From the
Increasing evidence shows that calnexin, a membrane-bound
chaperone in the endoplasmic reticulum, is a lectin that binds to newly
synthesized glycoproteins that have partially trimmed N-linked
oligosaccharides. It specifically attaches to core glycans from which
two glucoses have been removed by glucosidases I and II. Several recent
reports suggest, however, that it can also bind to proteins devoid of N-linked glycans. To investigate the extent of
glycan-independent binding, we have analyzed two mutant cell lines (Lec
23 and Pha 2.7) that are unable to process the core glycans
because they lack glucosidase I or glucosidase II, respectively. In
contrast to parental cell lines, calnexin binding of substrate proteins
was found to be virtually nonexistent in these cells. Neither cellular
nor viral proteins associated with the chaperone. It was concluded that
glycans are crucial for calnexin association and that the vast majority
of substrate proteins are therefore glycoproteins.
The lumen of the endoplasmic reticulum (ER) ()contains a large number of chaperones and folding enzymes
(see (1) and (2) ). They assist the folding,
oligomeric assembly, and quality control of newly synthesized proteins,
the majority of which are glycoproteins. Some of these folding factors,
such as BiP/GRP78 and GRP94, are members of classical chaperone
families while others are unique to the ER. The latter include
calnexin, a membrane-bound protein that interacts transiently with a
variety of soluble and membrane-bound substrate molecules (see (3) ).
Our studies have indicated that calnexin differs from
previously characterized chaperones in being a lectin; it binds to
glycoproteins with partially trimmed N-linked
oligosaccharides(4, 5) . The specificity for
monoglucosylated chains
(GlcMan
GlcNAc
) was recently
confirmed by Ware et al.(32) using purified N-linked oligosaccharides. These results are significant
because they link the folding of glycoproteins and their quality
control in the ER directly to the process of oligosaccharide trimming.
Based on these observations, we have proposed that the de- and
reglucosylation cycle, known to operate in the lumen of the
ER(6) , is connected to calnexin binding and that the cycle
plays a central role in the maturation of newly synthesized
glycoproteins(4, 7) .
Recent studies have confirmed
the requirement for glycans in substrate binding to calnexin. These
rely on glycosylation inhibitors (tunicamycin), -glucosidase
inhibitors castanospermine (CST) and 1-deoxynojirimycin, and
mutagenesis to remove consensus glycosylation
sequences(8, 9, 10) . It has also been
reported, however, that polypeptide chains that have no N-linked oligosaccharides can associate with calnexin. The
latter observations include the redistribution of the unglycosylated
CD3
subunit of the T cell receptor in cells expressing a mutant
version of calnexin, and the co-immunoprecipitation of unglycosylated
thyroglobulin, class I and II major histo compatibility complex
subunits and P-protein with
calnexin(11, 12, 13, 14, 15) .
It has therefore been suggested that calnexin binding may occur in more
than one way and may not require the presence of sugars.
To address the mode of substrate association with calnexin, we have analyzed mutant cells with glucosidase defects. Since they fail to remove glucoses from the core oligosaccharide, the glycoproteins do not reach the monoglucosylated form and are therefore unable to bind to calnexin via the lectin binding sites. Hence, these cells provide an opportunity to determine whether glycan-independent binding does take place under physiological conditions. Tests can be made without resorting to inhibitors and overexpression systems.
The two mutant cell lines used in this study were Lec 23 and
Pha 2.7. Lec 23 are mutant CHO cells defective in
glucosidase I(23) , and Pha
2.7 cells are mutants
of the BW 5147 mouse lymphoma cell line defective in glucosidase
II(24) . Previous work has established that glycoproteins are
synthesized in both mutant cell lines, but glucose trimming is
defective. Both mutant cell lines grow well in culture and synthesize
normal or slightly increased amounts of calnexin compared with the
parental lines (see below). Indirect immunofluorescence microscopy
using anti-calnexin antibodies showed that the calnexin is distributed
in a typical reticular ER pattern with nuclear rim staining (Fig. 1). The distribution of calnexin was identical to that
seen in the parental cells.
Figure 1:
Calnexin is localized to the ER in
glucosidase-deficient and parent cell lines. a, CHO; b, Lec 23; c, Pha 2.7; and d, BW
5147 cells. All cells were fixed in 3.7% formaldehyde. CHO and Lec 23
cells were permeabilized with 0.1% SDS, 0.5% Triton X-100, and
Pha
2.7 and BW 5147 cells with 0.05% saponin. Cells were
labeled with polyclonal anti-calnexin antibodies and detected with
fluorescein isothiocyanate-labeled anti-rabbit
antibody.
To analyze the interaction of newly
synthesized cellular proteins with calnexin in Lec 23 cells and in the
parental CHO cells, monolayers were pulse-labeled for 10 min with S-labeled ProMix. CST was included in some of the cultures
to prevent the action of glucosidases I and II. A CHAPS-solubilized
lysate was prepared and immunoprecipitated using anti-calnexin
antibodies, and the proteins were analyzed by SDS-polyacrylamide gel
electrophoresis and fluorography.
As reported previously(8) , several labeled protein bands were precipitated by the anti-calnexin antibodies from labeled wild type CHO cells (Fig. 2, lane 1). The band migrating at 90 kDa corresponded to calnexin itself, whereas the others were coprecipitating substrate proteins. When labeling was performed in the presence of CST (lane 2), only the calnexin band was seen, confirming the requirement for glucose trimming and the specificity of the antibodies.
Figure 2:
Calnexin binding of newly synthesized
glycoproteins is inhibited in glucosidase I-deficient and
castanospermine-treated cells. CHO and Lec 23 cells were labeled for 10
min with 0.1 mCi of S-labeled ProMix. The cells were
washed twice in ice-cold PBS with 20 mMN-ethylmaleimide and lysed in ice-cold 2% CHAPS in HBS
buffer with proteases inhibitors. Precleared (Zysorbin) lysates from
uninfected CHO (lanes 1 and 2) and Lec 23 cells (lanes 3 and 4) were immunoprecipitated with
anti-calnexin antibody. CHO and Lec 23 cells were infected by influenza
virus and VSV and immunoprecipitated with anti-calnexin antibodies (lanes 4-12). Immunoprecipitates from
castanospermine-treated cells are marked with a
+.
In Lec 23 cells, the coprecipitation of proteins was dramatically reduced (lane 3). Few if any substrate proteins associated with calnexin. The background was, however, further suppressed by the addition of CST (lane 4), suggesting that it was specific. The presence of small background staining was consistent with the report that Lec 23 cells are somewhat leaky, i.e. the glucosidase I activity is not completely inhibited(23) .
In infected wild type CHO cells, we have previously reported efficient binding of two viral glycoproteins, influenza HA and VSV G protein, to calnexin(4, 8) . This is also seen in lanes 5 and 9 (Fig. 2). Binding of these viral membrane proteins was blocked if they were synthesized in the presence of CST (lanes 6 and 10).
When Lec 23 cells were infected with the same viruses, calnexin binding of the two glycoproteins was virtually nonexistent (lanes 7 and 8 and 11 and 12). That the cells were, indeed, infected is shown in Fig. 3. The amounts of labeled HA and G protein after immunoprecipitation with anti-HA and G-protein antibodies show that the amounts were comparable with or without CST (compare lanes 1 and 3 and 5 and 6 and lanes 2 and 4 and 7 and 8). As expected, the apparent molecular weights of HA and G protein were slightly higher in Lec 23 cells due to the lack of glucose trimming.
Figure 3:
Glucosidase-deficient and parent cell
lines can be infected by influenza and vesicular stomatitis viruses.
CHO, Lec 23, BW 5147, and Pha 2.7 cells were infected by
vesicular stomatitis virus. In addition, CHO and Lec 23 cells were
infected by influenza virus. The cells were pulse-labeled and lysed as
in Fig. 2. Immunoprecipitations were performed using
anti-influenza antibodies (lanes 1-4) and anti-VSV G
antibody (lanes 5-8). Immunoprecipitates with anti-VSV G
protein antibody from BW 5147 and Pha
2.7 cell lysates are
shown in lanes 9 and 1,
respectively.
With the
glucosidase II-deficient Pha 2.7 cells, the results were
similar to those seen for Lec 23. Whereas numerous newly synthesized
proteins coprecipitated with anti-calnexin from the parental BW 5147
cells (Fig. 4, lane 3), virtually no labeled bands
(except for calnexin itself) were seen in the Pha
2.7 cells (lane 4). After infection of the wild type cells with VSV, the
G protein was coimmunoprecipitated with anti-calnexin as expected (lane 1), whereas coprecipitation from the mutant cells was
virtually undetectable (lane 2). That the cells were infected,
and that they expressed VSV G protein, was shown by immunoprecipitation
using anti-G protein antibodies (Fig. 1, lanes 9 and 10). Both the membrane-bound, intact G protein (G
)
and the cleaved (G
) forms were seen. Similar experiments
were attempted with influenza virus, but being lymphoid, these cells
could not be efficiently infected by this virus.
Figure 4:
Binding of newly synthesized glycoproteins
to calnexin is prevented in glucosidase II-deficient cells.
VSV-infected BW 5147 and Pha 2.7 cells were labeled for 10
min with 0.15 mCi of
S-labeled Promix, lysed, and
immunoprecipitated with anti-calnexin antibody (lanes 1 and 2). Anti-calnexin immunoprecipitates from noninfected BW 5147
and Pha
2.7 cells are shown in lanes 3 and 4, respectively.
Taken together, the results showed that the majority, if not all, of the substrate proteins that normally associate with calnexin in CHO and mouse lymphocytes must first be processed by glucosidases I and II. This confirms that substrate proteins were glycoproteins. Given that the results were obtained under normal tissue culture conditions without added inhibitors and without the use of overexpression, virus vectors, or mutant proteins, they probably reflect the normal binding specificity of calnexin.
The results are consistent with data obtained with CST
and other glucosidase inhibitors(4, 10) . As shown in Fig. 2such inhibitors effectively block the binding of virtually
all newly synthesized substrate proteins to calnexin. Pha 2.7 cells have recently been used by Kearse et al.(33) to probe the specificity of T cell receptor
and
chain binding to calnexin. Consistent with our results they found
that coimmunoprecipitation of these two glycoproteins was decreased
compared to the parental cell.
The reasons why association of calnexin with nonglycosylated proteins has been observed in several systems (11, 12, 13, 14) remain unclear. Some of the studies in question have been performed using tunicamycin as a glycosylation inhibitor or dithiothreitol as a folding inhibitors. Transient overexpression of glycoproteins has also been used. These experimental conditions are less physiological than those employed here. Tunicamycin causes global inhibition of N-linked glycosylation and massive aggregation of misfolded proteins in the ER lumen as well as a blockade in their transport to the Golgi complex (25, 26, 27) . Similar aggregation has been reported in the presence of dithiothreitol(28) . Mutant proteins and protein subunits that fail to oligomerize also often end up in large aggregates. It has, moreover, been our experience that transient expression of wild type proteins using the T7 vaccinia virus expression system (29) frequently results in aggregated products(27) . When the substrate proteins are aggregated, it is difficult to know whether the coimmunoprecipitation of a specific nonglycosylated substrate protein with calnexin occurs because of a direct contact between the two proteins or due to indirect association mediated by other proteins. Further studies using isolated proteins in vitro are needed to determine the various modes of calnexin binding to its substrate.
That Lec 23 and Pha 2.7 cells are viable
is interesting in view of their glucosidase defects and the lack of
calnexin-mediated folding. These cells have been selected for growth
and may therefore contain higher amounts of other folding factors. It
is known that many glycoproteins can fold correctly, albeit usually
less rapidly and less efficiently, in cells treated with glucosidase
inhibitors (see (30) and (31) ). Evidently, they can
fold and reach the mature conformation using other chaperones and
folding enzymes.