Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002
Calnexin and calreticulin are homologous molecular chaperones that promote proper folding, oligomeric assembly, and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER). Both are lectins that bind to substrate glycoproteins that have monoglucosylated N-linked oligosaccharides. Their binding to newly translated influenza virus hemagglutinin (HA), and various mutants thereof, was analyzed in microsomes after in vitro translation and expression in live CHO cells. A large fraction of the HA molecules was found to occur in ternary HA- calnexin-calreticulin complexes. In contrast to calnexin, calreticulin was found to bind primarily to early folding intermediates. Analysis of HA mutants with different numbers and locations of N-linked glycans showed that although the two chaperones share the same carbohydrate specificity, they display distinct binding properties; calreticulin binding depends on the oligosaccharides in the more rapidly folding top/hinge domain of HA whereas calnexin is less discriminating. Calnexin's binding was reduced if the HA was expressed as a soluble anchor-free protein rather than membrane bound. When the co- and posttranslational folding and trimerization of glycosylation mutants was analyzed, it was observed that removal of stem domain glycans caused accelerated folding whereas removal of the top domain glycans (especially the oligosaccharide attached to Asn81) inhibited folding. In summary, the data established that individual N-linked glycans in HA have distinct roles in calnexin/calreticulin binding and in co- and posttranslational folding.
THE ER of most eukaryotic cells contains two homologous lectin-like chaperones called calnexin and
calreticulin. Calnexin is a membrane protein and
calreticulin a soluble lumenal protein. They interact transiently with a variety of newly synthesized glycoproteins by
attaching to partially trimmed N-linked oligosaccharide moieties carrying a single glucose residue in the When transferred to growing nascent chains, the N-linked
core oligosaccharides carry three glucoses. ER glucosidases I and II rapidly remove two of them, thus generating
the monoglucosylated forms (Glc1Man7-9NAcGlc2) that
serve as ligands for calnexin and calreticulin binding. The
remaining single glucose residue is subsequently removed
by glucosidase II, resulting in the dissociation of the chaperone complex (Hebert et al., 1995 Although identical in their oligosaccharide specificity
(Hammond et al., 1994 In this paper, we have addressed the functional differences between calnexin and calreticulin using the well characterized influenza HA as a model substrate. After mutating the seven N-linked glycosylation consensus sequences
in different combinations, we observed that effects on calnexin and calreticulin binding were distinct. Calreticulin
binding depended on the glycans located in the more rapidly folding, globular top domain whereas calnexin appeared to be able to bind to glycans all over the molecule. Evidence was obtained for the presence of ternary HA-
calnexin-calreticulin complexes. We also analyzed the role
of individual oligosaccharides in the folding process, and
found that some of them affected both rate and efficiency.
Reagents
Components for the cell-free translation, translocation, and folding system (rabbit reticulocyte lysate, amino acids, DTT, and RNasin) were purchased from Promega Corp. (Madison, WI). The canine pancreas microsomes were a generous gift of R. Gilmore (University of Massachusetts
Medical Center, Worcester, MA). Radiolabeled [35S]methionine/cysteine,
oxidized glutathione (GSSG), and CHAPS (3-[3-cholamidopropyl]-dimethylammonino-1-propanesulfate), were purchased from Amersham Corp.
(Arlington Heights, IL), Fluka Chemical Corp. (Ronkonkoma, NY), and
Pierce Chemical Co. (Rockford, IL), respectively. All other chemicals
were purchased from Sigma Chemical Co. (St. Louis, MO).
HA Mutants
The various HA glycosylation mutants were created by changing the Asn
in the consensus glycosylation sequence to a Gln by standard molecular
biological techniques (Sambrook et al., 1989 Translation, Translocation, and Folding of HA
35S-Labeled HA was translated and translocated into canine pancreas microsomes as previously described (Hebert et al., 1995 Infection of CHO Cells and Metabolic Labeling
CHO cells were infected with influenza virus (X31) and labeled as described (Chen et al., 1995 For the infection of CHO cells with vaccinia virus, 6-cm dishes of nearly
confluent cells were incubated at 37°C with 1-ml serum-free DME containing 2.5 x 107 virus. 30 min after incubation, the infection medium was
aspirated, and 3 ml of transfection medium containing 20 µl lipofectamine and 4 µg HA DNA (plasmid carrying the HA mutant genes) were added and incubated at 37°C for 4 h. The cells were then starved in the cysteine-/
methionine-free medium for 30 min and then labeled with 200-250 µCi
[35S]cysteine/methionine for 2 min. Immediately after labeling, the cells
were washed twice with cold PBS containing 20 mM NEM, and lysed with
2.0% CHAPS in 50 mM Hepes, 200 mM NaCl, pH 7.5, containing 10 µg/ml
each of chymostatin, leupeptin, antipain, and pepstatin (CLAP), 1 mM
PMSF, 20 mM NEM, and 1 mM EDTA.
Immunoprecipitation and SDS-PAGE
Immunoprecipitation of 35S-labeled HA with anti-HA, -trimer specific
HA, -calnexin, or -calreticulin antibodies was performed as described previously (Hebert et al., 1995 Precipitation for cotranslational studies used the anti-peptide antibody
corresponding to the NHA only. The sequential precipitations and the
two-dimensional (2-D)-gel system used with the cotranslational studies
were described previously (Chen et al., 1995 Binding of Calnexin and Calreticulin to Partially
Folded Forms of HA
To analyze how calnexin and calreticulin bind to partially
folded forms of HA, we translated HA in vitro in the presence of canine pancreas microsomes and [35S]methionine/
cysteine. The redox conditions were adjusted so that the
translocated HA molecules would undergo disulfide bond formation and folding (Hebert et al., 1995 Our previous studies have shown that the newly synthesized, nonreduced, full-length HA runs as three bands
(Braakman et al., 1991 The coimmunoprecipitations in Fig. 1 (A-C, lanes 1-3)
indicate that calreticulin bound preferentially (but not exclusively) to the less oxidized folding intermediates IT1 and
IT2. In contrast, calnexin bound to all three forms, IT1,
IT2, and NT. When quantified by gel scanning, the average IT1/IT2/NT binding ratio in 10 experiments was 1.0:
0.65:0.63 for calnexin and 1.0:0.57:0.16 for calreticulin, with
normalization with anti-HA precipitations. Since folding of HA proceeds from IT1 and IT2 to NT (Braakman et al.,
1991
Differential binding of calnexin and calreticulin was also
observed when HA folding occurred posttranslationally.
In this case translation was performed in the presence of a
high concentration of the reducing agent DTT, and folding
began posttranslationally with the addition of GSSG (Fig.
1, lanes 4-9) (Marquardt et al., 1993 To determine whether the differences in calnexin and
calreticulin binding could be observed in live cells, we used
a 2-D SDS-PAGE system recently developed in our lab to
simultaneously monitor folding of nascent and full-length
HA chains. CHO cells infected with influenza virus were
pulse labeled for 1.5 min with [35S]methionine/cysteine.
Some experiments were performed in the presence of
castanospermine, a competitive inhibitor of the ER The three spots at the extreme right of Fig. 2 A represent the labeled IT1, IT2, and NT forms of the full-length
HA. The spurs on the left of the spots emanating diagonally correspond to the labeled nascent chains of variable
length (Chen et al., 1995
It is apparent from the gel patterns in Fig. 2, A-C that
both nascent and full-length HA molecules associate with
calnexin and calreticulin in living cells. Association was
dependent on the trimming of glucoses from the N-linked
oligosaccharides because it was blocked by castanospermine (data not shown) (Hammond et al., 1994 Here for the first time, we found that the soluble and lumenal calreticulin has access to the growing nascent chains
in the ER of living cells. These results also confirmed that
calreticulin participates mainly in the early stages of folding whereas calnexin is involved throughout. The capacity
of the chaperones to bind to nascent chains indicated,
moreover, that trimming of the two outermost glucoses
from the N-linked glycans can occur on the growing nascent chain in <1 min after addition of the glycan by the
oligosaccharyl transferase.
HA Forms Ternary Complexes with Calnexin
and Calreticulin
The HA monomer has seven N-linked glycans. Four are
located in the stem domain attached to asparagines 8, 22, 38, and 483. One is in the hinge region (Asn 285), and two
(Asn 81 and Asn 165) are in the top domain (Fig. 9). With
so many glycans distributed widely over the surface, the
protein has the potential of binding more than one calnexin or calreticulin molecule at a time.
To test whether ternary complexes containing HA and
both of the chaperones occurred in the ER-derived microsomes, sequential double immunoprecipitations were performed (Fig. 3, A and B). Primary precipitations with
When the radioactivity in the various bands was quantified using a phosphorimager (Fig. 3 B), it was found that
10% of the HA was present in calreticulin-HA complexes
and 8% in calnexin-HA complexes. As much as 40% of
the HA occurred in ternary calnexin-calreticulin-HA complexes. A large fraction of the HA was thus associated with both chaperones simultaneously. Consistent with calreticulin's preference for IT1 and IT2, the ternary complexes
and the binary calreticulin-HA complexes were enriched
in IT1 and IT2 (Fig. 3, lanes 6 and 5, respectively). The binary calnexin-HA complexes contained more HA of the
fully oxidized NT type (Fig. 3, lane 8). It could be concluded that the majority of the chaperone-bound HA occurred in complexes containing both calnexin and calreticulin.
Maturation and Calnexin/Calreticulin Binding
of Single Glycosylation Site Mutants
Since a large amount of HA was associated with both
chaperones, it was of interest to determine whether these
chaperones bound to different regions of the HA molecule. A series of mutants were constructed in which glycosylation sites were eliminated by point mutations in the
consensus glycosylation sites. Typically, the Asn in the consensus sequence was replaced with Gln. In specific cases,
other changes were also made to confirm that the changes were due to the loss of the oligosaccharide and not to the
amino acid change.
In the first series of mutants, glycosylation sequences
were eliminated one by one. The mutant proteins were
translated in the presence of microsomes and analyzed for
folding and oligomerization as well as calnexin/calreticulin
binding. We found that six out of seven mutants were able
to fold to the NT form (Fig. 4 A). The same mutant proteins also assembled into trimers, judging by immunoprecipitation, with a trimer-specific anti-HA monoclonal antibody (Fig. 4 B). Clearly, the oligosaccharide moieties in positions 8, 22, 38, 165, 285, and 483 were dispensable
when eliminated singly.
However, the folding of some of these six mutant molecules was not identical to the wild type in every respect.
For example, whereas the rate of NT formation for mutants Deletion of the COOH-terminal stem glycan in
Mutant When the calnexin and calreticulin binding to the mutant proteins was measured by coimmunoprecipitation, no
significant differences compared to wild-type HA were
seen for any of them, including Calreticulin Binds Preferentially to Top
and Hinge Glycans
Since we were unable to disrupt binding of calnexin or calreticulin with single glycan deletions, combinations of multiple deletions were tested. Depending on the combination,
differences in chaperone binding now emerged. Elimination of the three sites in the top/hinge domain (
Surprisingly, the reverse experiment in which three stem
glycans ( To correct for this effect, an additional mutation (C305S)
was introduced. This mutation prevents the formation of
disulfide bond C281-C305 which is needed to convert IT1
and IT2 to NT (Braakman, I., and A. Helenius, manuscript
in preparation). It effectively traps the protein in a form
equivalent to early folding intermediates. With this mutation in place, the protein stayed as IT1 (data not shown). In
this mutant background, the loss of the three-stem domain
glycans in positions 8, 22, and 38 had little or no effect on
calreticulin binding (Fig. 5 B). However, removal of the
three top/hinge domain glycans resulted in a 64% drop,
confirming the preferential binding of calreticulin to the
top/hinge glycans. Removal of all three glycans was required for calreticulin binding to be affected. Calnexin
binding was again virtually unaffected by the loss of glycans, suggesting that it can bind both to top/hinge and the
stem domain glycans.
Calnexin and Calreticulin Binding to HA Mutants in
Living Cells
To test whether elimination of multiple glycans would have
similar effects on calreticulin and calnexin binding in live
cells, wild-type HA and two mutants were expressed in
CHO cells using the T7 vaccinia virus system. In addition
to including the C305S mutation to prevent full oxidation,
the mutant proteins tested were devoid of top and hinge
oligosaccharides ( Calnexin binding to Calnexin and Calreticulin Binding to Mutants with
Decreasing Numbers of Oligosaccharide Moieties
That calnexin can bind to many parts of the HA molecule
was confirmed by a series of mutants in which glycans were
progressively removed one at a time from the NH2 terminus towards the COOH terminus (Fig. 6, lanes 2-8), or
conversely, from the COOH terminus to the NH2 terminus
(Fig. 6, lanes 9-15). The stepwise loss of glycans resulted
in a ladder of HA molecules with faster electrophoretic mobilities the fewer the oligosaccharides. Thus, with about
2-kD steps for each glycan removed, the HA band approached the mobility of the untranslocated HA (denoted
as UT, Fig. 6 A) present in the lysates. In the nonreduced
gels (Fig. 6, B-D), UT could not be seen because it formed
disulfide cross-linked aggregates under the oxidizing conditions used.
Immunoprecipitation with anti-calnexin antibodies showed
that calnexin binding to the various mutant HA molecules
did not diminish significantly with decreasing number of
glycans until a molecule was reached that had only a single
glycan (Fig. 6 C, compare lanes 1-6 with 7 and 8, and lanes
9-13 with 14 and 15). In other words, calnexin formed stable immunoprecipitable complexes with all HA molecules
except those that had less than two glycans. This finding
was consistent with observations using other substrate glycoproteins (Cannon et al., 1996 In the case of calreticulin (Fig. 6 D), binding already
started to drop significantly when three or four out of the
seven glycans were lost (Fig. 6, compare lanes 1-5 with 6-8,
and lanes 9-11 with 12-15). In this case interpretation was,
however, more complicated because some of the mutants
folded faster than others. If the early folding events are
rapid, less binding of calreticulin is expected to occur.
However, the results were consistent with the multiple glycan deletion mutants discussed above (Fig. 5, B and C)
that showed that the presence of at least one of the top domain glycans is required for calreticulin binding.
Folding of Glycosylation Mutants in Microsomes and
Live Cells
It was evident from Fig. 6 B, which shows the nonreduced
anti-HA precipitates, that the progressive loss of oligosaccharides not only affects calnexin and calreticulin binding,
but also influences how HA folds. The accelerated folding
of mutants lacking glycans in positions N8, 22, and 38 is
clearly visible (Fig. 6 B, compare lanes 3 and 4 with 1).
When more than half of the wild-type HA was still in the
form of IT1 and IT2 (Fig. 6 B, lane 1), most of mutants It was also evident that folding was severely suppressed
when four or more glycans were removed starting from either the NH2 or the COOH terminus (Fig. 6 B, lanes 5-8
and 12-15). This was not unexpected since the fourth glycosylation site from either terminus is N81, which, when
removed, alone caused inhibition of NT formation and
HA trimerization (Fig. 4, A and B).
To test whether folding of glycosylation mutants would
also be abnormal in live cells, we expressed the wild-type
HA and two mutants ( The
Such acceleration of HA folding has been previously observed in microsomes when calnexin and calreticulin binding is inhibited with castanospermine (Hebert et al., 1996 As expected, the Calnexin Binds to Membrane-bound Forms of HA
More Efficiently
An important difference between calnexin and calreticulin
is that one is membrane bound and the other is soluble in
the ER lumen. Although both are known to interact with
membrane glycoproteins and soluble proteins (Helenius
et al., 1997 We translated wild-type HA and its anchor-free truncated counterpart by in vitro translation in microsomes,
and assayed for folding and calnexin and calreticulin binding. The anchor
Our analysis of wild-type and mutant forms of HA confirmed the importance of the N-linked oligosaccharides in
the general process of glycoprotein folding calnexin and
calreticulin binding. Not only is the presence of N-linked
glycans necessary for HA folding as previously shown
(Hurtley et al., 1989 When growing nascent chains of wild-type HA were analyzed, it could be shown that both calnexin and calreticulin already begin to associate during translation and translocation. Association was first detected in cells when the
HA chain was about half finished, i.e., at a time when it
was still ~1 min from termination (Braakman et al., 1991 Removal of NH2-terminal glycans in positions N8,22,
and 38 resulted in HA molecules that reached the fully oxidized form more rapidly than wild-type HA. When assayed in living cells, cotranslational folding of the Our results indicate that as the folding of the HA chain
progresses, calreticulin associated preferentially with the
three top/hinge domain glycans, N81, 165, and 285 (Fig. 9).
The preferential binding to these glycans may have several
reasons. First, the oligosaccharides in the top part of the
molecule may be more easily accessed from the lumen of
the ER, thus attracting the soluble calreticulin more efficiently than the membrane-bound calnexin. Second, being
a membrane protein, calnexin may bind more efficiently to
the membrane-proximal stem domain glycans, with the
result that only the top domain glycans are left for calreticulin to bind to. Support for this notion comes from the
observed reduction in calnexin binding to anchor-free HA
compared to wild-type HA. Mere membrane binding that
affects substrate specificity is shown by the observations of
Wada and co-workers (Wada et al., 1995 It is also possible that the steric arrangement of the top
domain glycans may find a better fit in calreticulin's binding site. The glycan binding site(s) are thought to be located in the P-domains of calnexin and calreticulin. Although
highly homologous to calnexin's, the P-domain of calreticulin is smaller and has three double sets of sequence repeats instead of four (Wada et al., 1991 Finally, the distinct binding specificities could be determined by protein-protein contacts. Such interactions have
been postulated to occur between MHC class I and II heavy
chains and calnexin (Arunachalam and Cresswell, 1995 As a consequence of binding to the top/hinge glycans,
calreticulin dissociates sooner from the folding HA chain
than calnexin. This is consistent with results indicating that
folding of HA begins from the top domain and proceeds
from there towards the stem (Braakman et al., 1992a One possible reason why calreticulin dissociates before
the HA molecule is fully folded is that the top domain fails
to be reglucosylated by the UDP-glucose:glycoprotein glucosyltransferase. It may no longer be recognized as unfolded by this folding sensor. This would imply that the transferase senses unfold locally rather than globally. In other
words, in a protein with several domains, only the misfolded ones may serve as substrates. Further studies are
needed to define how close to a misfolded domain a glycan has to be to serve as a substrate for reglucosylation.
The elimination of most of the glycans singly did not affect the outcome of the folding process. It did, however,
affect the folding rate by either increasing (glycans 8, 22, 38, and 483) or decreasing it (glycans 81, 165, and 285). Of
the seven oligosaccharides only glycan N81 proved to be
essential for HA folding in microsomes. Mutant HA molecules devoid of this glycan were virtually incapable of acquiring intrachain disulfide bonds. They also failed to form the interchain disulfides typically observed when HA misfolds (Hurtley et al., 1989 It is somewhat surprising that glycan N81 should prove
to be so essential because it is not conserved among influenza strains. The conserved glycans are all in the stem domain and correspond to N22, 38, and 483 in the X31 HA.
However, Gallagher and co-workers (1992) have suggested
that the oligosaccahrides in positions 8 or 81 of the X31
HA may, in fact, be essential for maturation. They found
that the HA of the Japan strain, which does not have these
glycans, reaches a transport-competent conformation after tunicamycin treatment, whereas the HA of X31 HA that
has them is completely tunicamycin sensitive (Gallagher
et al., 1992 The differences observed among strains of influenza are
further illustrated by studies performed by Roberts and co-workers, who have found that two out of three of the conserved stem domain glycans are required for the proper
maturation and transport of HA in the strain of fowl
plague virus (A/FPV/Rostock/34[H7N1]) (Roberts et al.,
1993 In summary, we find that N-linked glycosylation on HA
influences the folding process in many complex ways. One
thing is clear, however; the N-linked glycans allow the
newly synthesized protein to interact with the calnexin-
calreticulin cycle. Though not totally essential for the folding of HA, this serves to decrease the rate of folding, prevent premature trimerization, inhibit degradation, increase
the efficiency of folding, and expose the protein to efficient
quality control (Hammond et al., 1994 It is also clear that glycans have effects on HA folding
that are independent of calnexin and calreticulin. For the
X31 HA, this is illustrated by the observation that the protein is completely misfolded in the presence of tunicamycin but only partially affected by castanospermine (Hurtley et al., 1989 1-3 antenna (Ou et al., 1993
; Hammond and Helenius, 1994
; Hammond
et al., 1994
; Hebert et al., 1995
; Peterson et al., 1995
; Tector and Salter, 1995
; Ware et al., 1995
; Spiro et al., 1996
).
The association of these chaperones with their substrate
glycoproteins promotes correct folding and oligomeric assembly, prevents degradation, and supports quality control (Rajagopalan and Brenner, 1994
; Hebert et al., 1996
;
Vassilakos et al., 1996
).
, 1996
; Rodan et al.,
1996
; Van Leeuwen and Kearse, 1996
). The monoglucosylated form of the oligosaccharides is also generated in the
ER by the action of UDP-glucose/glycoprotein glucosyltransferase. This lumenal enzyme selectively reglucosylates glycoproteins that possess high mannose glycans only
if the proteins are incompletely folded (Sousa et al., 1992
;
Trombetta and Parodi, 1992
). Thus, by adding and removing glucoses, glucosidase II and the glucosyltransferase drive their substrates through a cycle of calnexin/calreticulin binding and release (Hammond and Helenius, 1993
;
Hebert et al., 1995
; Van Leeuwen and Kearse, 1997
). Glycoproteins stay in the cycle as long as they have a nonnative conformation, with the glucosyltransferase serving as
a folding sensor (Suh et al., 1989
; Hammond et al., 1994
).
; Peterson et al., 1995
; Ware et al.,
1995
; Spiro et al., 1996
), recent studies suggest that calnexin and calreticulin may differ in their substrate selection.
Vesicular stomatitis virus G protein binds to calnexin but
not to calreticulin (Hammond and Helenius, 1994
; Peterson et al., 1995
). The bound proteins observed by coimmunoprecipitation from pulse labeled cells are not identical
(Peterson et al., 1995
; Wada et al., 1995
). The two chaperones bind to major histocompatibility complex (MHC) class I
antigens at different stages of maturation (Sadasivan et al.,
1996
; Van Leeuwen and Kearse, 1996
), and in the case of influenza virus hemagglutinin (HA),1 calreticulin dissociates more rapidly than calnexin as folding proceeds (Hebert et al., 1996
). It is possible that the two chaperones have distinct functions during glycoprotein biosynthesis.
Materials and Methods
). This included the use of a
site-directed mutagenesis kit (QuikChange; Stratagene, La Jolla, CA).
The truncated, soluble form of HA was generated by substituting the
codon for the first transmembrane residue (T514) with the stop codon,
TGA. Wild-type and mutant HA were cloned into a pBluescript expression system (Stratagene), and mRNA was transcribed (after linearization
at the SalI or KpnI sites) with T7 RNA polymerase (Boehringer Mannheim, Indianapolis, IN).
). HA was translated
in the presence of 4.0 mM GSSG at 32°C for cotranslational folding studies. For posttranslational folding, HA was translated in the absence of
GSSG. After 1 h of translation, protein synthesis was inhibited with 50 mM
cycloheximide and 4.5 mM GSSG was added to initiate synchronous oxidation. Samples were treated at the indicated time points with 20 mM
N-ethylmaleimide (NEM) to block free thiols by alkylation before immunoprecipitation.
). The recombinant vaccinia virus with the T7
polymerase and the cowpox hr gene was gift of R. Drillien (CisINSERM,
Strasbourg, France). The presence of cowpox hr gene allows the virus to
infect CHO cells. The virus was propagated in BHK cells on 15-cm plates
for 3 d after infection with 0.1 plaque formation units (pfu)/cell. The infected cells were homogenized and centrifuged at 2,000 g for 10 min. The
supernatant was titered for plaque formation units.
, 1996
). HA antibodies were a mixture of polyclonals raised to whole influenza virus and an anti-peptide polyclonal generated against a peptide corresponding to an NH2-terminal peptide of HA
(NHA). Calreticulin antibodies were obtained from Affinity BioReagents,
Inc. (Golden, CO), and calnexin antisera was raised against a peptide corresponding to the COOH-terminal cytosolic tail of calnexin. Immunoprecipitated HA was resolved on nonreducing and reducing (7.5%) SDS-PAGE.
). HA bands were quantified
by densitometry with a digital gel scanner or phoshorimager (Visage 200;
Molecular Dynamics, Inc., Sunnyvale, CA).
Results
, 1996
). After
synthesis, the microsomes were treated with NEM to alkylate-free sulfhydryl groups, and solubilized with nonionic
detergent. After immunoprecipitating with antibodies to
HA, calnexin, or calreticulin, the samples were analyzed
without reduction by SDS-PAGE and autoradiography.
; Marquardt et al., 1993
; Braakman,
I., and A. Helenius, manuscript in preparation). The band
called IT1 corresponds to an ensemble of incompletely
folded HA molecules that lack disulfide bonds C52-C277
and C14-C466. These two disulfide bonds form large loops in the HA molecule, and their presence considerably increases the electrophoretic mobility of the SDS complexes.
The IT2 band contains molecules that have disulfide C52-
C277 but do not contain C14-C466. The fastest migrating
band (NT, for native) is composed of monomeric molecules that have both of these disulfide bonds.
; Marquardt et al., 1993
), the results indicate that calreticulin participates mainly in early stages of folding whereas
calnexin association occurs throughout the folding of HA
monomers.
Fig. 1.
Calnexin and calreticulin differ in their association with
HA in microsomes. 35S-HA was translated in vitro in the presence of canine pancreas microsomes at 32°C under oxidizing conditions (A-C, Co, lanes 1-3), or under reducing conditions. At
time zero (lane 4), GSSG was added to initiate posttranslational
oxidation (lanes 5-9). Alkylated samples were divided into three
fractions for immunoprecipitation with anti-HA (A, HA), anti-calnexin (B, CNX), or anti-calreticulin antibodies (C, CRT), and
then subjected to nonreducing SDS-PAGE and visualized by autoradiography. Partially oxidized intermediates IT1 and IT2 and
the fully oxidized native HA (NT) were resolved.
[View Larger Version of this Image (57K GIF file)]
). The presence of
DTT not only inhibits formation of disulfide bonds in HA
but also prevents efficient binding of calnexin and calreticulin (Fig. 1, lane 4; Hebert et al., 1995
). When GSSG was
added, calnexin and calreticulin associated with HA. The
same preference of calreticulin for IT1 and IT2 was seen
again (Fig. 1, lanes 5-9), indicating that it was determined
by the intrinsic properties of the HA molecule. It did not
depend on the vectorial translocation of the growing chain
into the ER, or on limited accessibility of the HA polypeptide within the translocon complex.
-glucosidases, to inhibit calnexin and calreticulin binding to HA (Hammond et al., 1994
; Chen et al., 1995
; Hebert et al.,
1995
; Peterson et al., 1995
). The cells were alkylated, solubilized, and the lysates subjected to immunoprecipitation
with antibodies against the NH2-terminal peptide of HA, to
calnexin or to calreticulin. To separate the chaperone-bound
HA molecules from other substrates, the anti-calnexin and
-calreticulin precipitates were dissolved and reprecipitated
with the anti-HA antibodies. The samples were then subjected to 2-D SDS-PAGE in which the first dimension was without reduction and the second with reduction (Chen et
al., 1995
). Proteins that lack disulfide bonds run on the diagonal, proteins with intermolecular disulfides migrate
above the diagonal, and proteins with intramolecular disulfides, as a general rule, run below the diagonal.
). The spur connected to the IT1
spot contains nascent chains that lack disulfide bonds C52-
C277 and C14-C466. They may contain one or more of the
small intramolecular disulfide loops. The spur connecting
to the IT2 spot corresponds to nascent chains that had disulfide C52-C277 but not C14-C466.
Fig. 2.
Cotranslational association of HA with calnexin and
calreticulin. Influenza-infected CHO cells were pulsed for 1.5 min
with [35S]methionine/cysteine, and then the lysate was divided
into three, immunoprecipitated, and analyzed by 2-D SDS-PAGE (nonreducing in the first dimension and reducing in the
second dimension). (A) HA immunoprecipitated with an antibody against the NH2 terminus of HA (-NHA). (B) HA immunoprecipitated sequentially with antibody against calnexin and
antibody against HA NH2 terminus (
-CNX/
-NHA). (C) HA
immunoprecipitated sequentially with antibody against calreticulin and antibody against HA NH2 terminus (
-CRT/
-NHA). The
circles above the 2-D gels denote molecular markers of 66 and 45 kD. Exposure time for B and C was five times longer than A.
[View Larger Version of this Image (32K GIF file)]
; Chen et al.,
1995
; Peterson et al., 1995
). From the length of the main
spur, it could be estimated that binding occurred by the
time the length of the nascent chains was ~40 kD. Among the full-length forms, calreticulin bound preferentially to
the least folded molecules (IT1) whereas calnexin bound
to all forms (IT1, IT2, and NT).
Fig. 9.
Calnexin and calreticulin binding regions on HA. The
structure of the ectodomain of HA, modified from Wiley and co-workers (Wilson et al., 1981) is depicted with N-linked glycans
and the disulfide bonds designated by the circles and the ball-and-sticks, respectively. The large disulfide loop, Cys 14-466, is
represented by the unfilled ball-and-sticks. The numbers refer to
the Asn residue that contain the N-linked glycosylations.
[View Larger Version of this Image (28K GIF file)]
-HA,
-calnexin, or -calreticulin (Hebert et al., 1996
) showed that
53% of the 35S-labeled HA was associated with calnexin,
and 45% with calreticulin (Fig. 3, lanes 3 and 6, respectively). When the supernatants from these precipitations
were reprecipitated with the same antibodies, no additional HA was brought down (Fig. 3, lanes 2, 4, and 7), indicating that the primary precipitations were quantitative. In contrast, when aliquots of the supernatants were reprecipitated after the first precipitation with antibodies against
the second chaperone, additional HA was precipitated
(Fig. 3, lanes 5 and 8). This HA corresponded to molecules
that were associated only with one of the chaperones. The
relatively small amount of HA in these complexes suggested that most of the complexes contained both chaperones.
Fig. 3.
HA is found in calnexin-calreticulin ternary complexes.
The presence of ternary calnexin-calreticulin-HA complexes was
analyzed using double immunoprecipitations. (A) HA was translated as in Fig. 1 for 1 h at 32°C under oxidizing conditions. HA
was first immunoprecipitated with anti-HA (1°, HA, lanes 1 and
2), -calnexin (1°, CNX, lanes 3-5), and -calreticulin (1°, CRT,
lanes 6-8) antisera. The supernatants were cleared with protein
A and reprecipitated with the indicated antisera (2°). (B) Bands
from the autoradiogram in A were quantified by a digital densitometer and plotted as the fraction of total HA (IT1 + IT2 + NT
from lane 1).
[View Larger Version of this Image (30K GIF file)]
Fig. 4.
Folding, oligomerization, and calnexin/calreticulin binding of single glycan deletion mutants. (A) Wild-type and single glycosylation mutants of HA were translated under oxidizing conditions in the presence of canine pancreas microsomes at 32°C for 1 h. At the indicated times, samples were removed, alkylated, lysed, and immunoprecipitated with anti-HA antibodies. HA was resolved by nonreducing
SDS-PAGE and autoradiography. (B) Translations were performed as above with oxidation carried out for 8 h at 32°C. The alkylated
HA was immunoprecipitated with anti-trimer specific HA antibodies and resolved by reducing SDS-PAGE and autoradiography. (C)
Translation of 35S-labeled HA were performed as in A for 1 h at 32°C. After alkylation, HA was immunoprecipitated with anti-HA, -calnexin, and -calreticulin antibodies. HA was resolved upon nonreducing and reducing SDS-PAGE and autoradiography. The fraction HA
coprecipitating with calnexin or calreticulin was calculated as the amount of anti-calnexin or -calreticulin precipitable HA divided by the
total HA precipitated with anti-HA antibodies as described in Fig. 3.
[View Larger Versions of these Images (67 + 19K GIF file)]
8,
22 and
38 was similar to wild type, only minor
amounts of IT2 was seen as intermediates. Evidently, the
transition from IT2 to NT (i.e., the formation of the disulfide C14-C466) was accelerated.
483, on
the other hand, resulted in oxidation to NT at a rate faster
than the wild type (Fig. 4 A, below in Fig. 6 B, and data not
shown). Such differences indicated that although changes
in these six glycosylation sites did not affect the final outcome of the folding process, they did affect the kinetics of
folding and the expression of intermediate forms.
Fig. 6.
Determination of the number of glycans required for
calnexin or calreticulin binding. Consensus glycosylation sequences
were deleted by increasing, in single increments, from the NH2 to
the COOH terminus (lanes 2-8), and from the COOH to the NH2
terminus (lanes 9-15). 35S-HA was translated in vitro for 1 h at
32°C, alkylated, lysed, and precipitated with anti-HA, -calnexin
(CNX), or -calreticulin (CRT) antisera. The labeled HA was resolved by nonreducing (A, NR) and reducing (B-D, RD) SDS-PAGE and autoradiography. The band designated as UT corresponds to the untranslocated and unglycosylated HA.
[View Larger Version of this Image (86K GIF file)]
81, which lacks one of the two oligosaccharides
present in the top domain, was an exception. It was greatly
inhibited in its ability to fold beyond a form that was either
fully reduced or equivalent to IT1 with some of the small
disulfide bonded loops in place (Fig. 4 A). Surprisingly,
this protein seemed unable to form interchain disulfide
bonds which are common for misfolded forms of HA (Hurtley et al., 1989
; Braakman et al., 1992b
). Abolishing the
glycosylation site by a T83A mutation rather than the N81Q mutation resulted in the same folding phenotype (data not
shown) indicating that the effect was not due to a specific
amino acid change. The inability of
81 to oxidize was
likely due to disruption in the formation of disulfide C67-
C76, which is very close to this glycan (see Fig. 9), and
known to be crucial for formation of other intrachain disulfides in the HA monomer (Braakman, I., and A. Helenius, manuscript in preparation).
81 (Fig. 4 C). With exception of
483, they also bound normal levels of calreticulin.
The 40% reduction in calreticulin binding observed for
483 was likely caused by the rapid folding of this mutant
HA, resulting in a shorter exposure of the preferred calreticulin binding conformers, IT1 and IT2 (see below). We
concluded that no single glycan in HA served as the exclusive binding site for either calnexin or calreticulin.
81,165,285)
resulted in almost complete loss of calreticulin binding
(Fig. 5 A). In contrast, the mutant protein bound almost
normal amounts of calnexin. The top/hinge glycans were evidently needed for calreticulin but not for calnexin binding.
Fig. 5.
Mapping of calnexin and calreticulin binding sites by
mutating consensus glycosylation sequences. The consensus sequences for glycosylation were eliminated by changing the Asn to
Gln. The HA was translated under oxidizing conditions for 1 h at
32°C. (A) The fraction of total HA coprecipitating with the various antisera is plotted. (B) Binding to calnexin and calreticulin was
monitored as described above for a set of HA mutants which had,
in addition to mutations in the designated consensus glycosylation sites, an additional mutation, C305S, which arrested HA oxidation in the IT1 conformation. The disruption of disulfide 281-
305 by changing Cys305 to a Ser results in the accumulation of
nonaggregated IT1 (not shown). The values given are the average
of four experiments. (C) HA binding to calnexin and calreticulin
in CHO cells was quantified as in A and B. 35S-HA was expressed
in CHO cells by use of the T7-based vaccinia expression system. 4 h
after infection, cells were pulsed with [35S]methionine/cysteine
for 2 min at 37°C. Alkylated and lysed samples were then processed as above.
[View Larger Version of this Image (24K GIF file)]
8,22,38) were eliminated also showed reduced
binding of calreticulin (Fig. 5 A), whereas calnexin binding
was only marginally reduced. Further studies showed that
the apparent inability of calreticulin to bind to this particular mutant was caused by its accelerated folding (see below). The early oxidative intermediates IT1 and IT2 that
normally bind calreticulin were so short-lived in this mutant that the calreticulin-binding phase during folding could not be experimentally observed.
81,165,285/C305S) and stem glycans
(
8,22,38/C305S). 3 h after transfection, the cells were
pulse labeled for 2 min. After alkylation and solubilization (Braakman et al., 1991
), lysates were immunoprecipitated.
SDS-PAGE was performed and the HA bands quantified
using the phosphorimager.
8,22,38/C305S and
81,165,285/
C305S mutants was found to be 33% lower than to wild-type HA (Fig. 5 C). A similar decrease was observed in the
binding of calreticulin to
8,22,38/C305S, but its binding to
81,165,285/C305S was found to be dramatically decreased.
Taken together, the results suggest that in live cells, as well
as in microsomes, calreticulin binds preferentially, but not
exclusively, to the top domain glycans. Calnexin, on the
other hand, can use glycans in any part of the protein.
Since the stem disulfides are the last to form during folding (Braakman et al., 1991
), the results are consistent with the observation that calnexin binding continues for a
longer period during HA folding than calreticulin binding
(Fig. 1).
; Rodan et al., 1996
).
8,
22 (Fig. 6, lane 3) and
8,22,38 (Fig. 6, lane 4) had been
converted to NT. Folding was also accelerated in mutants in
which the most COOH-terminal glycan (
483; Fig. 6 B,
lane 9 or Fig. 4 A), or the two most COOH-terminal glycans
(
285,483, Fig. 6 B, lane 10), had been eliminated.
8,22,38 and
81) in CHO cells using the T7-based vaccinia virus expression. The 2-D SDS-PAGE system already described above was used to analyze the folding status of immunoprecipitated nascent and
full-length HA molecules after a 2-min pulse of [35S]methionine/cysteine.
8,22,38 mutant and the wild type displayed quite
different 2-D gel patterns (Fig. 7, A and B). Whereas the
IT1 spur was visible both in the wild type and in the mutant, the IT2 spur in the mutant was longer and more
prominent. This indicated that cotranslational formation of
disulfide C52-C277 occurred earlier and more efficiently.
The NT spot was also more prominent, suggesting more
rapid formation of the C14-C466 disulfide. We concluded
that co- and posttranslational folding of HA was accelerated when the three glycans in the NH2-terminal part of
the sequence were eliminated.
Fig. 7.
Cotranslational folding of wild-type and mutant HA,
8,22,38, and
81. CHO cells were infected with recombinant
vaccinia virus and then transfected with the various HA genes. 4 h
after transfection, the cells were starved in cysteine-/methionine-free medium for 30 min and then pulse labeled with [35S]cysteine/
methionine for 2 min. Immediately after labeling, the cells were
lysed and precipitated with antibodies raised against a peptide
corresponding to the NH2 terminus of HA. The precipitates were
subjected to 2-D SDS-PAGE as in Fig. 2.
[View Larger Version of this Image (35K GIF file)]
).
Taken together with the observed acceleration of folding
of mutants
8,22,
8,22,38,
483 and
285,483 in microsomes (Figs. 4 A and 6 B), the data suggest that calnexin binding to the oligosaccharides present in the stem
domain in positions 8, 22, 38, and 483 serves to slow down
HA folding and the formation of NT. Removal of some of
these glycans, or inhibition of calnexin binding to them, results in faster folding.
81 mutant folded inefficiently to the
NT form in the live cells (Fig. 7 C). Essentially no protein
reached the IT2 or NT forms after a 2-min pulse. A similar
result was obtained whether N81Q mutation or the T83A
mutation was used to eliminate the consensus glycosylation sequence (data not shown). The folding defect observed for these mutants confirmed that the oligosaccharide in position N81 is essential for proper folding of the
HA in the X31 strain of virus.
), it was of interest to determine whether the division of labor observed in their interaction with the membrane-bound wild-type HA would apply for a soluble anchor-free form of HA.
HA was found to undergo normal folding to NT, but as reported previously for live cells (Singh
et al., 1990
); it was unable to trimerize (data not shown).
Whereas anti-calreticulin antibodies coprecipitated equal
amounts wild-type and anchor
HA (50%), calnexin precipitated 82% of the wild type, but only 54% of anchor
HA (Fig. 8). The soluble HA thus associated somewhat
less efficiently with the membrane-bound chaperone. Presumably, the oligosaccharides in the stem region of the
anchor
HA were not as easily accessed by calnexin as in
the membrane-bound wild-type HA.
Fig. 8.
Binding of anchor HA to calnexin and calreticulin. A
soluble anchor-free form of HA was expressed truncated at the
first transmembrane residue by replacing the codon for T514 with
the stop codon, TGA. Wild-type and truncated (Anchor
) forms
of HA were translated for 1 h at 32°C under oxidizing conditions
in the presence of microsomes. After 1h, HA was alkylated, lysed,
and precipitated with anti-HA, -calreticulin, and -calnexin antisera.
HA was resolved, visualized, and quantified as in Fig. 3. Note the
levels of chaperone binding in this experiment were higher
throughout, likely due to the use of different antibody bleeds.
This experiment was repeated three times with similar levels observed.
[View Larger Version of this Image (15K GIF file)]
Discussion
; Gallagher et al., 1992
), but their number and location in the molecule are also crucial. The results showed that the glycans affect the structural maturation of the protein locally and globally. They influence the
rate of folding and the formation of disulfide bonds. By
determining the interactions with chaperones such as calnexin and calreticulin, they help to define the orderly progression of folding from one domain to the next. Calnexin
and calreticulin, although working according to the same
overall principles, were found to display distinct interactions with folding intermediates of HA.
).
At this time, five of the seven oligosaccharide chains (N8,
22, 38, 81, and 165) had been added. Since calnexin and
calreticulin binding was castanospermine sensitive, at least some of them must have been trimmed to the monoglucosylated form by glucosidase I and II. It is known that these
ER glucosidases have access to nascent chains, and that
glucose trimming is rapidly initiated (Hubbard and Ivatt,
1981
). No indication of disulfide bond formation was detectable at the time of initial calnexin and calreticulin association.
8,22,
and 38 mutant protein was also found to fold faster. We
have previously reported that inhibition of calnexin and
calreticulin binding by addition of castanospermine has a similar effect; the rate of NT formation is accelerated and
aberrant nonnative disulfide bonds are formed cotranslationally (Chen et al., 1995
; Hebert et al., 1996
). Based on
these observations, we hypothesize that by binding to the
NH2-terminal glycans, the chaperones sequester the NH2-terminal portion of the growing chain. This is likely to be
important because the sequence comprising residues 1-50
is predestined to be an integral part of the COOH-terminal stem domain (Fig. 9), and, therefore, has to wait before
it can begin to fold. For example, Cys 14 emerges from the
translocon in cells 1.5 min or more before its partner, Cys
466. By binding to the NH2-terminal glycans, the chaperones may serve to postpone the folding of the NH2-terminal segment and prevent Cys 14 from oxidizing with the
eight intervening cysteines in the polypeptide chain. This
could, in fact, explain why three glycans are located in the
first 38 residues of the X31 HA used in this study.
), who found that
the pattern of calreticulin-bound proteins in HepG2 cells
became similar to that of calnexin when the calreticulin was anchored to the membrane by calnexin's transmembrane region.
; Michalak et al.,
1992
; David et al., 1993
). These differences in the proteins
may contribute to selectivity in binding.
; Williams, 1995
; Zhang et al., 1995
). Although protein-protein
contacts are not required for the formation of stable complexes between substrate glycoproteins and the two chaperones (Rodan et al., 1996
; Zapun et al., 1997
), they cannot be ruled out as additional factors contributing to the
specificity of binding.
;
Braakman, I., and A. Helenius, manuscript in preparation).
The progression of folding is best illustrated by the orderly, hierarchical formation of disulfide bonds that starts
with the formation of either C67-C76 and C97-C139 in
the top domain, and proceeds to the hinge domain disulfide C281-C305, then disulfide C52-C277 (Fig. 9)(Braakman, I., and A. Helenius, manuscript in preparation). We
have, in this study, taken advantage of this stringent oxidation program by using the mutation C305S, which arrests
oxidation at IT1, to study calreticulin binding to glycosylation mutants that would otherwise fold too rapidly.
; Marquardt and Helenius, 1992
).
The protein remained essentially unoxidized for a long
time. The explanation for the poor folding may be the
close vicinity of N81 to a top domain disulfide bond, C67-
C76 (Fig. 9). Interestingly, when the cysteines of this disulfide are mutated to serines a similar oxidation-deficient
phenotype is observed (Braakman, I., and A. Helenius,
manuscript in preparation). Thus, the most likely explanation for the folding defect in
81 is that the formation of a
disulfide bond C67-C76 is inhibited when the glycan is
missing. This disulfide is important as it serves as an early
obligatory link in a chain of downstream oxidation events.
). However, when they analyzed single-site glycosylation mutants of X31 HA, they found that no single
glycan was essential for the acquisition of a transport-competent conformation in live cells. In their study, transport
ability was analyzed after extended times of chase, whereas
here we have monitored the folding or oxidation process.
In live cells the
81 mutant displayed a clear-cut cotranslational folding defect, but after a time some of it was able to
reach a folded conformation (data not shown) and therefore, would be expected to be competent for transport.
). However, the location of the top glycans in this
strain are very different from that of the X31 strain glycans. Furthermore, most HAs from avian and porcine influenza strains have no glycans in the top or hinge domain.
Depending on the strain, glycans can, therefore, be dispensable in the formation of the top domain disulfide bonds.
; Hebert et al., 1996
).
HA folding involves calnexin and calreticulin in an elaborate series of interactions starting on the nascent chain. Binding of calnexin and calreticulin to distinct oligosaccharides and different domains is apparently used by HA to organize the processes of folding. For example, it seems likely
that calnexin and calreticulin are used to temporarily sequester the NH2-terminal segment of HA, thus promoting
more efficient cotranslational folding of NH2-terminal and
COOH-terminal sequences to form the stem domain.
; Hammond et al., 1994
; Hebert et al., 1996
).
The requirement for a glycan in position N81 provides one
example of a direct effect. The importance of this oligosaccharide seems to be based on a local influence on the formation of a nearby disulfide bond. In addition to local effects, the presence of oligosaccharides is likely to have
more global influences on folding by increasing overall polarity of folding intermediates, thus counteracting their
tendency to aggregate (Kern et al., 1992
; Marquardt and
Helenius, 1992
; Kern et al., 1993
).
Received for publication 31 July 1997 and in revised form 28 August 1997.
We would like to acknowledge R. Gilmore (University of Massachusetts Medical Center), I. Braakman (University of Amsterdam, Amsterdam, The Netherlands), and T. Marquardt (University of Meunster, Muenster, Germany) for providing the canine pancreas microsomes and assistance with some of the glycosylation and truncation mutants, respectively. We would also like to thank N. Ayad (Yale University, New Haven, CT) and E.S. Trombetta (Yale University) for helpful discussions.This work was supported by the Patrick and Catherine Weldon Donaghue Medical Research Foundation to D.N. Hebert, and by National Institutes of Health grants to A. Helenius (5-RO1-GM38346 and 1-RO1-GM52972) and J.-X. Zhang (1-532-DKO9309).
GSSG, oxidized glutathione; HA, hemagglutinin; NEM, N-ethylmaleimide; NHA, NH2-terminal peptide of HA; 2-D, two-dimensional.
1. |
Arunachalam, B., and
P. Cresswell.
1995.
Molecular requirements for the interaction of class II major histocompatibility complex molecules and invariant
chain with calnexin.
J. Biol. Chem.
270:
2784-2790
|
2. | Braakman, I., H. Hoover-Litty, K.R. Wagner, and A. Helenius. 1991. Folding of influenza hemagglutinin in the endoplasmic reticulum. J. Cell Biol. 114: 401-411 [Abstract]. |
3. | Braakman, I., J. Helenius, and A. Helenius. 1992a. Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum. EMBO (Eur. Mol. Biol. Organ.) J. 11: 1717-1722 [Abstract]. |
4. | Braakman, I., J. Helenius, and A. Helenius. 1992b. Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum. Nature (Lond.). 356: 260-262 |
5. |
Cannon, K.S.,
D.N. Hebert, and
A. Helenius.
1996.
Glycan-dependent and -independent association of vesicular stomatitis virus G protein with calnexin.
J.
Biol. Chem.
271:
14280-14284
|
6. | Chen, W., J. Helenius, I. Braakman, and A. Helenius. 1995. Cotranslational folding and calnexin binding of influenza hemagglutinin in the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA. 92: 6229-6233 [Abstract]. |
7. |
David, V.,
F. Hochstenbach,
S. Rajagopalan, and
M.B. Brenner.
1993.
Interaction with newly synthesized and retained proteins in the endoplasmic reticulum suggests a chaperone function for human integral membrane protein
IP90 (calnexin).
J. Biol. Chem.
268:
9585-9592
|
8. | Gallagher, P.J., J.M. Henneberry, J.F. Sambrook, and M.-J. Gething. 1992. Glycosylation requirements for intracellular transport and function of the hemagglutinin of influenza virus. J. Virol. 66: 7136-7145 [Abstract]. |
9. | Hammond, C., and A. Helenius. 1993. A chaperone with a sweet tooth. Curr. Biol. 3: 884-885 . |
10. | Hammond, C., and A. Helenius. 1994. Folding of VSV G protein: sequential interaction with BiP and calnexin. Science (Wash. DC). 266: 456-458 |
11. | Hammond, C., I. Braakman, and A. Helenius. 1994. Role of N-linked oligosaccharides, glucose trimming, and calnexin during glycoprotein folding in the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA. 91: 913-917 [Abstract]. |
12. | Hebert, D.N., B. Foellmer, and A. Helenius. 1995. Glucose trimming and reglucosylation determines glycoprotein association with calnexin. Cell. 81: 425-433 |
13. | Hebert, D.N., B. Foellmer, and A. Helenius. 1996. Calnexin and calreticulin promote folding, delay oligomerization, and suppress degradation of influenza hemagglutinin in microsomes. EMBO (Eur. Mol. Biol. Organ.) J. 15: 2961-2968 [Abstract]. |
14. | Helenius, A., E.S. Trombetta, D.N. Hebert, and J.F. Simons. 1997. Calnexin, calreticulin, and the folding of glycoproteins. Trends Cell. Biol. 7: 193-200 . |
15. | Hubbard, S.C., and R.J. Ivatt. 1981. Synthesis and processing of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 50: 555-584 |
16. | Hurtley, S.M., D.G. Bole, H. Hoover-Litty, A. Helenius, and C.S. Copeland. 1989. Interactions of misfolded influenza hemagglutinin with binding protein (BiP). J. Cell Biol. 108: 2117-2126 [Abstract]. |
17. |
Kern, G.,
N. Schülke,
F.Z. Schmid, and
R. Jaenicke.
1992.
Stability, quaternary
structure, and folding of internal, external, and core-glucosylated invertase
form yeast.
Protein Sci.
1:
120-131
|
18. |
Kern, G.,
D. Kern,
R. Jaenicke, and
R. Seckler.
1993.
Kinetics of folding and association of differently glycosylated variants of invertase from Saccharomyces cerevisiae.
Protein Sci.
2:
1862-1868
|
19. | Marquardt, T., and A. Helenius. 1992. Misfolding and aggregation of newly synthesized proteins in the endoplasmic reticulum. J. Cell Biol. 117: 505-513 [Abstract]. |
20. |
Marquardt, T.,
D.N. Hebert, and
A. Helenius.
1993.
Posttranslational folding of
influenza hemagglutinin in isolated endoplasmic reticulum-derived microsomes.
J. Biol. Chem.
268:
19618-19625
|
21. | Michalak, M., R.E. Milner, K. Burns, and M. Opas. 1992. Calreticulin. Biochem. J. 285: 681-692 |
22. | Ou, W.-J., P.H. Cameron, D.Y. Thomas, and J.J.M. Bergeron. 1993. Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature (Lond.). 364: 771-776 |
23. | Peterson, J.R., A. Ora, P. Nguyen, Van, and A. Helenius. 1995. Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins. Mol. Biol. Cell. 6: 1173-1184 [Abstract]. |
24. | Rajagopalan, S., and M.B. Brenner. 1994. Calnexin retains unassembled major histocompatibility complex class I free heavy chains in the endoplasmic reticulum. J. Exp. Med. 180: 407-412 [Abstract]. |
25. | Roberts, P.C., W. Garten, and H.-D. Klenk. 1993. Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin. J. Virol. 67: 3048-3060 [Abstract]. |
26. | Rodan, A.R., J.F. Simons, E.S. Trombetta, and A. Helenius. 1996. N-linked oligosaccharides are necessary and sufficient for association of RNase B with calnexin and calreticulin. EMBO (Eur. Mol. Biol. Organ.) J. 15: 6921-6930 [Abstract]. |
27. | Sadasivan, B., P.J. Lehner, B. Ortmann, T. Spies, and P. Cresswell. 1996. Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP. Immunity. 5: 103-114 |
28. | Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 545 pp. |
29. | Singh, I., R.W. Doms, K.R. Wagner, and A. Helenius. 1990. Intracellular transport of soluble and membrane-bound glycoproteins: folding, assembly, and secretion of anchor-free influenza hemagglutinin. EMBO (Eur. Mol. Biol. Organ.) J. 9: 631-639 [Abstract]. |
30. | Sousa, M.C., M.A. Ferrero-Garcia, and A.J. Parodi. 1992. Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. Biochemistry. 31: 97-105 |
31. |
Spiro, R.G.,
Q. Zhu,
V. Bhoyroo, and
H.-D. Söling.
1996.
Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi.
J. Biol.
Chem.
271:
11588-11594
|
32. | Suh, P., J.E. Bergmann, and C.A. Gabel. 1989. Selective retention of monoglycosylated high mannose oligosaccahrides by a class of mutant vesicular stomatitis virus G proteins. J. Cell Biol. 108: 811-819 [Abstract]. |
33. |
Tector, M., and
R.D. Salter.
1995.
Calnexin influences folding of human class I
histocompatibility proteins but not their assembly with ![]() |
34. |
Trombetta, S.E., and
A.J. Parodi.
1992.
Purification to apparent homogeneity
and partial characterization of rat liver UDP-glucose:glycoprotein glucosyltransferase.
J. Biol. Chem.
267:
9236-9240
|
35. |
Van Leeuwen, J.E.M., and
K.P. Kearse.
1996.
Deglucosylation of N-linked glycans is an important step in the dissociation of calreticulin-class I-TAP complexes.
Proc. Natl. Acad. Sci. USA.
93:
13997-14001
|
36. |
Van Leeuwen, J.E.M., and
K.P. Kearse.
1997.
Reglucosylation of N-linked glycans is critical for calnexin assembly with T cell receptor (TCR) ![]() ![]() |
37. | Vassilakos, A., M.F. Cohen-Doyle, P.A. Peterson, M.R. Jackson, and D.B. Williams. 1996. The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules. EMBO (Eur. Mol. Biol. Organ.) J. 15: 1495-1506 [Abstract]. |
38. |
Wada, I.,
D. Rindress,
P.H. Cameron,
W.-J. Ou,
J.J. Doherty II,
D. Louvard,
A.W. Bell,
D. Dignard,
D.Y. Thomas, and
J.J.M. Bergeron.
1991.
SSR![]() |
39. |
Wada, I.,
S.-I. Imai,
M. Kai,
F. Sakane, and
H. Kanoh.
1995.
Chaperone function of calreticulin when expressed in the endoplasmic reticulum as the
membrane-anchored and soluble forms.
J. Biol. Chem.
270:
20298-20304
|
40. |
Ware, F.E.,
A. Vassilakos,
P.A. Peterson,
M.R. Jackson,
M.A. Lehrman, and
D.B. Williams.
1995.
The molecular chaperone calnexin binds Glc1Man9GlcNAc2
oligosaccharides as an initial step in recognizing unfolded glycoproteins.
J.
Biol. Chem.
270:
4697-4704
|
41. | Williams, D.B.. 1995. Calnexin: a molecular chaperone with a taste for carbohydrate. Biochem. Cell Biol. 73: 123-132 |
42. | Wilson, I.A., J.J. Skehel, and D.C. Wiley. 1981. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature (Lond.). 289: 366-373 |
43. | Zapun, A., S.M. Petrescu, P.M. Rudd, R.A. Dwek, D.Y. Thomas, and J.J.M. Bergeron. 1997. Conformation independent binding of monoglucosylated ribonuclease B to calnexin. Cell. 88: 29-38 |
44. |
Zhang, Q.,
M. Tector, and
R.D. Salter.
1995.
Calnexin recognizes carbohydrate
and protein determinants of class I major histocompatibility complex molecules.
J. Biol. Chem.
270:
3944-3948
|