(Received for publication, March 27, 1995; and in revised form, June 8, 1995)
From the
Class I major histocompatibility complex heavy chains bind to
calnexin before associating with -microglobulin
(
m) and peptides. Calnexin has been shown to retain in
the endoplasmic reticulum those class I heavy chains which have not
assembled properly and, thus, to serve as a quality control mechanism.
In addition, calnexin may direct the folding of class I subunits or
their subsequent assembly. We asked whether calnexin plays a role in
the initial folding of HLA-B*0702 heavy chains by assessing disulfide
bond formation in vivo. Our results show that class I heavy
chains form intrachain disulfide bonds very soon after translation, and
that calnexin is bound to both reduced and oxidized forms during this
process. When a cell-permeable reducing agent, dithiothreitol, was
added to cells, disulfide bond formation in newly synthesized heavy
chains was substantially blocked, as was their association with
calnexin. The reducing agent appeared to affect calnexin directly,
since binding was similarly abolished to a subset of proteins which do
not contain internal disulfide bonds. Addition of the glucosidase
inhibitor castanospermine to cells, shown previously to disrupt
calnexin binding to ligands, slowed formation of disulfide bonds but
did not decrease the amount of assembled heavy chain-
m
complexes that formed. Our data suggest that calnexin can promote
disulfide bond formation in class I heavy chains but does not directly
facilitate subsequent binding to
m.
Class I MHC ()molecules display intracellularly
derived peptides at the plasma membrane enabling the immune system to
recognize virally infected and tumor cells. These molecules are
composed of integral membrane glycoproteins called heavy chains, a
second soluble polypeptide called
-microglobulin
(
m), and peptides of 8-10 amino acids in length
(reviewed in (1) ). Class I heterotrimers form in an early
biosynthetic compartment and, once assembled, are transported through
the Golgi to the plasma membrane.
Assembly and transport of class I
molecules is a highly regulated process and has been studied
intensively. Newly synthesized class I heavy chains bind
calnexin(2, 3) , a protein found in the rough
endoplasmic reticulum (ER). Heavy chain-m dimers then
form and associate with TAP transporters(4, 5) , which
have been shown to translocate peptides from the cytosol into the lumen
of the ER(6, 7) . Upon peptide binding, class I
molecules dissociate from TAP and continue through the secretory
pathway.
One function of calnexin is to retain incompletely assembled multimeric proteins, preventing their premature release from the ER(3, 8, 9, 10, 11, 12) . In addition, however, calnexin may bind to individual polypeptides that have not folded properly(13, 14, 15, 16, 17, 18) . This raises the possibility that calnexin may promote folding and disulfide bond formation of many polypeptides(19) . Such a role has been directly demonstrated for VSV-G protein, which binds calnexin transiently during its biosynthesis. When its binding to calnexin is blocked, VSV-G does not fold properly, but instead remains in the ER in association with grp78(20) . Together these studies demonstrate that calnexin has multiple functions in the biogenesis of monomeric and multimeric proteins.
Mouse and human class I heavy chains associate
with calnexin before binding m. However, mouse class I
proteins remain bound to calnexin after binding
m(2, 21) , whereas human class I
heavy chains appear to dissociate from calnexin either before or while
binding
m(8, 21) . The reason for
this difference between class I molecules of the two species is
unclear, but in any case it is possible that the chaperone both
promotes folding of class I heavy chains and facilitates their binding
to
m. We have addressed this issue by analyzing the
formation of intrachain disulfide bonds, an indicator of heavy chain
folding, and subsequent assembly with
m under
conditions where calnexin is either active or inactive. We find that
calnexin promotes heavy chain folding, but not apparently association
with
m.
Figure 1: Identification of HLA-B*0702 folding intermediates. After radiolabeling for 3 min, cells were incubated during a chase period of 0 or 5 min with 0, 1, or 3 mM DTT. Cells were then lysed with CHAPS and class I heavy chains or calnexin with associated proteins isolated using UCSF#2 or AF8, respectively. Samples were then separated on non-reducing SDS-PAGE. Upper and lower class I bands correspond to reduced and oxidized heavy chains, respectively. Ichain, invariant chain.
Since oxidized heavy
chains are quite abundant immediately after 3 min of radiolabeling, we
think it is likely that one or two disulfide bonds form
co-translationally under normal circumstances. Post-translational
folding can be forced by adding DTT after radiolabeling, to cause
complete reduction of B*0702 heavy chains. After DTT removal, oxidation
and folding proceeds, apparently with normal kinetics. Under these conditions, calnexin rebinds heavy chains quite
rapidly, and before they associate with
m.
Figure 2: Calnexin contains intramolecular disulfide bond essential for interaction with other proteins. Cells were radiolabeled for 5 min, then incubated in the presence of unlabeled amino acids for 0 or 30 min. Samples were then lysed in lysis buffer containing 1% Triton X-100 and 20 mMN-ethylmaleimide either immediately(-) or after (+) incubation for an additional 5 min in 3 mM DTT. AF8 was then used to isolate calnexin and samples separated on non-reducing SDS-PAGE.
Figure 3:
Evidence that calnexin promotes disulfide
bond formation, but not assembly of HLA-B*0702 molecules. CIR cells
transfected with B*0702 were incubated in the presence or absence of
400 µg/ml castanospermine (CST) for 1 h prior to
radiolabeling for 3 min. After the indicated chase times, cells were
lysed in CHAPS. In A, lysates were split and subjected to
immunoprecipitation with AF8 (calnexin-specific), anti-H
(non-m-associated heavy chains), or w6/32
(
m-bound heavy chains). Samples were run on reducing
SDS-PAGE, and appropriate bands quantitated by scanning. Integrated
optical density values are shown on the y axis and were
normalized relative to the highest value obtained, which was seen at 0
min with anti-H and no castanospermine added. Chase times are shown on
the x axis. Incubation with castanospermine (opensymbols) or without (closedsymbols) is
indicated. In B, samples were obtained as in A, but
non-reducing SDS-PAGE was performed. Upper and lower class I bands
correspond to reduced and oxidized forms respectively. Migration of
invariant chain (Ichain) and calnexin is also
marked.
Figure 4:
Oxidation of HLA-B*0702 heavy chains
precedes binding to m. Cells were metabolically
labeled and incubated in 0,1, or 3 mM DTT for the indicated
chase periods, then lysed with CHAPS. Class I proteins were isolated
with UCSF#2 (H) or with 4E (C), which binds to
m-associated B*0702
molecules.
Fully folded class I MHC molecules contain two disulfide
bonds in the 2 and
3 domains, respectively. Disruption of
either bond markedly reduces the efficiency of transport of class I MHC
molecules to the cell surface(31, 32) . In vitro studies of mouse class I heavy chains showed that after
translocation into microsomes, the
3 domain disulfide forms
rapidly, even in the absence of
m(29) . The
disulfide bond in the
2 domain was detected only when
m was present, suggesting that it forms after the
3 domain interacts with
m. A study examining
heavy chain folding in vivo demonstrated that
m helps to form the second disulfide bond and that
both disulfide bonds can form before stable association with
m is demonstrable(30) .
As calnexin has
been shown to associate with class I molecules shortly after their
synthesis, we asked in the present study whether calnexin influenced
either disulfide bond formation or assembly of m with
class I heavy chains in human cells. First we established that
formation of disulfide bonds could be measured in B*0702 heavy chains
from CIR transfectants. Heavy chain oxidation was very rapid and
occurred before detectable binding to
m. Both reduced
and oxidized heavy chains could be co-purified with calnexin. This
suggested either that calnexin bound to both reduced and oxidized heavy
chains or that reduced heavy chains bound calnexin and remained
associated while becoming oxidized.
To distinguish between these
possibilities, we asked whether heavy chains became oxidized at the
same rate when calnexin binding was blocked by using the drug
castanospermine, which inhibits trimming of glucose residues from N-glycans. Previous studies demonstrated that calnexin binds
to monoglucosylated N-glycans on proteins and that calnexin
binding can be inhibited by drugs which alter normal processing of
glycans(28, 33) . Treatment with castanospermine
decreased the total amount of non-m-associated heavy
chains and also significantly diminished the percentage of heavy chains
which were oxidized. This shows that calnexin participates in folding
and disulfide bond formation of class I heavy chains, but it does not
appear to be absolutely required. Furthermore, these results strongly
suggest that calnexin binds to reduced heavy chains and remains
associated until heavy chain folding is complete.
Several
alternative explanations for our results were considered. Class I heavy
chains might form aggregates that constrain folding in the absence of
calnexin. No evidence for this was seen on non-reducing SDS-PAGE.
Mislocalization of class I heavy chains might occur without calnexin,
but this seems unlikely since cells were lysed immediately after 3 min
of labeling. Rapid degradation of heavy chains after castanospermine
treatment was expected, as it was observed in an earlier study using
CMT-cK1 cells(34) . In our experiments, incubation
in the drug decreased heavy chain levels by about 20%. However, the
altered ratio of reduced to oxidized heavy chains noted after calnexin
binding is inhibited is independent of overall heavy chain levels and
suggests that calnexin directly facilitates heavy chain folding.
Since calnexin has a prolonged association with heavy chains in
cells lacking m(35) , it seemed possible that
calnexin might directly promote heavy chain binding to
m. Our results suggest that this is not the case, but
rather that calnexin helps heavy chains fold properly and thereby
increases the pool of fully conformed heavy chains available to bind
m. Heavy chain folding and disulfide bonding can occur
independently of calnexin, however, since oxidized heavy chains formed
after treatment with castanospermine, albeit less efficiently (Fig. 3B). Furthermore, the overall amount of class I
subunits that assembled and could bind w6/32 was unaffected, presumably
because other components required for class I assembly, such as
m and peptides, further limit the process.
Our data demonstrate that calnexin has one or more disulfide bonds essential for function. In support of this, we observed that addition of DTT to cells rapidly disrupted interactions between calnexin and a variety of proteins, including the invariant chain, which has no internal disulfide bonds, and both reduced and oxidized class I heavy chains (Fig. 1). These results are consistent with the hypothesis that calnexin, which is a monomer, contains intramolecular disulfide bonds. A similar conclusion was reached recently by Helenius and co-workers (36) . It should be noted that binding of the antibody AF8 appeared not to be affected by DTT treatment, ruling out the possibility of a conformational change in calnexin causing the antibody epitope to be lost. In contrast to our data, two studies have demonstrated increased binding of individual proteins to calnexin after DTT treatment, namely gp80 in MDCK cells and thyroglobulin in bovine thyrocytes(14, 37) . There is not a clear explanation for these disparate results, but they may be a reflection of the cell types used. We would also suggest that DTT treatment might cause aggregation of certain proteins in the absence of essential chaperones, as shown previously(38) . Such aggregates might include many unfolded proteins, including calnexin, and lead to an apparent increase in association between calnexin and other molecules in the aggregate.