(Received for publication, September 9, 1994; and in revised form, November 22, 1994)
From the
Calnexin is a molecular chaperone that resides in the membrane
of the endoplasmic reticulum. Most proteins that calnexin binds are N-glycosylated, and treatment of cells with tunicamycin or
inhibitors of initial glucose trimming steps interferes with calnexin
binding. To test if calnexin is a lectin that binds early
oligosaccharide processing intermediates, a recombinant soluble
calnexin was created. Incubation of soluble calnexin with a mixture of
GlcMan
GlcNAc
oligosaccharides resulted in specific binding of the
Glc
Man
GlcNAc
species. Furthermore,
Glc
Man
GlcNAc
oligosaccharides bound relatively poorly, suggesting that, in addition
to a requirement for the single terminal glucose residue, at least one
of the terminal mannose residues was important for binding. To assess
the involvement of oligosaccharide-protein interactions in complexes of
calnexin and newly synthesized glycoproteins,
-antitrypsin or the heavy chain of the class I
histocompatibility molecule were purified as complexes with calnexin
and digested with endoglycosidase H. All oligosaccharides on either
glycoprotein were accessible to this probe and could be removed without
disrupting the association with calnexin. Furthermore, the addition of
1 M
-methyl glucoside or
-methyl mannoside had no
effect on complex stability. These findings suggest that once complexes
between calnexin and glycoproteins are formed, oligosaccharide binding
does not contribute significantly to the overall interaction. However,
it is likely that the binding of
Glc
Man
GlcNAc
oligosaccharides is a
crucial event during the initial recognition of newly synthesized
glycoproteins by calnexin.
Calnexin (previously known as p88 or IP90) is a resident protein
of the endoplasmic reticulum (ER) ()that was originally
identified by virtue of its transient association with assembling class
I histocompatibility molecules(1) . Subsequently, calnexin has
been found associated with folding and assembly intermediates of a wide
array of soluble and membrane
proteins(2, 3, 4) . These include subunits of
the T cell receptor(2, 5) , membrane
immunoglobulin(2) , class II histocompatibility
molecules(6, 7) ,
integrins(8) , influenza hemagglutinin (HA)(9) ,
vesicular stomatitis virus G protein(9) , as well as many
monomeric secretory glycoproteins such as
-antitrypsin
and transferrin(4, 10) .
Binding of calnexin to
most proteins occurs rapidly following (and possibly during) their
synthesis. Its dissociation appears to correlate with folding or
assembly events. In the case of transferrin and influenza HA, calnexin
binds to incompletely oxidized folding intermediates and dissociates at
about the time fully disulfide-bonded molecules are
formed(4, 9) . For the major secretory glycoprotein of
Madin-Darby canine kidney cells, gp80, calnexin dissociation correlates
with gp80 precursor folding as judged by the differential
susceptibility to proteinase K of calnexin-bound versus released
molecules(11) . Furthermore, the dissociation of calnexin from
the gp80 precursor can be blocked by modulating disulfide bond
formation with either dithiothreitol or diamide. In addition to
folding, subunit assembly can occur while polypeptides are associated
with calnexin. Assembly of the heavy chain,
-microglobulin (
m), and peptide
ligand of mouse class I histocompatibility molecules takes place on
calnexin. Formation of the complete ternary complex is required for
efficient dissociation of calnexin since incomplete complexes lacking
m or peptide exhibit prolonged binding to
calnexin(12) . By contrast, in human cells, assembly of class I
heavy chain-
m heterodimers appears to be sufficient to
trigger calnexin dissociation (13, 14) . Class II
histocompatibility molecules assemble into a large complex consisting
of three invariant chains and two
dimers while associated
with calnexin. Addition of the final
dimer correlates with
calnexin dissociation(7) . The consistent observation that
calnexin interacts with incompletely folded or assembled proteins, but
is absent from native (or nearly native) structures, suggests a
molecular chaperone function for calnexin.
Although direct evidence
demonstrating that calnexin facilitates protein folding or assembly
events is lacking, it is clear that calnexin is a component of the
quality control system that retains misfolded or incompletely
folded/assembled proteins in the ER. For both class I and class II
histocompatibility molecules, dissociation of calnexin correlates
closely with the transport of these molecules out of the
ER(1, 7) . Furthermore, incompletely assembled forms
of class I molecules(12) , the T cell receptor(5) , and
integrins (8) remain stably associated with calnexin and are
not transported. Misfolded mutant proteins such as those produced by
the metabolic incorporation of amino acid analogs(4) , the
ts045 mutant of vesicular stomatitis virus G protein(9) , and a
truncated variant of -antitrypsin (10) are
also retained as complexes with calnexin. A direct demonstration of
calnexin's capacity to retain incompletely assembled proteins was
provided by co-expressing calnexin along with free class I heavy chains
or heavy chain-
m heterodimers in Drosophila cells(15) . The aberrant transport of these assembly
intermediates out of the ER that occurs in Drosophila cells
was impeded when calnexin was co-expressed. In a separate study,
retention by calnexin was demonstrated by expressing either full-length
calnexin or a truncated variant that lacks an ER localization signal in
cells that also express the T cell receptor
subunit or class I
heavy chain subunit. Whereas these subunits were retained in the ER in
association with intact calnexin, their association with truncated
calnexin resulted in redistribution to the Golgi complex or cell
surface(16, 17) .
The most extensively studied
molecular chaperones are soluble ATPases that are members of the Hsp 60
and 70 families of heat shock proteins. Through ATP-driven cycles of
binding and release these chaperones act to stabilize unfolded proteins
and prevent their aggregation(18) . Calnexin differs
substantially from Hsp 60 and 70 chaperones in that it is an integral
membrane protein of 574 residues containing a single type 1
transmembrane domain(5, 19) . Its unglycosylated ER
luminal domain (464 amino acids) contains several regions with homology
to calreticulin, and its cytoplasmic domain contains phosphorylation
sites for casein kinase II (20, 21) as well as an ER
localization signal (-RKPRRE) at the C terminus(16) . Like
calreticulin, the major calcium binding protein of the ER lumen,
calnexin binds calcium(11) . This property could potentially be
involved in regulating calnexin associations because chelation of
calcium in vitro has been shown to disrupt complexes of
calnexin and a truncated variant of
-antitrypsin(10) . Similarly, treatment of
cells with the Ca
ionophore A23187 prevents binding
of calnexin to the class I H-2L
molecule(22) . By
sequence analysis, calnexin has no apparent nucleotide binding sites,
and induction by stress has not been demonstrated(23) . All of
these differences from Hsp chaperones may reflect a unique function
and/or mechanism of action of calnexin in protein biogenesis in the ER.
One of the most conspicuous characteristics of calnexin is its
apparent specificity for glycoproteins that possess Asn-linked
oligosaccharides. Ou et al.(4) originally showed that
pretreatment of human hepatoma cells with tunicamycin prevents the
formation of complexes between calnexin and many newly synthesized
secretory glycoproteins. This finding was subsequently reproduced with
the integral membrane glycoproteins, influenza HA and vesicular
stomatitis virus G. Furthermore, pretreatment with the
-glucosidase I and II inhibitors, castanospermine and
1-deoxynojirimycin, blocks the binding of calnexin to HA or G
proteins(9) . In contrast, the
-mannosidase inhibitor
1-deoxy-mannojirimycin has no effect. The data obtained with the
oligosaccharide processing inhibitors suggests that glucose trimming of
newly synthesized glycoproteins is a requirement for calnexin binding.
Additional studies showed that the HA glycoprotein isolated from a
complex with calnexin likely contains oligosaccharides with one or two
terminal glucose residues and that the vesicular stomatitis virus G
ts045 mutant, which possesses monoglucosylated oligosaccharides for
extended periods in the ER, exhibits prolonged association with
calnexin. All of these observations led to the proposal that for
calnexin binding to occur, a glycoprotein must possess oligosaccharides
that have undergone partial trimming from the initial
Glc
Man
GlcNAc
structure to one
containing either two or, more likely, a single glucose
residue(9, 24) .
One interpretation of these
results is that calnexin is a lectin with specificity for
monoglucosylated oligosaccharides. However, as discussed
recently(23) , other interpretations are equally plausible. In
fact some observations are difficult to reconcile with recognition of
monoglucosylated oligosaccharide being a prerequisite for calnexin
binding. For example, both the T cell receptor subunit that lacks
Asn-linked oligosaccharides and a recombinant form of the multidrug
resistance P glycoprotein in which N-glycosylation sites are
absent, form stable and long-lived complexes with
calnexin(16, 25) . Conversely, removal of the
transmembrane and cytoplasmic domains from the T cell receptor
subunit almost completely eliminates calnexin binding, but this
truncated subunit still possesses its full complement of N-linked oligosaccharides(26) . Finally, cross-linking
experiments have indicated that Asn-linked oligosaccharides are
unlikely to be the sole mode of association between calnexin and class
I heavy chains(26) . This latter study identified a region
encompassing the transmembrane domain and three flanking amino acids of
the heavy chain as a site of interaction with calnexin.
In an effort
to clarify the involvement of Asn-linked oligosaccharides in the
binding of calnexin to newly synthesized glycoproteins, we tested the
ability of calnexin to function as a lectin by assaying its ability to
bind to a series of oligosaccharide-processing intermediates.
Additionally, the relative contribution of protein-carbohydrate and
protein-protein interactions in maintaining the association between
calnexin and newly synthesized soluble or transmembrane glycoproteins
was assessed. Our findings indicate that calnexin is indeed a lectin
with specificity for the GlcMan
GlcNAc
oligosaccharide. They also lead us to propose a model in which
binding to this oligosaccharide is a critical event that occurs during
initial recognition of newly synthesized glycoproteins by calnexin.
However, once calnexin-glycoprotein complexes are formed,
protein-protein interactions become predominant and oligosaccharide
binding contributes minimally, if at all, to the overall association.
To analyze the oligosaccharides that bound
to the immobilized proteins, the nickel-agarose beads were subjected to
a series of sequential washes. The first wash was performed briefly
(1-2 min) at 4 °C with 100 µl of binding buffer followed
by centrifugation for 5 min at 2,600 g and recovery of
supernatant. Four subsequent washes were then performed, each for 1 h
at 23 °C with agitation at 200 rpm, consisting of 100 µl of
binding buffer alone followed by binding buffer supplemented with (in
order) 0.1 M
-methyl D-galactopyranoside, 0.1 M
-methyl D-mannopyranoside, or 0.1 M
-methyl D-glucopyranoside (Aldrich). The sixth and
final wash was performed with agitation overnight at 23 °C with 100
µl of 0.1 M
-methyl D-glucopyranoside in
binding buffer. An aliquot of each supernatant (10%) was analyzed by
liquid scintillation counting, and the remaining six wash supernatants
for each immobilized protein were pooled and treated with Dowex and
Amberlite beads as described above. The entire eluate sample was then
analyzed by HPLC with a 2.5-ml detector cell, which gives greater
detector sensitivity relative to a 1.0-ml detector cell, but also
increases peak widths and decreases peak resolution. To detect
oligosaccharides, which remained bound to the proteins after the
elution procedure, the nickel-agarose beads were boiled for 15 min in
100 µl of binding buffer. No tritium was detected in the resulting
supernatants for either H-2K
-agarose or calnexin-agarose.
Figure 3:
Binding of calnexin to
mannosidase-treated glucosylated oligosaccharides. A mixture of
[H]Glc
Man
GlcNAc
oligosaccharides was incubated with calnexin-agarose. Unbound
material was collected in the supernatant fraction following
centrifugation, and bound material was collected by eluting the pellet
with 0.1 M
-methylglucoside. The starting, unbound, and
bound fractions were analyzed by HPLC. In each case, the flow rate was
1 ml/min, and fractions were collected every 0.5 min. Fraction 20
corresponds to 35 min in the solvent program. The elution positions of
the various oligosaccharides are indicated by the arrowheads.
Figure 1:
Structure of the
full-length dolichol-linked oligosaccharide. This oligosaccharide is
transferred to Asn-X-Ser(Thr) sequences during translocation
of nascent polypeptides into the ER lumen. It is subsequently processed
through the action of -glucosidases I and II within the ER and by
-mannosidases in both the ER and Golgi apparatus. The sites of
cleavage by the glucosidases are indicated. The
Glc
Man
GlcNAc
oligosaccharide that
is bound by calnexin is enclosed by the box. Mannose residues
removed by digestion with Jack bean
-mannosidase (see Fig. 3) are shown in italics.
Figure 2:
Selective binding of
GlcMan
GlcNAc
oligosaccharide by
immobilized calnexin. A mixture of
Glc
Man
GlcNAc
oligosaccharides was dissolved in binding buffer (see
``Experimental Procedures''), divided into three equal
portions, and incubated for 1 h at 23 °C either alone, with
H-2K
-agarose, or with calnexin-agarose. PanelA, HPLC analysis of the oligosaccharide mixture incubated
in the absence of immobilized protein. The arrowheads indicate
the elution times of the various oligosaccharides in the mixture. PanelB, HPLC analysis of oligosaccharides eluted
from calnexin-agarose (closedcircles). The elution
profile of a mixture of the following oligosaccharide standards is also
included (opencircles):
Man
GlcNAc
, Man
GlcNAc
,
Glc
Man
GlcNAc
,
Glc
Man
GlcNAc
, and
Glc
Man
GlcNAc
.
Solutions of
[H]mannose-labeled
Glc
Man
GlcNAc
were incubated
with a soluble His
-tagged form of calnexin, or, as a
control, a soluble His
-tagged form of the class I
H-2K
histocompatibility protein, each immobilized on
nickel-agarose. After 1 h, the agarose was removed by centrifugation,
and the supernatants were analyzed by HPLC. An incubation without
agarose beads was also included. As shown in Fig. 2A,
four distinct oligosaccharide species
(Glc
Man
GlcNAc
) were present
in the sample incubated without agarose. After incubation with
H-2K
-agarose, the amount of each oligosaccharide remaining
in solution was not significantly altered (data not shown). In
contrast, the Glc
Man
GlcNAc
oligosaccharide was selectively depleted after incubation with
calnexin-agarose. In several experiments, the amount of
Glc
Man
GlcNAc
oligosaccharide
recovered was reduced by 20-45%, whereas no depletion of other
oligosaccharides from the mixture by calnexin could be detected (data
not shown).
To examine material which specifically bound to
calnexin, a six-step elution procedure (Table 1) was employed
with the H-2K- and calnexin-agarose samples recovered after
the experiment. Each successive step involved a condition expected to
be more effective for elution of bound oligosaccharide from calnexin.
In total, about 80 cpm was recovered from H-2K
-agarose,
whereas approximately 560 cpm was recovered from calnexin-agarose.
Surprisingly, a large fraction of the radioactive material eluted from
calnexin-agarose in the initial steps that employed buffer alone or
buffer plus
-methyl galactoside, a compound not expected to
inhibit calnexin. These data suggested that the oligosaccharide was not
tightly bound to calnexin. After the sequential elution procedure, no
additional radioactivity was recovered by boiling the agarose. The
fractions eluted from calnexin-agarose were then pooled and analyzed by
HPLC (Fig. 2B). A single peak was observed that
co-eluted with Glc
Man
GlcNAc
. In
other experiments, the small amount of radioactivity eluting from
H-2K
-agarose was analyzed by HPLC, but no discrete peaks
were observed. In contrast, radioactivity recovered from
calnexin-agarose always eluted as
Glc
Man
GlcNAc
.
Calnexin-heavy chain complexes were
immunoisolated from digitonin lysates of metabolically radiolabeled Drosophila transfectants using an antibody raised against the
carboxyl-terminal 14 amino acids of calnexin followed by collection on
protein A-agarose. The agarose-bound calnexin-K or -D
complexes were incubated with either 1 M
-methyl
glucoside or
-methyl mannoside and then separated into supernatant (S) and agarose bead (B) fractions (Fig. 4).
Following this treatment, nearly all heavy chains remained in the bead
fraction associated with calnexin. Small amounts of heavy chain and
calnexin were detected in the supernatant fraction at approximately the
same ratio as observed in the agarose bead fraction. The amounts in the
supernatant were variable from experiment to experiment and likely
reflect the difficulty in sedimenting agarose beads efficiently through
viscous solutions of 1 M glycoside. Similar results were
obtained when the experiment was repeated using Nonidet P-40 rather
than digitonin for cell lysis and immune isolation (data not shown).
The inability to dissociate calnexin-heavy chain complexes under
conditions known to be effective in dissociating other
lectin-glycoprotein complexes (33) suggests that
oligosaccharide-protein interactions may not contribute substantially
to the calnexin-heavy chain association once complexes are formed.
Alternatively,
-methyl glucoside and
-methyl mannoside may be
inefficient competitors of the binding of calnexin to the
Glc
Man
GlcNAc
oligosaccharide.
Figure 4:
Incubation of calnexin-glycoprotein
complexes with -methyl glycosides. Drosophila cells
expressing H-2K
or D
heavy chains and calnexin
were radiolabeled with [
S]Met for 10 min. Cells
were lysed in buffer containing 0.5% digitonin and calnexin-heavy chain
complexes were isolated with anti-calnexin antibody followed by protein
A-agarose. The agarose beads were incubated with either 1 M
-methyl glucoside (
-methylGlc) or
-methyl
mannoside (
-methylMan) and then were centrifuged to form
bead-bound (B) and supernatant (S) fractions.
Radiolabeled proteins in these fractions were analyzed by SDS-PAGE. PanelA, effect of
-methyl glycosides on
calnexin-K
heavy chain complexes; panelB, effect of
-methyl glycosides on calnexin-D
heavy chain complexes. The mobilities of calnexin (cnx),
the K
and D
heavy chains, as well as molecular
mass standards are indicated.
Figure 5:
Digestion of calnexin-glycoprotein
complexes with endo H. PanelsA and B,
radiolabeled calnexin-heavy chain complexes were isolated with
anti-calnexin antibody followed by protein A-agarose as described in
the legend to Fig. 4. The agarose beads were incubated in the
absence or presence of endo H and then were centrifuged to form
bead-bound (B) and supernatant (S) fractions (lanes3-6). In addition, separate samples of
calnexin-heavy chain complexes were dissociated in SDS and incubated in
the absence or presence of endo H to provide mobility standards of
glycosylated and deglycosylated heavy chains, respectively (lanes1 and 2). To confirm that the heavy
chain standards were completely deglycosylated, limited endo H digests
were performed to visualize all partially deglycosylated species (data
not shown). The major heavy chain bands in lane2, panelsA and B, do indeed represent the
K heavy chain lacking its two oligosaccharides and the
D
heavy chain lacking its three oligosaccharides,
respectively. PanelC, HepG2 cells were radiolabeled
for 10 min with [
S]Met. Cells were lysed in
buffer containing 2% sodium cholate, and calnexin-glycoprotein
complexes were isolated, digested with endo H, and separated into
supernatant and bead-bound fractions as in A and B.
Subsequently, the fractions were boiled in PBS containing 0.2% SDS,
adjusted to 1% Nonidet P-40, and subjected to a second round of
immunoprecipitation using anti-
-antitrypsin antibody (lanes3-6). Lanes1 and 2 contain standards of glycosylated and deglycosylated
-antitrypsin, respectively. Complete deglycosylation
of the
-antitrypsin standard was confirmed as
described for the class I heavy chains.
Calnexin
binds not only to membrane-associated proteins such as class I
molecules but also to a number of soluble, secretory
glycoproteins(4) . Since secretory glycoproteins lack
transmembrane segments that have been implicated in the binding of
membrane proteins to calnexin(26) , they may rely more heavily
on oligosaccharide-protein interactions to maintain stable associations
with calnexin. Furthermore, experiments involving endo H digestion of
complexes containing calnexin and secretory glycoproteins avoid the
potential complication of the deglycosylated glycoprotein being unable
to dissociate from calnexin due to immobilization within the same
detergent micelle. Thus, to evaluate the role of N-linked
oligosaccharides in the interaction between calnexin and a secretory
glycoprotein, anti-calnexin immunoprecipitates from sodium cholate
lysates of metabolically radiolabeled HepG2 cells were treated with
endo H. Previous studies have shown that such immunoprecipitates
contain complexes between calnexin and many secretory glycoproteins
including -antitrypsin,
-antichymotrypsin,
-fetoprotein, transferrin,
complement component C3, and apoB-100 (4) . Following
separation into supernatant and bead fractions, the endo H-digested
samples were boiled in SDS to disrupt calnexin-glycoprotein complexes
(and to inactivate endo H) and then were subjected to a second round of
immunoprecipitation with antibodies against
-antitrypsin (Fig. 5C). At least 50%
of
-antitrypsin molecules present in complexes with
calnexin could be completely deglycosylated by endo H (compare the endo
H-digested agarose bead fraction, lane6, with the
deglycosylated
-antitrypsin standard, lane2). Furthermore, the deglycosylated molecules remained
associated with calnexin in the bead fraction (compare the endo
H-digested S and Blanes). These results
were consistent with those obtained using calnexin-class I heavy chain
complexes and suggest that once calnexin-glycoprotein complexes are
formed, N-linked oligosaccharides are dispensable in
maintaining an association with calnexin. The less efficient
deglycosylation observed with
-antitrypsin relative to
class I heavy chains may reflect the fact that a large number of
substrates that compete for endo H are present in calnexin
immunoprecipitates from HepG2 cells, whereas calnexin-heavy chain
complexes constitute the major species immunoprecipitated from Drosophila transfectants.
Our findings indicate that the ER luminal domain of calnexin
has the capacity to bind to the early N-linked oligosaccharide
processing intermediate,
GlcMan
GlcNAc
. The binding is
specific because calnexin selected this oligosaccharide from a mixture
containing Glc
Man
GlcNAc
species. Calnexin also preferentially selected this
oligosaccharide from a mixture containing
Glc
Man
GlcNAc
. Thus, in
addition to recognizing a single terminal glucose, calnexin also
appears to recognize at least 1 terminal mannose residue in
Glc
Man
GlcNAc
(Fig. 1). More
extensive specificity studies and a detailed analysis of the binding
kinetics were precluded by the limited quantities of purified, soluble
calnexin available.
In none of our experiments was hydrolysis of any
oligosaccharide detected, suggesting that calnexin is a lectin rather
than an enzyme that modifies oligosaccharide structure, e.g. a
glycosidase. However, unlike most lectins that bind large
oligosaccharides with micromolar dissociation constants(33) ,
calnexin binds the GlcMan
GlcNAc
oligosaccharide weakly; extensive dissociation of the complex is
detectable following short incubations in buffers lacking sugar
haptens. Furthermore, primary sequence comparisons revealed that
calnexin lacks the carbohydrate-recognition domains characteristic of
each of the three major groups of animal lecins. These include the C-
(Ca
-dependent) type, the galectins (S-type), and the
P- (mannose 6-phosphate) type lectins(35) . Although some
lectins, such as concanavalin A, are capable of binding to processing
intermediates of Asn-linked oligosaccharides, none exhibit the binding
specificity associated with calnexin(35, 36) .
Consequently, we conclude that calnexin constitutes a new type of
lectin with unique specificity for the
Glc
Man
GlcNAc
oligosaccharide.
Treatment of cultured cells with either tunicamycin or the
glucosidase inhibitors, castanospermine or 1-deoxynojirimycin, results
in a dramatic block in the formation of complexes between calnexin and
a large array of newly synthesized
glycoproteins(4, 9) . Consistent with previous
speculation(9, 24) , the demonstration that calnexin
binds selectively to the
GlcMan
GlcNAc
-processing
intermediate provides a clear molecular explanation for the action of
these drugs. The remarkable efficacy of tunicamycin and the glucosidase
inhibitors also underscores the crucial role that oligosaccharide
binding must play in the formation and/or maintenance of
calnexin-glycoprotein complexes.
Two observations lead us to propose
that the binding of GlcMan
GlcNAc
oligosaccharide is important in the initial formation of
complexes with calnexin, but it cannot be responsible for maintaining
complexes once they are formed. First, the apparent affinity of
calnexin for the oligosaccharide is low and it is unlikely that
complexes maintained through this interaction alone would survive the
prolonged immune isolation procedures used for their purification. It
is possible that calnexin could possess more than one carbohydrate
binding site or could exist as a homooligomer (features that might
increase the avidity of the interaction), but such properties would not
be effective for the many glycoproteins having a single oligosaccharide
chain. Second, in purified complexes of calnexin with either class I
heavy chains or
-antitrypsin, all oligosaccharides are
accessible to endo H, and their removal is not accompanied by complex
dissociation. Consequently, we envision a two-step mechanism for the
interaction of newly synthesized glycoproteins with calnexin (Fig. 6).
Figure 6: Two-step model for binding of calnexin to unfolded glycoproteins. Following removal of two glucose residues, newly synthesized glycoproteins initially contact calnexin via their monoglucosylated oligosaccharide chains (Step1). Having been placed in proximity to calnexin by this first interaction, the unfolded polypeptide associates directly with additional sites on calnexin (Step2). The oligosaccharide chains are accessible to exogenous probes at this stage. Reglucosylation by UDP-glucose:glycoprotein glucosyltransferase may play an important role in recovering proteins that have lost all three glucoses prior to any contact with calnexin or in facilitating rebinding to calnexin during cycles of folding. Asterisks indicate steps blocked by castanospermine and 1-deoxynojirimycin.
In this model, the initial interaction between
nascent glycoproteins and calnexin occurs through binding of the
GlcMan
GlcNAc
oligosaccharide. This
intermediate first appears on nascent chains (37) and, given
the rapid association observed between calnexin and some
glycoproteins(1, 7, 9, 11) , it is
possible that the initial binding of calnexin could occur
co-translationally. Having been brought in proximity to calnexin
through this initial interaction, the unfolded glycoprotein then binds
to calnexin through segments of its polypeptide chain. The physical
features recognized by calnexin in this second interaction are largely
unknown, but they may be hydrophobic segments or patches as is thought
to be the case for chaperones of the Hsp 60 and Hsp 70
families(18) . Due to the reversibility of carbohydrate
binding, dissociation of the oligosaccharide likely occurs as the
polypeptide chain and calnexin interact, perhaps aided by new steric
constraints placed on the oligosaccharide and calnexin. Subsequently,
the polypeptide folds in association with calnexin and in conjunction
with folding enzymes until sites for calnexin binding are buried in the
folded molecule. Whether folding (and assembly) occurs while the
polypeptide is tethered to calnexin or during cycles of calnexin
binding and release is unknown. Although calnexin does not possess
consensus sequences for nucleotide binding, it has recently been
demonstrated that the ER luminal domain of calnexin binds ATP in
vitro(38) . This raises the possibility that calnexin
could undergo cyclic interactions with unfolded glycoproteins in a
manner analogous to other chaperones. Conceptually, the two step
binding model is reminiscent of the mechanism that regulates leukocyte
localization in the vasculature. Circulating leukocytes are brought
into proximity with endothelial cells via transient
selectin-carbohydrate interactions followed by tight adhesion mediated
by integrins and Ig superfamily adhesion receptors(39) .
The existence of monoglucosylated oligosaccharides on newly synthesized glycoproteins is prolonged by a cycle of deglucosylation by glucosidase II and reglucosylation via an ER enzyme known as UDP-glucose:glycoprotein glucosyltransferase(40, 41) . The latter enzyme reglucosylates only nonnative glycoproteins and it has been suggested by Helenius that the purpose of the cycle is to ensure that nonnative glycoproteins continually oscillate between calnexin-bound and unbound states. Once a glycoprotein folds, it is no longer a substrate for reglucosylation and it dissociates from calnexin(24) . Although attractive, this model requires that oligosaccharide structure is the main regulator not only of calnexin binding but of release as well. This is inconsistent with our observation that oligosaccharides do not participate in maintaining calnexin-glycoprotein complexes. Rather, we suggest that the function of reglucosylation may be to provide newly synthesized glycoproteins with additional opportunities to bind calnexin on those occasions when all three glucoses are removed before an initial interaction with calnexin can take place (Fig. 6, RecoveryPathway). This may explain the increased level of association with calnexin that occurs for some glycoproteins during the 5-10-min period postsynthesis(4, 9) . Reglucosylation followed by two-step binding may also be the means whereby a folding glycoprotein can rebind to calnexin if dissociation from calnexin occurs before folding is complete (Fig. 6).
Why
has calnexin evolved to utilize oligosaccharide binding for its initial
interaction with unfolded glycoproteins? Unlike soluble chaperones of
the ER, calnexin is constrained within the ER membrane and, at least in
some cell types, it may be associated with components of the
translocation apparatus(19) . Given this disposition,
polypeptide binding sites on calnexin may have limited access to
nascent glycoproteins. Oligosaccharide addition, being among the first
covalent modifications that occur on nascent chains, ensures that a
conserved and well exposed site for calnexin binding is present at an
early stage in the folding of nascent chains. Calnexin could even be
associated with glucosidase II, poised to capture the monoglucosylated
oligosaccharide as soon as it is formed. For some proteins, however,
this initial stage of oligosaccharide capture can be bypassed since
they bind to calnexin even though they are unglycosylated. This is the
case for the T cell receptor subunit (16) and also for
variants of the multidrug resistance P glycoprotein (25) and
the class I H-2 L
heavy chain (42) that lack
consensus sequences for N-glycosylation. Presumably these
proteins have accessible sites on their polypeptide chains for
interaction directly with calnexin.
As suggested
previously(24) , the finding that calnexin binds to the
GlcMan
GlcNAc
processing
intermediate may explain why eukaryotes from yeasts to humans initiate N-glycosylation with a common, preassembled oligosaccharide (Fig. 1). If the purpose of early attachment of oligosaccharide
was solely to ensure that segments of a folding polypeptide remained
exposed to solvent, then oligosaccharides of diverse size and
composition would likely suffice. Calnexin is an abundant protein in
virtually all eukaryotic cell types examined including yeast (in which
it has an essential function), (
)plants, worms, and mammals
(see (23) for references). Its intimate association with N-linked oligosaccharides as part of its quality control and
chaperone functions may be the major factor responsible for preserving
the enbloc mode of glycosylation that originated in
early eukaryotes.