(Received for publication, September 27, 1994; and in revised form, November 21, 1994)
From the
Proper folding of nascent polypeptides is essential for their
function and is monitored by intracellular ``quality
control'' elements. The molecular chaperone calnexin participates
in this process by retaining in the endoplasmic reticulum a variety of
unfolded proteins, including class I major histocompatibility complex
molecules. We transfected human B cell lines with genes encoding either
wild-type HLA-A2 heavy chains or mutant heavy chains lacking sites for
glycosylation or deficient in binding to
-microglobulin (
m). In CIR cells,
calnexin did not associate detectably with wild-type heavy chains but
bound strongly to mutant heavy chains unable to bind
m. Removal of the glycosylation addition site by
further mutagenesis prevented binding of mutant heavy chains to
calnexin. In Daudi cells, deficient in synthesis of
m,
wild-type HLA-A2 heavy chains, but not a nonglycosylated mutant, bound
calnexin. Castanospermine, which blocks trimming of glucose residues
from asparagine-linked glycans, inhibited association of calnexin with
heavy chains encoded by a second class I gene, HLA-B*0702. Although
initiation of calnexin binding appears to depend on the presence of
oligosaccharide on the substrate, removal of the glycan from
calnexin-associated heavy chains by digestion with endoglycosidase H
did not disrupt the interaction. These results suggest that calnexin
first recognizes carbohydrate on substrate proteins and then binds more
stably to peptide determinants, which disappear upon folding.
The molecular chaperone calnexin binds incompletely folded
proteins and prevents their premature export from the endoplasmic
reticulum (ER)()(1, 2, 3) .
Calnexin may also facilitate directly folding and disulfide bond
formation in nascent polypeptides(4) . Not all proteins
interact with calnexin, however, and it is known that some proteins
that normally interact with calnexin can fold properly in vivo in its absence(5) . This supports the proposal that
calnexin functions primarily as a quality control mechanism in the ER.
As many of its substrates are glycoproteins, it has been suggested that calnexin recognizes carbohydrate. Tunicamycin, which inhibits transfer of the core saccharide to asparagine residues, blocks binding of many proteins to calnexin(4) . Castanospermine and other drugs that inhibit trimming of glucose residues from high mannose glycans also block calnexin binding, leading to the suggestion that calnexin recognizes monoglucosylated glycans on substrates(6) . This implies that calnexin may be a lectin, although it does not appear to have homology to known lectins. Alternatively, calnexin may recognize determinants of proteins that disappear upon further glycan modification, as previously proposed(6) .
Our experiments
addressed the structural features of class I major histocompatibility
complex (MHC) heavy chains, which are important for recognition by
calnexin. Previous studies demonstrated that calnexin binds to class I
MHC heavy chains prior to binding -microglobulin
(
m) in the ER and then dissociates before class I MHC
molecules enter the Golgi. We found that calnexin bound only to
non-
m-associated heavy chains, which were glycosylated
at asparagine 86. Our results suggest that calnexin recognizes both
oligosaccharide and unfolded regions of proteins.
To further investigate binding of class
I HLA heavy chains to calnexin, we studied a mutant (Q242K) of HLA-A2
with substitution of lysine for glutamine at position 242, a residue in
the interface between the 3 domain and
m(17) . The mutation weakens association of
the heavy chain with
m and inhibits
m
binding and subsequent transport at 37 °C(10) . The class I
HLA phenotype of CIR cells transfected with Q242K is thus similar to
Daudi cells, as heavy chains do not assemble with
m
and are retained in the ER.
Q242K heavy chains co-isolate with
calnexin at 37 °C, in contrast to wild-type HLA-A2 molecules (Fig. 1). This indicates that the mutation both inhibits
association with m and results in strong binding to
calnexin. Q242K heavy chains remain bound to calnexin after 1 h,
whereas class II MHC-associated invariant chains dissociate more
rapidly, suggesting that Q242K heavy chains are stabilized by calnexin.
Figure 1:
Substitution at position 242 in the
3 domain of HLA-A*0201 results in prolonged association with
calnexin. CIR cells transfected with Q242K (A) and wild-type (B) genes were radiolabeled for 30 min (A) or 10 min (B) and then incubated in chase medium for the times indicated
at 37 °C. After lysis, proteins were isolated with antibodies and
separated on SDS-PAGE. In A, bands migrating below Q242K in
the AF8-precipitated lanes correspond to invariant
chain.
Figure 2: HLA-A*0201 encoded heavy chains in Daudi cells associate with calnexin. Transfected or untransfected Daudi cells were radiolabeled for 2 h. After lysis, proteins were isolated with anti-calnexin (AF8) or anti-H chain (UCSF#2) antibodies and analyzed on isoelectric focussing gels, with separation from acidic (left) to basic (right) followed by SDS-PAGE. Endogenous Daudi heavy chains are indicated by largertriangles. Smallertriangles mark A2 heavy chains in A2-Daudi or the corresponding position in Daudi samples. Invariant chains co-precipitating with calnexin are labeled I.
In
transfected CIR cells, S88A assembled with m, although
somewhat less efficiently than the wild-type molecule, and was
expressed at the cell surface at approximately 50% of wild-type levels
(data not shown). No binding to calnexin was apparent (Fig. 3),
as observed for the wild-type molecule. The double mutant, S88A/Q242K,
which is not glycosylated and does not assemble with
m
at 37 °C, was synthesized in transfected CIR cells, but in contrast
to singly mutated Q242K, it did not associate with calnexin (Fig. 3B).
Figure 3: Class I HLA heavy chains mutated at the glycosylation site do not bind calnexin. Transfectants expressing mutants S88A (A) or S88A/Q242K (B) were radiolabeled for 10 min (A) or 30 min (B) and then incubated in chase medium for the indicated times. Proteins were isolated from cell lysates with antibodies and separated by SDS-PAGE. Heavy chains lacking glycans migrate faster than endogenous HLA-C*0401 and HLA-B*3503 molecules expressed at low levels in CIR cells(21) .
In transfected Daudi cells, HLA-A2 heavy
chains have a prolonged association with calnexin, due apparently to a
lack of m. The nonglycosylated mutant S88A did not
bind calnexin (Fig. 4), although the protein was stably
expressed, and could be isolated with UCSF#2 or anti-56-69 sera.
These results provide strong evidence that the N-linked glycan
at position 86 is important for class I binding to calnexin.
Figure 4: HLA-A2 heavy chains mutated at the site of glycosylation do not associate with calnexin in Daudi cells. Daudi cells and transfectants were radiolabeled for 2 h before lysis and isolation of proteins with indicated antibodies. Positions of full-length endogenous heavy chains (CLASSI) and the glycan-deficient mutant heavy chains (88A) are marked.
Figure 5: Castanospermine inhibits the interaction between calnexin and substrate proteins. CIR cells transfected with HLA-B*0702 were incubated for 45 min with the indicated amounts of castanospermine before labeling for 3 min. Cells were lysed, and proteins were isolated with antibodies as indicated. SDS-PAGE analysis is shown in A, with positions of HLA-B7 heavy chains (B7) and invariant chains (I) marked. In B, scanning densitometry of the results in A are shown. Circles and squares indicate invariant chain and B7 proteins, respectively. Opensymbols show calnexin-associated molecules, and closedsymbols show total amounts of each protein. Relative optical densities (R.O.D.) were calculated from individual optical density values as a percentage of the value obtained with no drug added.
It is notable that calnexin associates with slower migrating invariant chains induced by castanospermine, a result consistent with partial inhibition of trimming. There is no indication, however, that HLA-B7 molecules, which contain a single glycan, show a similar effect. This suggests that trimming of glucose residues from the glycan of class I HLA heavy chains occurs before binding calnexin.
Figure 6: Endo H digestion indicates that glycans are accessible on class I HLA heavy chains bound to calnexin. Daudi cells (panelA) and Q242K CIR cells (panelB) were radiolabeled for 2 h and lysed, and proteins were isolated with either UCSF#2 or AF8 antibodies. Samples bound to protein A-beads were then incubated for 16 h without Endo H at 4 °C (lanesA), 37 °C (lanesB), or with Endo H at 37 °C (lanesC), before elution and separation on SDS-PAGE. The positions of Endo H resistant and sensitive forms of class I heavy chains and invariant chains are marked.
Calnexin is a calcium-binding transmembrane protein found in
the ER, which associates with many newly synthesized, unfolded
polypeptides (4) . Upon proper folding, proteins dissociate
from calnexin, leave the ER, and continue through the secretory
pathway. This suggests that calnexin functions to prevent export of
unfolded proteins and perhaps to assist in the folding or assembly
process as a chaperone. Strong evidence that calnexin functions to
retain incompletely folded proteins in the ER comes from studies
showing that truncation of the cytoplasmic domain of calnexin does not
interfere with its binding to substrate proteins but causes
mislocalization of complexes within the cell(1) . In addition,
mouse class I heavy chains and m, which form dimers
and are transported in Drosophila cells, move to the plasma
membrane more slowly in the presence of calnexin(5) .
We
investigated the structural basis for calnexin binding to class I HLA
heavy chains in CIR and Daudi cells. HLA-A2 heavy chains in CIR cells
do not interact detectably with calnexin, an observation that can be
explained by rapid folding and assembly with m.
Substitution at position 242 of the heavy chain, shown previously to
inhibit pairing with
m(10) , resulted in
strong binding to calnexin, consistent with this interpretation.
Likewise, HLA-A2 heavy chains in Daudi cells, which lack
m, associate with calnexin. A mutant with
substitutions at positions 88 and 242, which interfere with
glycosylation and
m binding, respectively, did not
bind calnexin, and the single S88A mutant did not bind calnexin
detectably in Daudi transfectants. These data support the hypothesis
that calnexin is sensitive to both folding and glycosylation of
substrate proteins.
Drugs that block trimming of glucose residues from high mannose glycans on substrate proteins were shown by others and now by us to inhibit interaction with calnexin(6) . This led Hammond and co-workers (6, 19) to propose a model for calnexin function in which monoglucosylated substrate proteins that bind to calnexin are released upon partial folding and then are reglycosylated by the enzyme UDP-glucose:glycoprotein glycosyltransferase(6, 19) . After trimming to the monoglucosylated form, the protein would rebind calnexin and continue to cycle through the pathway. Once folding is complete, a protein is no longer a substrate for UDP-glucose:glycoprotein glycosyltransferase (20) and therefore would not bind calnexin and be retained in the ER. In this model, calnexin need serve only as a lectin, since folded proteins are not substrates for UDP-glucose:glycoprotein glycosyltransferase. Alternatively, calnexin might recognize both carbohydrate and unfolded regions of proteins.
Our data suggest that calnexin is not simply a lectin, since the glycan of class I heavy chains is accessible to Endo H digestion after binding. This implies that calnexin interacts with determinants on the protein. Furthermore, since the folding and glycosylation double mutant S88A/Q242K does not interact with calnexin, while the folding single mutant Q242K does, it seems unlikely that calnexin recognizes only determinants that disappear concomitantly with folding and further glycan processing. We propose that calnexin functions both as a lectin and as a chaperone with a binding site for peptides. Cleavage by Endo H of the glycan on substrate proteins after calnexin binding suggests that the interaction might be initiated via the glycan, and then maintained by recognition of determinants on the protein.