(Received for publication, April 20, 1995; and in revised form, May 12, 1995)
From the
The type I membrane protein calnexin functions as a molecular
chaperone for secretory glycoproteins in the endoplasmic reticulum with
ATP and Ca Calnexin is an integral membrane protein of the endoplasmic
reticulum (ER) The function of calnexin as a
molecular chaperone initially arose from the demonstration that it is
associated in the ER with newly synthesized major histocompatibility
complex class I heavy chain and other cell surface receptors (see
Bergeron et al.(1994) for review). Subsequently the repertoire
of membrane protein substrates for calnexin was expanded to soluble
monomeric secretory glycoproteins (Ou et al., 1993; Le et
al., 1994; Wada et al., 1994). It was observed that there
is a requirement for glycosylation for proteins to bind to calnexin in vivo, because the N-linked glycosylation inhibitor
tunicamycin prevented the association of newly synthesized,
incompletely folded substrates with calnexin (Ou et al.,
1993). In addition, deoxynojirimycin and castanospermine, inhibitors of
glucosidase I and II, also abrogated the association of newly
synthesized, incompletely folded viral membrane glycoproteins with
calnexin. These observations led to the proposal that calnexin was a
lectin with specificity for monoglucosylated N-linked
oligosaccharides (Hammond et al., 1994). Support for this
hypothesis has come from the recent demonstrations of direct binding
between a recombinant soluble calnexin and the free oligosaccharide
Glc However, in vivo calnexin can also bind non-glycosylated proteins
(Rajagopalan et al., 1994; Loo and Clarke, 1994; Kim and
Arvan, 1995). The N-linked oligosaccharide can also be removed
by endoglycosidase H from coimmunoprecipitates of substrates bound to
calnexin without disrupting their association (Ware et al.,
1995). These studies indicate that the N-linked
oligosaccharide may be preferred for the initial interaction of newly
synthesized glycoproteins with calnexin but is not necessary to
maintain the interaction. There are several different regions of
calnexin that have been argued to be responsible for binding
substrates. The transmembrane and immediate juxtamembrane regions of
newly synthesized substrate membrane proteins have been indicated to be
required for binding to calnexin (Margolese et al., 1993). A
role for cytosolically oriented phosphorylation of calnexin has also
been proposed in the association of different allotypes of major
histocompatibility complex class I heavy chains with calnexin (Capps
and Ziga, 1994).
Furthermore, an ATP requirement has been demonstrated for the
association of the newly synthesized soluble glycoprotein gp80 with
calnexin in Madin-Darby canine kidney cells (Wada et al.,
1994). However, it is clear that secretory glycoproteins can only
interact directly with the luminal domain of calnexin and that membrane
substrates also have the opportunity for this interaction. Taken
together, these studies indicate multiple mechanisms of presentation
and binding of substrates to calnexin (Bergeron et al., 1994). In order to establish the mechanism of association of calnexin with
its substrates and to determine the cofactors involved, we have
expressed a soluble recombinant derivative of calnexin in the
baculovirus-infected Sf9 expression system. We demonstrate here that
soluble calnexin can associate with the newly synthesized soluble
glycoprotein substrate HIV-1 gp120 when they are coexpressed in Sf9
cells. We further show that soluble calnexin purified from the
extracellular medium of recombinant baculovirus-infected Sf9 cells
undergoes conformational changes as revealed by the formation of a
Ca
Figure 6:
Protease digestion of calnexin. A, purified calnexin
Figure 1:
A, protease
sensitivity of calnexin. 10 µl of 0.5 M KCl-washed canine
microsomal membranes (1 mg/ml) were incubated with the indicated
concentrations of proteinase K in 0.25 M sucrose, 1 mM dithiothreitol, 50 mM KCl, 50 mM Tris-HCl (pH
7.5) at 20 °C for 30 min in the presence (lane 9) or
absence (lanes 1-8) of 0.5% CHAPS. In all cases
Ca
Figure 10:
Direct binding of ATP to
calnexin
Figure 2:
Secretion of calnexin
We devised an
experiment that clearly shows that calnexin
Figure 3:
Expression and association of
calnexin
Figure 4:
Purification of calnexin
Figure 5:
Figure 7:
Alignment of four cysteine residues among
calnexins from various sources. Four conserved cysteines (indicated by arrows) and their surrounding sequences are shown. Identical
residues are boxed. On the right side are the amino
acid residue numbers from the NH
Figure 8:
Disulfide bonding of calnexin
Figure 9:
Calcium- and ATP-dependent conformational
change of calnexin
Because
both EGTA and ATP induced oligomerization of calnexin
We have shown that calnexin is a type I membrane protein as
predicted from its sequence, that the luminal domain can bind in
vivo to a secretory glycoprotein, and that the luminal domain and
full-length calnexin share several properties. When calnexin was
originally identified, it was apparent that it was related by sequence
to the major Ca There is evidence from in vivo experiments
that calnexin can transiently interact with newly synthesized secretory
glycoproteins and membrane proteins during their maturation in the ER.
Although there are indications that membrane proteins such as major
histocompatibility complex class I can interact with residues within or
near the transmembrane domain or even perhaps with the cytosolically
directed COOH-terminal domain (Margolese et al., 1993; Capps
and Ziga, 1994), it is
clear that secretory glycoproteins can only interact with the luminal
domain. The orientation of the protein was experimentally determined by
peptide-specific antibodies and protease digestion of rough
microsomes, confirming that the NH The secretion from insect cells of calnexin In the presence of Ca The detail in conformational changes of calnexin as
related to its substrate binding remains to be studied. However, it is
clear that these conformational changes induced by Ca Current models for the actions of several chaperones including the
GroEL family and BiP involve a cyclical association and dissociation of
protein-folding intermediates with the chaperones correlated with
changes in the chaperone quaternary structures regulated by ATP binding
and hydrolysis (Freiden et al., 1992; Blond-Elguindi et
al. 1993; Todd et al., 1994; Weissman et al. 1994). One model for chaperone function of calnexin (Hammond et al., 1994) proposes that glycoprotein-folding intermediates
associated with calnexin undergo a cyclical reglucosylation as the
mechanism of cyclical substrate presentation to calnexin. Calnexin and
BiP have recently been proposed to act sequentially and cooperate in
the folding of newly synthesized glycoproteins in vivo (Hammond and Helenius, 1994; Kim and Arvan, 1995). ATP binding and
hydrolysis are likely to regulate the cooperation between
calnexin/calreticulin and BiP in protein folding in the ER lumen in
vivo. The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
X53616[GenBank Link].
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
as two of the cofactors involved in
substrate binding. Protease protection experiments with intact canine
rough microsomes showed that amino acid residues 1-462 of
calnexin are located within the lumen of the endoplasmic reticulum.
Expression using the baculovirus Sf9 insect cell system of a
recombinant truncated calnexin corresponding to residues 1-462
(calnexin
TMC) revealed an association in vivo with a
coexpressed secretory glycoprotein substrate, human immunodeficiency
virus type I gp120. For the in vitro characterization of
calnexin
TMC, we purified this secreted form to homogeneity from
the medium of Sf9 cells. We demonstrate that the properties of the
purified calnexin
TMC correspond to those of full-length calnexin
in canine microsomes with at least one intramolecular disulfide bond
and binding to
Ca
. Calnexin
TMC
underwent a marked and reversible conformational change following
Ca
binding as measured by its resistance to
proteinase K digestion of a 60-kDa fragment and also by the change from
an oligomeric form of calnexin
TMC to a monomeric form. We also
found that calnexin bound Mg-ATP leading to a conformational change
from a monomeric to an oligomeric form that coincided as with markedly
increased proteinase sensitivity. Our results identify the luminal
domain of calnexin as responsible for binding substrates,
Ca
, and Mg-ATP. Because Ca
and ATP
are required in vivo for the maintenance of calnexin-substrate
interactions, conformational changes in the luminal domain of calnexin
induced by Ca
and Mg-ATP are relevant to the in
vivo function of calnexin as a molecular chaperone.
(
)that functions as a molecular
chaperone and as a constituent of the quality control apparatus of the
ER (reviewed in Bergeron et al.(1994)). Calnexin was
originally identified as a major integral membrane phosphoprotein of
the ER (Wada et al., 1991). Cloning of the cDNA and sequencing
predicted that calnexin is a type I membrane protein with the predicted
luminal domain revealing regions of sequence similarity with the major
Ca
-binding protein of the ER lumen, calreticulin.
Based on the binding of
Ca
to ER
membrane proteins as well as the isolated protein, it was shown that
calnexin is one of the two major Ca
-binding resident
proteins of the membrane of canine rough ER (Wada et al.,
1991; Cala et al., 1993).
Man
GlcNAc
(Ware et al.,
1995) and of binding of glucosylated thyroglobulin and soybean
agglutinin in a cell free system.
(
)
-dependent proteinase-resistant core and a
Ca
-dependent change from an oligomeric to a monomeric
state, whereas Mg-ATP promoted a change from a monomeric to an
oligomeric state that coincided with increased proteinase sensitivity.
Finally, we show that secreted soluble calnexin is at least partially
disulfide-bonded intramolecularly and can bind ATP directly but does
not have detectable ATPase activity. These findings extend the
functional significance of calnexin as an ATP-binding molecular
chaperone with properties distinguishable from those of the ER luminal
Ca
-binding and ATP-binding chaperone, BiP.
Recombinant Baculovirus and Infection of Sf9
Cells
cDNA encoding a truncated canine calnexin (amino acid
residues 1-462) followed immediately by an in-frame stop codon
was generated by polymerase chain reaction and subsequently cloned into
the transfer vector pETL1 at the BamHI site filled in with
Klenow polymerase. The recombinant baculovirus was isolated by the
-galactosidase screening and used to infect Sf9 cells that were
grown in Sf9000 II serum-free medium as described by Vialard et
al.(1990). The sequence of the calnexin construct in the
baculovirus vector was confirmed, and the following differences were
observed with the published sequence (Wada et al., 1991).
Nucleotides 199 and 201 are determined to be A and A and not T and C,
respectively, as previously determined. This changes the codon for
residue 67 from TCC to ACA, and the corresponding amino acid change is
Ser
to Thr
. There is also a threonine at this
position in human calnexin (David et al., 1993). The sequence
of a peptide of canine calnexin from this region also predicted a
threonine at this position. Thus we conclude that the original canine
calnexin nucleotide sequence is incorrect in this region. We also found
one other difference with the published canine calnexin nucleotide
sequence at nucleotide 201, which is a change of the originally
predicted C to an A; this does not change the amino acid sequence.
Expression of HIV-1 gp120 in Sf9 cells by recombinant baculovirus has
been described previously (Li et al., 1994).
Purification of Calnexin
Sf9 cells were
infected with recombinant baculovirus expressing calnexinTMC
TMC and
grown for 72 h. PMSF and p-hydroxymercuriphenylsulfonic acid
(final concentrations, 1 mM and 0.1 mM, respectively)
were added to the medium. All of the buffers used for purification of
calnexin contained 1 mM CaCl
. Ammonium sulfate
(70% saturation) was added to the medium, and the supernatant was
passed through a phenyl-Sepharose column (2.7
5 cm). The
flow-through was desalted using a G-25 Sephadex column and loaded onto
a DEAE-Sepharose column (2.7
10 cm) (Pharmacia Biotech Inc.)
equilibrated with 10 mM Tris-HCl (pH 7.5), 50 mM
NaCl. The column was washed with the same buffer, and the proteins were
eluted with a linear gradient of 50-600 mM NaCl. The
calnexin
TMC-containing fractions were pooled, concentrated with a
Centriprep 30 (Pharmacia Biotech Inc.), and further purified on a fast
protein liquid chromatography Mono-Q column (10/10) equilibrated with
10 mM Tris-HCl (pH 7.5), 200 mM NaCl. The proteins
were eluted with a gradient of 200-800 mM NaCl.
Calnexin
TMC was detected by immunoblotting with anti-CN1, a rabbit
polyclonal antibody made against a synthetic peptide encompassing
residues 30-48 of the NH
terminus of calnexin. This
antibody recognizes mammalian calnexin but not insect calnexin.
Proteinase K Digestion
Purified calnexinTMC
(1 mg/ml) was incubated with increasing concentrations of proteinase K
(Boehringer Mannheim) in 20 mM Hepes (pH 7.4), 50 mM NaCl buffer at 30 °C for 30 min. Protease digestion of native
calnexin was carried out using solubilized canine pancreatic ER
membranes (2 mg/ml) in the presence of 1% CHAPS (Sigma). After the
addition of 1 mM PMSF to the proteinase K incubations,
proteins were separated on SDS-PAGE (5%-15% gel) followed by Coomassie
Blue staining for the purified calnexin
TMC (see Fig. 6A) or by immunoblotting with anti-CN1 for the ER
membranes (see Fig. 6B).
TMC (1 mg/ml) was incubated with the
indicated concentrations of proteinase K in the presence of 2 mM CaCl
(lanes 1-7) or 5 mM EGTA (lanes 8-13), or calnexin
TMC was preincubated with
5 mM EGTA for 5 min and subsequently treated with 10 mM CaCl
for 5 min. The treated calnexin
TMC was then
incubated with proteinase K (lanes 14-19). The
proteinase K digestion was terminated by the addition of 1 mM PMSF, and the mixtures were analyzed on SDS-PAGE (5-15% gel)
followed by Coomassie Blue staining. The significance of the fragments
at
30 kDa in lanes 7, 13, and 19 are
uncertain. B, canine pancreatic ER membranes (2 mg/ml) were
incubated with increasing concentrations of proteinase K in the
presence of 1% CHAPS under the same conditions as described in A. The proteins were separated on SDS-PAGE followed by
immunoblotting with anti-CN1. Lane 20 in B is the
fragment of calnexin
TMC produced by 100 µg/ml proteinase K
treatment in the presence of CaCl
(lane 6 in A). The fragment matches exactly the fragment of native
calnexin produced by the same amount of proteinase K.
CN,
calnexin
TMC; CN, native calnexin. C, purified
calnexin
TMC (lane 1) was incubated with proteinase K (PK, lanes 2 and 3) or trypsin (lanes 4 and 5) in the presence of 1 mM CaCl
at 30 °C for 30 min. After PMSF was added to stop the
reactions, proteins were analyzed on SDS-PAGE followed by Coomassie
Blue staining. PK, proteinase K;
CN,
calnexin
TMC.
ATP Binding Assay
Purified calnexinTMC (0.1
mg/ml) was incubated with 20 µCi/ml (25 µM)
[
-
P]ATP (800 Ci/mmol; DuPont Canada,
Mississauga, Ontario) in 20 mM Hepes-NaOH (pH 7.5) buffer
containing 50 mM NaCl, 10 mM MgCl
, 2
mM CaCl
at 20 °C for 10 min and then exposed
to UV radiation for 5 min. For inhibition experiments, reagents were
incubated with calnexin prior to the addition of
[
-
P]ATP. The
P-labeled protein
was separated on SDS-PAGE and stained with Coomassie Blue followed by
radioautography.
Metabolic Labeling and Immunoprecipitation
24 h
after infection with recombinant baculoviruses, Sf9 cells were labeled
with 100 µCi/ml [S]methionine (1000 Ci/mmol,
Amersham Corp.) for 15 min at 25 °C. The labeled cells were washed
with cold phosphate-buffered saline and lysed in 50 mM
Hepes-NaOH buffer (pH 7.4) containing 200 mM NaCl, 2% CHAPS, 1
mM CaCl
, 1 mM PMSF, and 5 µg/ml each
leupeptin and aprotinin. The cell lysate was precleared with protein
A-Sepharose and immuno-precipitated with anti-CN1 or anti-gp120
antibodies as described previously (Ou et al., 1993).
Analytical Methods
Protein concentration was
determined as described by Bradford(1976). The Ca
overlay assay was as described in
Wada et al.(1991). SDS-PAGE was performed as described by
Laemmli(1970). Nondenaturing gel electrophoresis was carried out in the
absence of SDS using the Laemmli system. Protein sequencing of
calnexin
TMC or its protease-cleaved fragments was determined using
an Applied Biosystems 470A protein sequencer equipped with an on-line
phenylthiohydantoin amino acid analyzer.
Orientation of Calnexin in Vivo
From its primary sequence calnexin is predicted to be a type
I integral membrane protein of the ER (Wada et al., 1991). In
this study we confirm the orientation of calnexin in the ER membrane by
using proteinase K digestion of canine microsomes and antibodies to
peptides in different regions of calnexin (Fig. 1). Digestion of
intact microsomes revealed a 70-kDa proteinase-resistant fragment,
indicating that the majority of the protein was intraluminal (Fig. 1A). The result is consistent with the
demonstration that in dog cardiac sarcoplasmic reticulum vesicles a
70-kDa fragment of calnexin is protected from digestion by proteases
(Cala et al., 1993). Both the native and protease-resistant
fragment of calnexin were recognized by antibodies to residues
30-48 of calnexin (anti-CN1). Antibodies to residues
555-573 (anti-CN4) only recognized full-length calnexin in intact
microsomes, and proteinase treatment abolished this recognition (Fig. 1B). These results confirm that in vivo calnexin is a type I membrane protein and that residues
1-462 of calnexin are luminally oriented followed by a
hydrophobic membrane-spanning domain and a cytosolically oriented
COOH-terminal tail (Fig. 1C).
was omitted from the incubations. The reaction was
terminated by the addition of 1 ml of 1% PMSF and processed for
immunoblotting with antibody made to residues -8 to +268 of
bacterially expressed calnexin as described in Ahluwalia et
al.(1992). The mobility of intact calnexin and the proteinase
resistant fragment is indicated. B, membrane orientation of
calnexin. Dog pancreatic stripped rough microsomes (25 µg) were
incubated with trypsin (200 µg/ml) at 20 °C for 30 min either
in the absence (lanes 2 and 5) or presence (lanes
3 and 6) of 0.2% Triton X-100. The reaction was
terminated by the addition of 1 mM PMSF. Lanes 1 and 4 are control samples incubated in the absence of trypsin or
detergent. Samples were subjected to SDS-PAGE followed by
immunoblotting with anti-CN1 (NH
terminus; lanes
1-3) or anti-CN4 (COOH terminus; lanes 4-6). C, deduced orientation of canine calnexin (modified from Fig. 10of Wada et al.(1991)). Shown are the positions
of the residues to which anti-CN1 (
-calnexin 1) and
anti-CN4 (
-calnexin 4) were raised, the predicted
transmembrane segment, and the luminal region of calnexin with the four
regions (A-D) of high sequence similarity to
calreticulin.
TMC. Calnexin
TMC (lanes 1-4) or bovine
serum albumin (lanes 5) was incubated with
[
-
P]ATP (20 µCi/ml, 25 µM)
in the presence of 10 mM MgCl
(lanes 1 and 4), 2 mM CaCl
(lanes
2), or 10 mM EDTA (lanes 3) for 10 min at room
temperature. The mixtures were irradiated with UV radiation (lanes
2-5) for 5 min or not exposed (lanes 1). The
proteins were analyzed on SDS-PAGE and stained with Coomassie Blue (A) followed by radioautography (B). C.B.,
Coomassie Blue. C, ATP binding assays were carried out as
described under ``Experimental Procedures'' except that,
prior to the addition of [
-
P]ATP,
calnexin
TMC was preincubated with the indicated reagents for 5
min. Proteins were analyzed on SDS-PAGE followed by radioautography.
The bands corresponding to calnexin
TMC were excised from the gels,
and their radioactive content was determined by liquid scintillation
counting. The values are expressed as percentages of the control. DTT, dithiothreitol; IAA, iodoacetamide; NEM, N-ethylmaleimide.
Heterologous Coexpression and Association of a Truncated
Soluble Calnexin with Newly Synthesized HIV-1 gp120
Because calnexin can transiently bind both membrane and
soluble secretory proteins, the luminal domain is predicted to be
responsible for this interaction. With type I membrane proteins removal
of the membrane spanning domain can lead to their secretion from cells.
Introduction of a stop codon at residue 463 (i.e. immediately
preceding the predicted transmembrane domain of calnexin) (Fig. 1C) led to the secretion of calnexinTMC. 24
h after recombinant baculovirus infection (Fig. 2, lanes 3 and 4), intracellular calnexin
TMC with a mobility of
70 kDa was recognized by the anti-CN1 antibody with increasing amounts
of the protein being secreted at 48, 72, and 96 h postinfection (Fig. 2, lanes 5-10). Whereas two polypeptides of
70- and 69-kDa were observed for intracellular calnexin
TMC (Fig. 2, lanes 5, 7, and 9),
extracellularly only the 70-kDa form was observed (Fig. 2, lanes 6, 8, and 10).
TMC from Sf9
cells. Sf9 cells were infected with recombinant baculovirus expressing
calnexin
TMC as described under ``Experimental
Procedures.'' At the indicated times postinfection, cells (C) and medium (M) were separately harvested and
analyzed on SDS-PAGE followed by immunoblotting with anti-CN1.
CN, calnexin
TMC.
TMC can interact in
vivo with a viral glycoprotein. We compared the association of
calnexin
TMC and that of full-length calnexin with a calnexin
substrate, HIV-1 gp120.
(
)HIV-1 gp120 is
normally processed from the precursor gp160 by proteolytic cleavage,
generating soluble gp120 and the COOH-terminal membrane attached gp41;
but in this experiment gp120 alone was expressed by inserting a
termination codon at the site of cleavage.
As shown in Fig. 3, 24 h after infection of Sf9 cells with recombinant
baculovirus containing calnexin
TMC (lane 1), HIV-1 gp120 (lane 2), or full-length calnexin (lane 5), they
could be detected by immunoprecipitation of pulse-labeled cells with
either anti-CN1 (lanes 1 and 5) or anti-HIV-1 IgG (Fig. 3, lane 2). Coinfection with baculoviruses
containing either calnexin
TMC (Fig. 3, lane 3) or
full-length calnexin (Fig. 3, lane 6) with HIV-1 gp120
revealed their association because they could be coimmunoprecipitated
with anti-CN1. The lack of radiolabeled calnexin in the
immunoprecipitates with HIV-1 IgG (Fig. 3, lanes 4 and 7) is due to the large pool of pre-existing calnexin (Ou et al. 1993). The gp120 molecules that were associated with
calnexin (Fig. 3, lanes 3 and 6) were
incompletely folded, because they were not recognized by the receptor
CD4, as has been recently shown for full-length calnexin
and was also shown for gp120 in association with calnexin
TMC
(data not shown). Thus, full-length calnexin and calnexin
TMC
appear to be equally capable of interacting with a soluble glycoprotein
substrate.
TMC or full-length canine calnexin with HIV-1 gp120. Sf9
cells were infected with recombinant baculovirus expressing either
calnexin
TMC (lane 1) or HIV-1 gp120 (lane 2) or
coinfected with recombinant baculovirus containing calnexin
TMC and
gp120 (lanes 3 and 4) or viruses containing
full-length calnexin alone (lane 5) or coinfection with
recombinant baculoviruses containing full-length calnexin and gp120 (lanes 6 and 7). 24 h after infection, the cells were
labeled with 100 µCi/ml [
S]methionine for 15
min, and the labeled cells were lysed and immunoprecipitated with
anti-CN1 (lanes 1, 3, 5, and 6) or
anti-gp120 (lanes 2, 4, and 7) antibodies.
The immunoprecipitates were analyzed on SDS-PAGE followed by
fluorography.
CN, calnexin
TMC; CN,
full-length calnexin.
Purification of Soluble Secreted Calnexin
The association of intracellular soluble calnexinTMC
TMC
with gp120 suggested that the luminal domain alone interacts with
substrates. Thus we used this expression system to purify
calnexin
TMC for in vitro characterization. At 72 h
postinfection, calnexin
TMC was about 1% of the total protein in
the medium from Sf9 cells, and it was purified to homogeneity (see
``Experimental Procedures''). The final preparation showed a
single band for calnexin
TMC with an apparent molecular mass of 70
kDa on SDS-PAGE (Fig. 4A). We confirmed the
NH
-terminal sequence of the first 20 residues of the
purified protein as HEGHD(D)(D)MID(I)ED(D)LD(D)VIE (residues in
parentheses indicate the inherent difficulty in identifying consecutive
residues), which is identical to that for calnexin purified from
microsomes (Wada et al., 1991).
TMC.
Purification of calnexin
TMC from the culture medium of infected
Sf9 cells was carried out as described under ``Experimental
Procedures.'' The proteins from each purification step were
separated on SDS-PAGE followed by Coomassie Blue (C.
B.)staining (A) or by immunoblotting with anti-CN1 (B). Lane 1, culture supernatant; lane 2,
supernatant of 70% saturated ammonium sulfate; lane 3,
phenyl-Sepharose fraction; lane 4, DEAE-Sepharose fraction; lane 5, Mono-Q fraction.
CN,
calnexin
TMC.
Characterization of Purified Calnexin
TMC
Calcium Binding
The luminal domain of calnexin
shows 4 regions of sequence similarity with calreticulin, the major ER
luminal calcium-binding protein (Fig. 1C and Wada et al. (1991)). The results in Fig. 5show that the
detergent phase of Triton X-114-extracted canine pancreatic ER
membranes contains a major calcium-binding protein corresponding to
calnexin (90 kDa) as well as the other major calcium-binding ER
membrane protein pgp35 (35 kDa) (Fig. 5, lane 1). We
were also able to show that purified soluble calnexinTMC can bind
calcium using the
Ca
overlay technique,
whereas in a control experiment bovine serum albumin did not (Fig. 5, lane 3). Thus both full-length calnexin from
ER membranes and purified calnexin
TMC show similar behavior in
their ability to bind calcium.
Ca binding to
calnexin
TMC. Triton X-114 detergent phase extracted from 50 µg
of canine pancreatic ER membrane (lanes 1), 2 µg of
purified calnexin
TMC (lanes 2), or 5 µg of bovine
serum albumin (lanes 3) were separated on SDS-PAGE followed by
Coomassie Blue (C. B.) staining (A) or by
electrotransfer to a nitrocellulose membrane. The membrane was then
incubated with
Ca
followed by
radioautography as described under ``Experimental
Procedures'' (B).
CN, calnexin
TMC. The
90- and 35-kDa proteins that displayed calcium binding in lane 1 are native calnexin and pgp35, respectively (Wada et al.,
1991).
Proteinase Sensitivity
Sensitivity to proteinase
digestion was used to evaluate protein domain structure. When
calnexinTMC was incubated with low concentrations (1-10
µg/ml) of proteinase K in the presence of calcium, a 65-kDa
fragment was generated (Fig. 6A, lanes
2-4). With increasing concentrations of the proteinase
(100-300 µg/ml), a fragment of 60 kDa was generated (Fig. 6A, lanes 6 and 7). The
proteinase digestion of native calnexin in the solubilized ER membrane
resulted in a similar profile (Fig. 6, compare A, lanes 2-7, with B, lanes 2-7).
Low (1-10 µg/ml) and high (100-300 µg/ml)
concentrations of the proteinase-produced fragments of 65 and 60 kDa,
respectively (Fig. 6, compare A and B, lanes 19 and 20). Sequence analysis of the 60-kDa
fragment revealed the major sequence to be K
SKPDTSAPTSP,
and therefore it includes the sequence used to generate the anti-CN1
antibody (residues 30-48). Digestion of calnexin
TMC with
trypsin in the presence of calcium also generated the 60-kDa fragment (Fig. 6C, lanes 4 and 5); but the
sequence was S
KPDTSAPTSP, just one residue downstream from
the proteinase K cleavage site. Our data indicate that
proteinase-sensitive sites exist at both the carboxyl and the amino
termini of calnexin. But there is a proteinase-resistant core structure
that requires calcium to maintain resistance, because, in the presence
of EGTA, both calnexin
TMC and native calnexin became highly
sensitive to a low concentration (10 µg/ml) of proteinase K (Fig. 6, A and B, lanes 8-13).
To determine if the proteinase-resistant and -sensitive forms of
calnexin are reversible, calnexin
TMC was first incubated with
EGTA, leading to the proteinase-sensitive conformation, and then excess
calcium was added to the protein followed by the addition of proteinase
K. This procedure restored the proteinase-resistant conformation (Fig. 6A, lanes 14-19). Full-length
calnexin also displayed the same reversible conformational change of
calnexin
TMC (Fig. 6B, lanes 14-19).
Thus both full-length calnexin from ER membranes and purified
calnexin
TMC can be digested with protease to generate the same
resistant core fragment.
Disulfide Bonded Calnexin
Calnexin has four
cysteine residues (161, 195, 361, and 367 for canine calnexin), which
are conserved in calnexins from all species (Fig. 7). To
investigate the redox state of secreted purified calnexin, we compared
the mobility of the protein on SDS-PAGE under reducing and nonreducing
conditions (Fig. 8). Usually monomeric proteins with
intramolecular disulfide bonds migrate faster on nonreducing gels than
on reducing gels. Under nonreducing conditions, the mobility of both
calnexinTMC and native calnexin in isolated ER membranes revealed
an increased mobility (Fig. 8, compare lanes 1 and 2 and compare lanes 3 and 4), indicating
that calnexin contains at least one internal disulfide bond. As a
positive control, we used albumin, which contains 17 disulfide bonds
(Simon and Dugaiczyk, 1981), and detected a larger increase in mobility
under nonreducing conditions (Fig. 8, lanes 5 and 6). Because calnexin can only contain at the most two
disulfide bonds, the mobility shift found was correspondingly less.
Hence, at least one and perhaps both potential disulfide bonds in
calnexin are formed intramolecularly.
termini.
TMC.
Purified calnexin
TMC (lanes 1 and 2), canine
pancreatic ER membranes (lanes 3 and 4), or bovine
serum albumin (BSA, lanes 5 and 6) were
separated on SDS-PAGE under reducing (lanes 1, 3, and 5) or nonreducing (lanes 2, 4, and 6) conditions followed by immunoblotting with anti-CN1 (lanes 1-4) or staining with Coomassie Blue (C.
B.) (lanes 5 and 6). Small differences in
mobility were observed for disulfide bonded (ox) and reduced (red) forms of calnexin
TMC (
CN) or native
calnexin (CN).
Calcium- and ATP-dependent Conformational Change of
Calnexin
Purified calnexinTMC
TMC was analyzed on
native gels to investigate its state of oligomerization (Fig. 9, A and B). In the presence of calcium,
calnexin
TMC was a monomer (Fig. 9, A and B, lanes 2). However, after incubation with EGTA,
dimeric and oligomeric calnexin
TMC were observed (Fig. 9, A and B, lanes 3). These oligomers were also
visualized by immunoblotting (Fig. 9B, lane 3)
with anti-CN1, which reacted more strongly with oligomeric calnexin
than with monomeric calnexin. Remarkably, ATP also induced the
formation of calnexin oligomers, suggesting the direct interaction of
the nucleotide with calnexin
TMC (Fig. 9, A and B, lanes 4). The stronger binding of anti-CN1
antibody to ATP-induced oligomers of calnexin is also consistent with a
conformational change exposing the epitope. When native calnexin was
analyzed on nondenaturing gels, the protein migrated as large
aggregates on the top part of the gels (data not shown).
TMC. Purified calnexin
TMC (lanes
1) was treated with 1 mM CaCl
(lanes
2), 10 mM EGTA (lanes 3), or 1 mM
Mg
ATP (lanes 4) at 25 °C for 10 min.
The protein was analyzed on 5-15% nondenaturing polyacrylamide
gels followed by Coomassie Blue (C. B.) staining (A)
or immunoblotting with anti-CN1 (B). The oligomeric states of
calnexin
TMC are indicated on the right. M,
monomer; D, dimer; T, tetramer. The preferential
reaction of anti-CN1 with oligomeric calnexin is revealed in B. C, purified calnexin
TMC was incubated at 30
°C for 30 min with 20 µg/ml (lanes 2, 4, 6, 8, and 10) or 100 µg/ml (lanes
3, 5, 7, 9, and 11) of
proteinase K (PK) in the presence of 5 mM CaCl
(lanes 2, 3, 8, and 9), 10
mM EGTA (lanes 4, 5, 10, and 11), or 1 mM ATP (10 mM MgCl
) (lanes 6-11). After 1 mM PMSF was added to
terminate the reactions, the proteins were separated on 10% SDS-PAGE
followed by Coomassie Blue staining. Lane 1 is purified
calnexin
TMC without protease
digestion.
TMC, we
tested the protease sensitivity of calnexin
TMC in the presence of
ATP. When purified calnexin
TMC was treated first with Mg-ATP and
then with proteinase K (Fig. 9C, lanes 6 and 7), calnexin
TMC was completely digested. Addition of
excess calcium to the ATP-treated calnexin
TMC partially restored
proteinase K resistance (Fig. 9C, lanes 8 and 9), but, compared with calcium alone (Fig. 9C, lanes 2 and 3), calnexin
TMC was much more
sensitive to proteinase K digestion. Because 10 mM MgCl
was present in the reaction mixtures, the possibility that ATP
effect is as a chelator can be excluded. Hence the Mg-ATP effect on
oligomerization can occur in the presence of calcium.
ATP Binding
Several molecular chaperones bind ATP
(Gething and Sambrook, 1992), and their function is regulated by ATP
(Munro and Pelham, 1986; Flynn et al., 1989). In vivo in Madin-Darby canine kidney cells, ATP is required to maintain
the interaction of the newly synthesized soluble glycoprotein gp80 with
calnexin (Wada et al., 1994). We established that purified
calnexinTMC can bind ATP. Purified calnexin
TMC was incubated
with [
-
P]ATP in the absence or presence of
magnesium or calcium, and the samples were analyzed by SDS-PAGE
followed by radioautography. Binding of ATP was
Mg
-dependent and required UV-enhanced cross-linking (Fig. 10B, lane 4), suggesting that ATP does
not form a covalent interaction with the protein and that UV radiation
promoted the cross-linking of ATP and the protein. The nucleotides,
ATP, ADP, and AMP were all able to compete for binding (Fig. 10C). ATP
S was the most potent competitor
for ATP binding. GTP also showed strong inhibition of ATP binding,
which has also been observed for other chaperone proteins (Csermely and
Kahn, 1991). The reducing reagent, dithiothreitol, strongly inhibited
ATP binding, whereas sulfydryl reagents, such as iodoacetamide and N-ethylmaleimide, had little effect on the binding activity,
suggesting that the oxidized disulfide bonds in calnexin are required
for ATP binding. Using a variety of conditions, which included the
presence of denatured proteins, we were unable to detect any ATPase
activity in purified calnexin
TMC.
-binding protein of the ER lumen,
calreticulin (Wada et al., 1991). The generation of soluble
calnexin
TMC further extends the similarity to calreticulin (32%
identical); we suggest that this is because of the high degree of
sequence conservation between calnexin
TMC and calreticulin that
our observations on calnexin
TMC are also directly applicable to
calreticulin.
-terminal domain is in
the ER lumen and would interact with substrates. Thus it is probable
that this is the region that is responsible for the recognition and
molecular chaperone activity for all substrate proteins. We were able
to demonstrate by coexpressing the two genes in Sf9 insect cells that, in vivo, this region does interact with the secretory
glycoprotein HIV-1 gp120. This was tested by expressing soluble
truncated calnexin deleted for the transmembrane and cytosolic domains,
calnexin
TMC, and assessing its interaction with the soluble
glycoprotein HIV-1 gp120, a known substrate for full-length
calnexin.
By the criterion of coimmunoprecipitation, newly
synthesized gp120 was found to be associated with soluble calnexin. The
choice of the Sf9 cell to assess expression of both proteins was
especially appropriate because our anti-CN1 antibody did not recognize
insect cell calnexin. Hence, the coimmunoprecipitation studies
indicated a direct interaction in vivo between
calnexin
TMC and HIV-1 gp120. Although models exist for the
interaction of calnexin with its substrates via recognition features in
its transmembrane or cytosolic domains (Margolese et al.,
1993; Capps and Ziga,
1994), our in vivo data with calnexin
TMC and HIV-1 gp120
indicate that substrate recognition is luminal as predicted from the
studies of Ou et al.(1993), Wada et al.(1994), and Le et al.(1994). Our studies define the luminal domain as the
region responsible for the molecular chaperone activity of calnexin.
TMC and its
subsequent ease of purification enabled us to examine that this form of
calnexin, which functions as a molecular chaperone, shares other
properties with the full-length calnexin. Soluble calnexin
TMC,
when purified from the extracellular medium, was found to be monomeric
but to contain at least one intramolecular disulfide bond. All members
of the calnexin family from humans to plants reveal sequence
conservation about the 4 invariant cysteine residues (Fig. 7).
Based on the different mobilities of calnexin in reducing and
nonreducing gels, we conclude that at least one potential disulfide
bond is formed internally.
,
both native calnexin and calnexin
TMC formed a core structure
resistant to proteases. Calcium was required to maintain the core
structure because it was destroyed by EGTA. All substrates evaluated by
Ou et al.(1993), Wada et al.(1994), and Le et
al.(1994) required Ca
for their association with
calnexin. Soluble calnexin
TMC bound Ca
, and
Ca
addition led to the conversion of oligomeric
calnexin
TMC to monomeric calnexin
TMC. Remarkably,
Mg
ATP led to the conversion of monomeric
calnexin
TMC to oligomeric calnexin
TMC. Oligomerization was
accompanied by a change in the conformation of individual calnexin
molecules as observed by an increased sensitivity to proteinases. The
opposite effects of Mg-ATP and Ca
in regulating
calnexin oligomerization is reminiscent of the inverse effects of these
same agents in promoting the association (Ca
) or
dissociation (ATP) of incompletely folded substrate proteins with BiP
(Suzuki et al., 1991; Gaut and Hendershot, 1993). Wada et
al.(1994) demonstrated that ATP is required to maintain the
association of the substrate gp80 with calnexin in vivo in
Madin-Darby canine kidney cells. This observation may now be explained
by our demonstration of ATP binding to calnexin and the ATP-dependent
conformational change in calnexin. The direct binding of ATP to
calnexin in vitro was specific because adenosine nucleotides (e.g. ATP, ADP, AMP, and the ATP analogue ATP
S) were able
to compete for binding, binding was Mg
-dependent, and
binding was inhibited by dithiothreitol. However, sulfydryl reagents
that reacted with free sulfydryl groups did not affect the ATP binding
activity. Hence, we conclude that it is the disulfide bond(s) that act
to maintain a specific protein conformation in calnexin competent for
binding ATP, and it is not free sulfydryl groups that are involved in
ATP binding.
and ATP are relevant to its chaperone function in vivo.
The luminal content of Ca
in the ER has been
estimated to be at concentration of approximately 5 mM (Somlyo et al., 1985), and the translocation of ATP into the ER and
its binding to ER proteins have also been documented (Clairmont et
al., 1992). The requirement of both calcium and ATP for the
binding of substrate proteins to calnexin suggests that the binding of
substrate proteins may accompany conformational changes in calnexin.
Another molecular chaperone of the ER, BiP also binds Mg-ATP. Mg-ATP
and Ca
are also regulatory to BiP function and effect
conformational changes in BiP (Suzuki et al., 1991;
Blond-Elguindi et al., 1993; Brot et al., 1994). BiP
is a potent ATPase during the step of substrate release from the
chaperone (Suzuki et al., 1991). Although we have been unable
to detect ATPase activity thus far of the purified calnexin
TMC,
this is likely regulated by substrate binding to calnexin and/or an
associated protein. Direct binding of purified calnexin
TMC in
vitro with peptides or denatured proteins has only been
demonstrated when they are glucosylated with UDP-Glc:glycoprotein
glucosyltransferase on Man
N-linked glycosylation
(Parodi et al., 1984).
However, ATPase activity
can be detected in immunoprecipitates of full-length calnexin with
associated secretory glycoproteins.
(
)Hence, the
possibility still exists that the presence of a substrate-calnexin
interaction is required for ATPase activity or perhaps, more probably,
that a calnexin-associated protein may be required for ATPase activity.
Good candidates for these protein are the ER membrane proteins that we
originally found associated with calnexin (Wada et al., 1991).
S, adenosine
5`-O-(3-thiotriphosphate); BiP, binding protein.
We thank Dr. Ikuo Wada (Sapporo, Japan) and Pamela
Cameron (McGill) for participating in the experiment of Fig. 1.
We also thank Dr. Alexander Bell (McGill) for constructive criticism of
the manuscript and France Dumas for protein sequencing. We thank
Roseanne Tom and Dr. Amine Kamen for the insect cell culture service at
the Biotechnology Research Institute, Daniel Tessier for establishing
the time course of baculovirus infection, and Daniel Dignard for
confirming the sequence of calnexinTMC in the recombinant
baculovirus. Human anti-HIV-1 immunoglobulin was obtained from Dr.
Alfred Prince through the AIDS Research and Reagent Program, NIAID,
NIH.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.