to purified
Glc3Man9GlcNAc or
Glc2Man9GlcNAc oligosaccharides in
vitro (3), the possibility remained that the CNX-substrate
complexes observed in CAS-treated or glucosidase-deficient cells could
be mediated through lectin interactions with di- or triglucosylated
oligosaccharides. To address this issue, we tested the association of
CNX with Kb H chains mutated to lack Asn-linked
oligosaccharides. In addition, we examined the consequence of varying
the number of N-linked glycans on CNX binding. The various
Kb glycosylation mutants containing 0, 1, 2, or 3 glycans
are depicted in Fig. 4A. The
wild type Kb H chain and glycosylation mutants were stably
expressed in murine L cells (which contain
The various L cells transfectants were radiolabeled and lysed in
digitonin buffer, and CNX-associated proteins were isolated with
anti-CNX Ab. Kb H chains were then recovered from the
anti-CNX immunoprecipitate in a second round of immune isolation with
anti-H chain antiserum. As demonstrated in Fig. 4C,
left panel, all of the Kb glycosylation mutants
associated with CNX, including the nonglycosylated mutant. The same
result was obtained when Kb H chains were isolated from
nonradiolabeled cell lysates, and the presence of associated CNX was
detected by immunoblotting (Fig. 4C, right
panel). However, a comparison of the relative amounts of
Kb mutants synthesized with the relative amounts recovered
in association with CNX indicated that the nonglycosylated mutant
formed complexes with CNX somewhat less efficiently than the
glycosylated forms (compare Fig. 4, B with C,
left panel). This suggests that the presence of one or more
N-linked glycans increases the stability of the
CNX-Kb interaction.
Because aggregation is a common fate for glycoproteins that have been
treated with tunicamycin to block N-glycosylation or that
have been mutated to lack Asn-linked glycans, it was essential to
determine whether the co-isolation of the nonglycosylated
Kb protein with CNX occurred because of a specific
protein-protein interaction or was due merely to the nonspecific
inclusion of CNX and possibly many other glycoproteins in a large
aggregate of unglycosylated H chains. To test this possibility,
transfectants expressing the wild type Kb H chain or its
nonglycosylated mutant were radiolabeled and lysed in digitonin lysis
buffer, and the lysates were subjected to sedimentation through
glycerol density gradients. Fractions were collected, and
Kb molecules were isolated from each fraction. As shown in
Fig. 4D, the wild type Kb H chain was detected
mainly in fractions 5 and 6. A similar distribution was observed for
the nonglycosylated Kb H chain; there was no evidence of
large aggregates that sediment near the bottom of the gradient. When
CNX-H chain complexes were isolated from fractions with anti-CNX Ab,
the nonglycosylated H chain was again found to sediment primarily in
fractions 5 and 6, indicating that CNX-unglycosylated H chain complexes
are not found in large aggregates (data not shown). Therefore, we
conclude that the association between CNX and nonglycosylated
Kb H chains is a specific polypeptide-based interaction
that can occur independently of lectin-oligosaccharide binding.
Our findings indicate that when care is taken to preserve
weak interactions during metabolic radiolabeling and immune isolation, complexes between CNX and a diverse array of proteins lacking monoglucosylated oligosaccharides can be detected. This was observed when the formation
of Glc1Man7-9GlcNAc2
oligosaccharides was blocked with the glucosidase inhibitor CAS or
through the use of cells lacking either glucosidase I or glucosidase
II. Complexes were not likely due to weak interactions between di- and
triglucosylated oligosaccharides on the glycoprotein substrate and the
CNX lectin site because interactions were maintained even when a
nonglycosylated substrate was tested. Also, CNX does not bind
detectably to these oligosaccharides in vitro (3).
Furthermore, the association of CNX with proteins lacking
monoglucosylated oligosaccharides was not due to the nonspecific
inclusion of CNX into protein aggregates nor was it due to nonspecific
adsorption of proteins onto anti-CNX immune precipitates. Consequently,
we conclude that CNX is capable of associating with diverse proteins
in vivo through polypeptide-based interactions in addition
to its well characterized lectin-oligosaccharide interaction.
It is conceivable that the association between CNX and various proteins
that lack monoglucosylated oligosaccharides is indirect, being mediated
by the thiol oxidoreductase ERp57, which is known to bind to CNX and
CRT. Previous studies have shown that CNX- or CRT-bound ERp57 forms
mixed disulfides with various glycoproteins during their oxidative
folding (49). However, we consider such an indirect association to be
unlikely for three reasons. First, it has been demonstrated that
ERp57-glycoprotein-mixed disulfides fail to form when cells are treated
with CAS (49), the same conditions we use to detect lectin-independent
CNX-substrate complexes. Second, under nonreducing SDS-PAGE conditions
we have consistently been unable to detect covalent complexes between
various defined glycoproteins and ERp57 at a level that could account
for the amount of CNX-associated glycoprotein observed in CAS-treated cells. For example, note that in Fig. 2C there is a strong
CNX band co-isolating with the Db H chain in CAS-treated
cells, but there is no significant band corresponding to a covalent
ERp57-H chain complex (~105 kDa) that could have mediated this
association with CNX. Finally, we and others (30-34) have readily
detected associations between CNX or CRT and nonglycosylated proteins
or peptides in vitro in the complete absence of any added
ERp57. Consequently, we favor the view that the interactions we observe
in vivo between CNX and proteins lacking monoglucosylated
oligosaccharides are directly mediated through a polypeptide binding
site on CNX.
It is noteworthy that the interactions between various proteins and CNX
were affected to markedly different extents when the formation of
monoglucosylated oligosaccharides was blocked. Examination of the
patterns of proteins recovered in anti-CNX immune complexes (Figs. 1,
2A, and 3A) revealed that some proteins were
completely lost, others appeared to be present at reduced levels, and
others seemed unaffected. This situation was also observed when
complexes between CNX and specific glycoproteins were examined. The
murine class I molecules, H-2Kb and H-2Db,
could not be recovered in association with CNX after CAS treatment of
EL4 cells, although some interaction could be detected when overexpressed in Drosophila cells. However, unglycosylated
H-2Kb remained firmly associated with CNX. This likely
reflects different conformational states between the glycosylated and
unglycosylated proteins with the accompanying presentation of different
polypeptide determinants to CNX. In contrast to the glycosylated murine
class I molecules, the human class I molecule, HLA-B27, and the human MHC class II invariant chain were largely unaffected in their interactions with CNX after CAS treatment. Collectively, these findings
suggest that different glycoproteins exhibit different dependencies on
lectin-oligosaccharide interactions for their stable association with
CNX. In other words, in the absence of lectin-oligosaccharide binding,
different proteins exhibit differences in the strength of their
polypeptide-based interactions with CNX.
Consistent with the above in vivo findings,
substrate-specific differences have also been observed when the
interactions of CNX or CRT with unglycosylated protein or peptide
substrates were studied in vitro. For example, when the
ability of CNX to suppress the aggregation of various unfolded proteins
was assessed, equimolar amounts of CNX effectively suppressed the
aggregation of the naturally unglycosylated proteins citrate synthase
and malate dehydrogenase (33). However, more than a 3-fold molar excess
was required to fully suppress the aggregation of enzymatically
deglycosylated soybean agglutinin (33), and a 30-fold molar excess was
only partially effective in preventing the aggregation of enzymatically deglycosylated
Differences in the strength of polypeptide-based interactions between
CNX or CRT and various glycoprotein substrates coupled with variations
in the stringency of isolation conditions can account for many of the
conflicting results reported in the literature concerning the
association of these chaperones with specific glycoproteins after
treatment with glucosidase or glycosylation inhibitors. For example, if
glycosylation is prevented or if glucosidases are inhibited, complexes
are not detected between CNX or CRT and the The preceding survey illustrates how conflicting conclusions can arise
when generalizations are made on the basis of studying the association
of a single glycoprotein with either CNX or CRT or when only a single
type of isolation condition is employed. It is exceedingly unlikely
that all of the examples of complexes between CNX or CRT with
unglycosylated or non-glucose-trimmed proteins can be dismissed on the
basis of their nonspecific inclusion in aggregates, trapping within
detergent micelles, and insolubility of folding intermediates, as has
frequently been claimed (1, 35-37). Indeed, in the present study, we
have eliminated the possibilities of insolubility and aggregation
through the use of high speed sedimentation and glycerol density
gradient ultracentrifugation analyses. Furthermore, it is not possible
to explain our results by the mere "trapping" of membrane proteins
within the same detergent micelles as CNX. Such a phenomenon should be
applicable to all of the specific membrane proteins we studied, and yet
we observed no such trapping of CNX with mouse class I molecules
isolated from CAS-treated EL4 cells (Fig. 2A).
Rather, a view more consistent with the data is that CNX and CRT
possess both a lectin site and a polypeptide binding site and that the
latter binds with varying affinities to polypeptide segments of
different glycoproteins. Such a dual binding model (3) has the
advantages that it accommodates the in vitro demonstrations
of polypeptide interactions between diverse substrates and either CNX
or CRT (32-34) and it evokes the possibility of an enhanced avidity of
chaperone-glycoprotein interactions via contacts through two binding sites.
In fact, a lectin-only type of interaction is rather difficult to
rationalize in light of the stable complexes that occur between CNX or
CRT and various monoglucosylated glycoproteins. Recent studies have
demonstrated that the affinity of CRT for IgG carrying a single
Glc1Man9GlcNAc2 oligosaccharide is
1-2 µM (56). Glycans with dissociation constants greater
than about 1 µM are typically retarded on immobilized
lectin columns rather than binding tightly (57). Such chromatographic
behavior has been documented for the interaction of monoglucosylated
oligosaccharides with immobilized CRT (4) and is consistent with our
observation that Glc1Man9GlcNAc2
bound to immobilized CNX is readily released upon washing (3). An
increased affinity could result if CNX and CRT are oligomeric and
capable of binding to multiple oligosaccharides on a glycoprotein
substrate. Indeed there are several reports of enhanced CNX
interactions when a glycoprotein is converted from a singly to a doubly
glycosylated form (35, 37, 54). This could be due either to oligomeric
CNX or to a bivalent CNX interaction that is induced by the
precipitating anti-CNX antibody. Although we and others provide gel
filtration evidence suggestive of CNX or CRT oligomers (33, 34, 58),
this appears not to be the case. First, no increase in apparent
affinity was observed when CRT was incubated in vitro with
IgG possessing two monoglucosylated oligosaccharides (56). Second,
recent biophysical studies demonstrate that CRT is monomeric and
possesses a highly asymmetric structure that accounts for its anomalous
gel filtration behavior (59). Third, CNX behaves as a monomer, as
evidenced by sedimentation through a sucrose density gradient (60), and
it is highly asymmetric, with a single lectin site as revealed by its
recently solved x-ray crystallographic
structure.3 Finally, using
epitope-tagged variants of CNX or CRT in transfected cells, we have
consistently been unable to co-isolate these proteins as mixed
oligomers with the endogenous
chaperones.4 Despite the weak
lectin-oligosaccharide binding affinity and the apparent lack of CNX or
CRT oligomers, complexes of these chaperones even with singly
glycosylated glycoproteins (such as the human class I molecule,
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit
(25), coagulation factors V and VIII (26), acid phosphatase (27), and
the
subunit of the nicotinic acetylcholine receptor (28). Fourth, both CNX and CRT have been shown to bind specifically to
nonglycosylated peptides both in vitro and in
vivo (29-32). Fifth, using purified components in
vitro, it has been demonstrated that CNX and CRT are capable of
functioning as molecular chaperones to suppress the aggregation and
enhance the folding not only of glycoproteins bearing monoglucosylated
oligosaccharides but of nonglycosylated proteins as well (33, 34).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimum essential medium. Stably transfected
Drosophila melanogaster Schneider cells that express CNX
along with H-2Kb heavy (H) chains in the presence of mouse
2-microglobulin (41) were maintained in Schneider's
insect medium (Sigma). All media were supplemented with 10% fetal
bovine serum and antibiotics.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Association of newly synthesized proteins
with CNX in the absence of glucosidase activity. A,
BW5147 and PhaR2.7 cells were incubated for 1 h at
23 °C in the absence (lanes 1-6) or presence (lane
7) of 1 mM CAS in Met-free medium, radiolabeled at
23 °C for 10 min with [35S]Met, and lysed in 1%
digitonin lysis buffer. Lysates were incubated with preimmune serum
(PI) or with two polyclonal anti-CNX antisera, anti-N CNX
and anti-C CNX, as indicated. Immune complexes were analyzed by
reducing SDS-PAGE. B, CHO-K1 and CHO-Lec23 cells were
radiolabeled and lysed as in A and then immuno-isolated
either with preimmune serum or with anti-N-CNX or anti-C-CNX antisera
as indicated. C, HA-tagged CNX (CNX(HA)) and a truncated ER
luminal segment of CNX (CNX-(1-387(HA))) were transiently expressed in
L cells. The cells were radiolabeled as in A and then lysed
with buffer containing 1% CHAPS. The HA-tagged molecules and
associated proteins were immuno-isolated with mAb 12CA5. D,
lanes 1-4, CHO-K1 and CHO-Lec23 cells were radiolabeled at
23 °C for 10 min with [35S]Met, lysed in 1% digitonin
lysis buffer, and then immuno-isolated either with preimmune serum or
with anti-C-CNX antiserum as indicated (for details see "Experimental
Procedures"). Lanes 5-8, CHO-K1 and CHO-Lec23 cells were
radiolabeled at 37 °C for 10 min with [35S]Met and
lysed in 50 mM Hepes buffer, pH 7.6, containing 0.1 M NaCl, 2% CHAPS, and protease inhibitors as described by
Ora and Helenius (16). Cell lysates were precleared for 30 min with
fixed S. aureus cells and then were immuno-isolated either
with preimmune serum or with anti-C-CNX antiserum overnight as
indicated. Immune complexes were washed three times in 50 mM Hepes buffer, pH 7.6, containing 0.1 M NaCl
and 0.5% CHAPS before SDS-PAGE analysis.
2-microglobulin subunit, and an 8-10-residue peptide
ligand. EL-4 cells were incubated with or without CAS and screened for
proteins associated with CNX by immuno-isolation with anti-CNX
antibodies. As expected, many proteins co-isolated as complexes with
CNX (Fig. 2A, lane 3). After CAS treatment, many of these complexes were lost, but others were preserved (Fig. 2A, lane 4). To
confirm that CAS was effective in inhibiting glucosidase activity in
this and preceding experiments, the H-2Kb and
H-2Db proteins were isolated directly, and their H chain
subunits were shown to exhibit the characteristic decrease in mobility
after CAS treatment that reflects unprocessed N-linked
oligosaccharides (Fig. 2A, compare lanes 5 and
6 and lanes 7 and 8). The interactions of H-2Kb and H-2Db H chains with CNX were
completely blocked after CAS treatment. This was evidenced by the loss
of the intense 46-kDa species in the anti-CNX immunoprecipitate from
CAS-treated cells (Fig. 2A, compare lanes 3 and
4) and also by the absence of H chains after their immune
isolation from solubilized anti-CNX immunoprecipitates (Fig.
2A, compare lanes 9 and 10 and
lanes 11 and 12). Thus, murine class I molecules
in EL4 cells appear to depend extensively on lectin-oligosaccharide
interactions for their stable association with CNX.
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Fig. 2.
Castanospermine inhibits CNX interaction with
Kb H chains in EL4 cells but not in Drosophila
cells. A, EL-4 cells were incubated for 1 h
in the absence or presence of 1 mM CAS as indicated and
then radiolabeled as in Fig. 1. Digitonin lysates were treated either
with preimmune serum (PI; lanes 1 and
2), anti-N CNX antiserum (lanes 3 and
4), anti-8 antiserum to isolate Kb H chains
(lanes 5 and 6), or mAb 28-8-14S to isolate
Db H chains (lanes 7 and 8). To
recover Kb or Db H chains that were associated
with CNX, CNX and associated proteins were first isolated with anti-N
CNX antiserum. Immune complexes were then dissociated in SDS and
subjected to a second round of immune isolation with anti-8 or 28-14-8S
antibodies (lanes 9-12). Isolated proteins were analyzed by
SDS-PAGE. B, Drosophila cells expressing
Kb H chains, mouse 2-microglobulin, and CNX
were incubated for 1 h in the absence or presence of 1 mM CAS, radiolabeled with [35S]Met for 10 min, and lysed in digitonin lysis buffer. Lysates were treated with
anti-C-CNX or anti-8 antiserum as indicated, and immune complexes were
either analyzed directly or after digestion with endoglycosidase H
(Endo H) to remove immature N-linked glycans.
C, Drosophila cells expressing Db H
chains, mouse
2-microglobulin, and CNX were incubated
for 1 h in the absence or presence of 1 mM CAS,
radiolabeled with [35S]Met for 10 min, and lysed in
digitonin lysis buffer. Lysates were treated with 28-14-8S mAb and
immune complexes were heated in sample buffer at 55 °C before
analysis by SDS-PAGE under nonreducing conditions.
2-microglobulin, and CNX in D. melanogaster Schneider cells. These cells possess the glucosidases
and glucosyltransferase required for a functional
deglucosylation-reglucosylation cycle (45-47), but they lack the
specialized transporter associated with antigen processing that
transports peptide ligands for class I molecules from the cytosol to
the lumen of the ER. We showed previously that under these conditions
the H chain assembles with
2-microglobulin but remains
peptide-deficient, and the heterodimer is retained in the ER in
association with CNX (14, 48). In transfected Drosophila
cells, the Kb molecule is the major substrate associated
with CNX, and it can be identified directly in anti-CNX
immuno-isolates. When these cells were incubated in the absence or
presence of CAS, the Kb H chain mobility was reduced in
response to CAS treatment, reflecting a block in glucose trimming (Fig.
2B, lanes 5 and 6). However, when
CNX-Kb complexes were recovered with anti-CNX Ab, a
significant fraction of Kb molecules remained associated
with CNX after CAS treatment (Fig. 2B, lanes 1 and 2), and these molecules possessed the slower
electrophoretic mobility indicative of unprocessed oligosaccharides. To
confirm that the change in mobility of the CNX-associated
Kb H chain after CAS treatment was due to carbohydrate
modification, the immune complexes were digested with endoglycosidase H
to remove Asn-linked oligosaccharides, after which they possessed the
same mobility with or without CAS treatment (Fig. 2B,
lanes 3 and 4). It is noteworthy that
CNX-Kb complexes could also be detected in the absence or
presence of CAS by immuno-isolating Kb molecules and
visualizing the associated CNX band (Fig. 2B, lanes 5 and 6). Similar results were obtained with the
Db H chain (Fig. 2C). It is conceivable that the
altered glycans resulting from CAS treatment promoted aggregation of
Kb and Db molecules and the nonspecific
inclusion of CNX in such aggregates. We showed previously that a mix of
disulfide-linked and non-disulfide-linked aggregates of Kb
or Dd H chains can readily be detected under conditions of
nonreducing SDS-PAGE when samples are heated only to 55 °C (14).
Under these conditions, no aggregated Db molecules could be
detected at the top of the separating gel (Fig. 2C). Thus,
in Drosophila cells, incompletely assembled class I H chains
are capable of associating with CNX in the apparent absence of
monoglucosylated oligosaccharides on the H chain. The difference
relative to EL4 cells is probably related to detection sensitivity
since H chains are the major substrates associated with CNX in
Drosophila cells.
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Fig. 3.
HLA-B27 and invariant chain remain associated
with CNX in CAS-treated C1R-B27 cells. A, C1R-B27 cells
were incubated in the absence or presence of 1 mM CAS and
then were radiolabeled with [35S]Met for 5 min and lysed
in digitonin lysis buffer. CNX and associated proteins were
immuno-isolated either with anti-N-CNX (lanes 2 and
5) or anti-C-CNX (lanes 3 and 6)
antisera. Lanes 1 and 4 depict lysates treated
with preimmune (PI) serum. The mobilities of CNX and
invariant chain are indicated. B, C1R-B27 cells were
incubated with various amounts of CAS as indicated before radiolabeling
and lysis as in panel A. To isolate CNX-associated HLA-B27
molecules, CNX-containing complexes were first recovered with
anti-N-CNX serum, and after dissociation in SDS, HLA-B27 H chains were
isolated with UCSF#2 anti-serum (left panel). In addition,
one-tenth the amount of each lysate was treated directly with UCSF#2
antiserum (center panel). To test for the presence of
insoluble H chain aggregates, radiolabeled cell lysates were
centrifuged at 100,000 × g for 30 min, and HLA-B27 H
chains were immuno-isolated from supernatant (S) and
solubilized pellet (P) fractions (right panel).
C, radiolabeled lysates of C1R-B27 cells were prepared as in
panel B, and CNX-associated invariant chains were recovered
using anti-N-CNX antibody followed by a second round of immune
isolation with the anti-invariant chain mAb PIN1.1 (left
panel). Invariant chains were also isolated directly from
one-tenth the amount of each lysate using mAb PIN1.1 (center
panel). Radiolabled cell lysates were also centrifuged at
100,000 × g as described in panel B to
detect any invariant chain aggregates (right panel)
2-microglobulin and all other components required for
normal assembly of class I molecules). Fig. 4B shows that
the various forms of the Kb H chain were synthesized in L
cells and possessed the electrophoretic mobilities expected based on
their different numbers of oligosaccharide chains. We also established
that the differentially glycosylated H chains associated normally with
2-microglobulin and that the singly, doubly, and triply
glycosylated proteins were all transported from the ER to the Golgi
apparatus at comparable rates (data not shown). In the case of the
nonglycosylated molecule, its stability was similar to wild type
Kb, and it could be detected at the cell surface by flow
cytometry, albeit at lower levels than observed for the wild type
protein (data not shown). Collectively, these findings suggest that
changes in the number of N-glycans do not cause major
misfolding of the Kb molecule.
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Fig. 4.
Association of Kb H chain
glycosylation mutants with CNX. A, the locations of
glycosylation sites in the various Kb H chain glycosylation
mutants are indicated by residue number (CHO
represents the nonglycosylated protein). The Kb H chain is
depicted in black, and the
2-microglobulin
subunit is shown in gray. B and C,
untransfected L cells (lane 1) or L cells expressing wild
type Kb with two glycosylation sites (lane 2),
Kb with a single glycan (lane 3),
nonglycosylated Kb (lane 4), or Kb
with three glycans (lane 5) were radiolabeled with
[35S]Met for 10 min and lysed in digitonin lysis buffer.
Kb H chains were immuno-isolated with anti-8 antiserum
(panel B), and CNX-associated Kb H chains were
recovered by immuno-isolating first with anti-N-CNX
antiserum followed by complex dissociation and isolation of
Kb H chains with anti-8 antiserum (panel C,
left). In panel C, right,
CNX-associated Kb H chains were also detected by isolating
the various Kb glycosylation mutants from lysates of
transfected cells using anti-8 antiserum. After separation of proteins
by reducing SDS-PAGE, proteins were transferred to nitrocellulose
membrane, probed with anti-N-CNX antiserum, and visualized with a
chemiluminescence-based detection system. Lane 1,
untransfected cells; lane 2, wild type Kb with
two glycosylation sites; lane 3, unglycosylated
Kb; lane 4, Kb with a single glycan;
lane 5, Kb with three glycans. Lane 6 represents one-hundredth of the cell lysate used for
immunoprecipitation and serves as a positive control for CNX
detection. D, L cells expressing either wild type or
unglycosylated Kb H chains were radiolabeled and lysed in
digitonin-containing buffer, and the lysates were applied to the top of
a 10-40% glycerol density gradient. After centrifugation for 15 h at 34,000 rpm in an SW41 rotor, the gradients were fractionated, and
Kb H chains were isolated from each fraction with anti-8
antiserum. Isolated proteins were analyzed by reducing SDS-PAGE.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase.2 CNX did not
bind to any of these proteins without their prior denaturation. An
analysis of the binding of 39 different peptides to CRT also revealed
its marked preference for certain peptides. In general, hydrophobic
peptides lacking acidic residues were favored, and there also appeared
to be a minimum length requirement (32). A preference for binding to
hydrophobic peptides is a common characteristic among molecular
chaperone families, and for some chaperones such as the cytosolic Hsp90
and Tric proteins, there are clear preferences for certain protein
substrates over others (50).
and
subunits of the
T cell receptor (51), influenza hemagglutinin (2), vesicular stomatitis
virus G glycoprotein (52), ribonuclease B (35), myeloperoxidase (9),
cruzipain (53), and tyrosinase (13). However, complexes can readily be
detected at normal or reduced levels under conditions of
deglycosylation or glucosidase inhibition with the
and
subunits
of the T cell receptor (20, 25), P glycoprotein (23), erythrocyte
AE1 (54), acid phosphatase (27), major histocompatibility complex class
II-
and -
chains (19), major histocompatibility complex class II
invariant chain (24), major histocompatibility complex class I H chain
(this study), and human immunodeficiency virus gp160 (10). Other
interesting examples of variability include the finding that CAS
treatment almost completely prevented the formation of complexes
between CNX and coagulation factors V and VIII but only partially
inhibited the formation of complexes with CRT (26). Furthermore, CAS
prevented the formation of complexes between CNX and the
subunit of
the acetylcholine receptor in one study (55) but had little effect on
complex formation in another (28). The main difference appeared to be
the use of Triton X-100 for cell lysis in the former study as opposed
to the milder CHAPS detergent in the latter. In addition to these studies that focused on complexes of specific glycoproteins with either
CNX or CRT, variable results have also been reported when the entire
spectrum of CNX- or CRT-associated proteins were examined. In agreement
with our current study, Kearse et al. (51) observe strong
association of many proteins with CNX in the glucosidase II-deficient
PhaR2.7 cell line and in CAS-treated wild type cells even
though associations with T cell receptor-
and -
were virtually
eliminated. In contrast, Helenius and co-workers (12, 16) observe an
almost complete elimination of CNX- or CRT-associated proteins in
PhaR2.7 cells and in CAS-treated wild type cells. Likewise,
in glucosidase I-deficient Lec23 cells, we observed very few changes in
the number of CNX-associated proteins relative to wild type CHO,
whereas Ora and Helenius documented a substantial loss of these
complexes (16). The opposite outcomes of the latter two studies can
clearly be attributed to differences in the radiolabeling, lysis, and immuno-isolation conditions employed (Fig. 1D). The milder
conditions used in our study preserved interactions that were lost
after the more stringent procedures used by Ora and Helenius
(16).
-fetoprotein, and the nicotinic acetylcholine receptor
subunit)
are stable to detergent solubilization and immune isolation. Such
stable associations can most readily be explained by a dual binding
mechanism that encompasses both lectin-oligosaccharide and
polypeptide-based interactions. Further proof of this model must await
the identification of the polypeptide binding sites on CNX and CRT and
the elucidation of their fine binding specificities.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Rosalind Kornfeld, Peter Cresswell, Ari Helenius, Brian Barber, Frances Brodsky, Tania Watts, Ikuo Wada, and Paul Hamel for generous gifts of cell lines and antibodies. We also thank Michael Leach and Dr. Yoichiro Noguchi for helpful comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Studentship from the National Cancer Institute of Canada.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Medical Sciences Bldg., University of Toronto, Toronto, Ontario, Canada M5S 1A8. Tel.: 416-978-2546; Fax: 416-978-8548; E-mail: david.williams@utoronto.ca
Published, JBC Papers in Press, May 3, 2001, DOI 10.1074/jbc.M100270200
2 V. S. Stronge and D. B. Williams, unpublished observations.
3 J. D. Schrag, J. J. M. Bergeron, Y. Li, S. Borisova, M. Hahn, D. Y. Thomas, and M. Cygler, manuscript submitted.
4 U. G. Danilczyk and D. B. Williams, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: ER, endoplasmic reticulum; CAS, castanospermine; CNX, calnexin; CRT, calreticulin; HA, influenza hemagglutinin; H chain, heavy chain of class I histocompatibility molecule; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; mAb, monoclonal antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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