(Received for publication, August 1, 1995; and in revised form, September 27, 1995)
From the
Recent evidence indicates that efficient expression of major
histocompatibility complex (MHC) complexes requires their interaction
with the resident endoplasmic reticulum (ER) chaperone calnexin, which
for certain proteins functions as a lectin specific for
monoglucosylated glycans. In the current report, we studied the
expression of MHC class I proteins in BW wild type thymoma cells (BW
WT) and glucosidase II-deficient BW PHAR2.7 cells. Consistent with a
requirement for glucose (Glc) trimming for interaction of class I
proteins with calnexin, we found that nascent H-2K proteins
associated with calnexin in untreated BW WT cells, but not in BW WT
cells treated with the glucosidase inhibitor castanospermine (cas), or
in untreated glucosidase II-deficient BW PHAR2.7 cells. Suprisingly, we
found that H-2K
expression occurred with similar efficiency
in BW PHAR2.7 cells as in BW WT cells and that formation of nascent
H-2K
complexes was perturbed by cas treatment in BW WT
cells but not in BW PHAR2.7 cells. Finally, it was noted that
expression of the molecular chaperone Bip was markedly increased in BW
PHAR2.7 cells relative to BW WT cells, which is suggested to play a
role in regulating the expression of H-2K
complexes in BW
PHAR2.7 cells. The current study demonstrates that Glc trimming is
required for efficient interaction of nascent H-2K
proteins
with calnexin; that expression of MHC class I proteins can, under
certain conditions, proceed effectively in the absence of Glc trimming
and calnexin association; and that Bip expression is markedly increased
under conditions where diglucosylated glycans persist on nascent
glycoproteins within the ER. These data are consistent with the
hypothesis that alternative oligomerization pathways exist for class I
proteins within the quality control system of the ER that have
differential requirements for removal of Glc residues from nascent
glycan chains.
Most major histocompatibility complex (MHC) ()class I
molecules expressed at the cell surface consist of heavy chains (HC)
noncovalently associated with
-microglobulin proteins
and processed peptides(1, 2) . The molecular chaperone
calnexin plays an important role in the assembly of nascent
HC
peptide complexes within the endoplasmic
reticulum (ER) and moderates their transport to the Golgi
compartment(3, 4, 5, 6) . Assembly
of nascent murine MHC class I complexes is believed to occur by: (i)
association of newly synthesized HC with calnexin to form HC-calnexin
protein complexes, (ii) interaction of
-microglobulin
proteins with calnexin
HC proteins to form
calnexin
HC
complexes; and finally (iii)
addition of peptide to calnexin-associated HC
proteins to form HC
peptide
complexes, which rapidly dissociate from calnexin and exit the
ER(7, 8, 9) .
A growing body of evidence
signifies that removal of glucose (Glc) residues from nascent
oligosaccharide chains is important for association of certain
glycoproteins with calnexin, including clonotypic chains of
the T cell antigen receptor complex(10) , viral gene
products(11, 12) , and many unidentified
proteins(10) . Removal of Glc residues from immature
Glc
Man
GlcNAc
(Glc = glucose,
Man = mannose; and GlcNAc = N-acetylglucosamine)
glycan chains is accomplished by the sequential action of ER
glucosidase I and glucosidase II enzymes, which remove the outermost
and two proximal Glc residues, respectively(13) . Since
calnexin associates with glycoproteins containing monoglucosylated
(Glc
Man
GlcNAc
) glycan
chains(12, 14) , both glucosidase I and glucosidase II
activities are necessary to create glycan substrates for calnexin
binding. Oligosaccharide chains are not strictly required for calnexin
association, however, as several nonglycosylated molecules interact
stably with calnexin, including recombinant multidrug resistance P
glycoprotein lacking N-linked addition sites (15) and
the CD3
subunit of the T cell antigen receptor
complex(16) .
To further our understanding of the role of
Glc trimming and calnexin association in the assembly and expression of
immune receptor molecules, we studied the expression of MHC class I
proteins in BW wild type thymoma cells (BW WT) and the glucosidase
II-deficient BW variant cell line, PHAR2.7. We found that expression of
H-2D proteins was markedly reduced on BW PHAR2.7 cells
relative to BW WT cells, and, unexpectedly, that expression of
H-2K
proteins was equivalent on both cell types. Our
results show that Glc trimming and calnexin association are required
for efficient formation of nascent H-2K
complexes in BW WT
cells but not BW PHAR2.7 cells, indicating that alternative folding
pathways exist within the ER quality control system for assembly of MHC
class I protein complexes. Lastly, Bip expression was observed to be
significantly increased in BW PHAR2.7 cells relative to BW WT cells,
which is implicated to play a role in regulating H-2K
expression in BW PHAR2.7 cells.
Figure 1:
Surface
expression of H-2D and H-2K
proteins on BW WT
cells and BW PHAR2.7 cells. A, surface expression of
H-2D
and H-2K
complexes was examined by
indirect staining with anti-H-2D
(15-5-5) and
anti-H-2K
(36-7-5) mAbs, followed by fluorescein
isothiocyanate (FITC)-conjugated goat anti-mouse IgG2a Ab
molecules (GAM). Control staining was performed with FITC-GAM
Ab only. The relative expression of MHC class I proteins on BW PHAR2.7
cells relative to BW WT cells was 0.35 for H-2D
proteins
and 1.1 for H-2K
proteins. The data shown are
representative of at least five independent experiments; variation
between experiments was less than 10%. B, BW WT and BW PHAR2.7
cells were surface-labeled by biotinylation, solubilized in 1% Nonidet
P-40, and lysates were precipitated with anti-H-2D
(15-5-5)
and anti-H-2K
(36-7-5) mAbs preadsorbed to protein
A-Sepharose. Immunoprecipitated material was analyzed on
one-dimensional SDS-PAGE gels under reducing conditions and visualized
by chemiluminescence techniques. The positions of H-2D
and
H-2K
proteins are indicated.
To study the
requirement for glucosidase activity for expression of MHC class I
complexes on BW WT cells, experiments were performed using the
glucosidase inhibitor castanospermine (cas)(13) . As shown in Fig. 2, surface expression of H-2D complexes on BW
WT cells was markedly reduced by overnight culture in cas (Fig. 2). Not suprisingly, surface density of H-2D
complexes was similar on untreated and cas-treated BW PHAR2.7
cells (Fig. 2), as expression of H-2D
complexes was
already decreased on glucosidase II-deficient BW PHAR2.7 cells. Most
importantly, these data show that cas treatment dramatically reduced
expression of H-2K
complexes on BW WT cells, but not BW
PHAR2.7 cells (Fig. 2). Reduced expression of MHC class I
complexes on cas-treated BW WT cells was specific in that expression of
other surface molecules (CD45 proteins, thymic shared antigen proteins,
and peanut agglutinin lectin-binding proteins) was unaffected by cas
treatment, and no change in class I expression was observed following
culture with the mannosidase inibitor deoxymannojirimycin (data not
shown). These results show that impaired glucosidase activity is
correlated with decreased expression of H-2D
complexes on
both BW WT and BW PHAR2.7 cells and that BW WT and BW PHAR2.7 cells
exhibit differential requirements for glucosidase activity in the
surface expression of H-2K
complexes.
Figure 2:
Effect of cas treatment on surface
expression of H-2D and H-2K
proteins on BW WT
cells and BW PHAR2.7 cells. Surface expression of H-2D
and
H-2K
complexes was examined by cell surface staining as
described in Fig. 1A. Where indicated, cells were
cultured overnight at 37 °C in the presence of the glucosidase
inhibitor castanospermine (Cas) at a concentration of 100
µg/ml. The data shown are representative of at least three
independent experiments; variation between experiments was less than
10%. Viability was identical in media and cas-treated groups (data not
shown).
Figure 3:
Intracellular transport of nascent
H-2K complexes in BW WT and BW PHAR2.7 cells. A,
BW WT and BW PHAR2.7 cells were metabolically pulse-labeled with
[
S]methionine for 30 min, chased for the time
period indicated, solubilized in 1% Nonidet P-40 detergent, and lysates
immunoprecipitated with anti-H-2K
mAb -(5) .
Precipitates were either mock-treated or digested with Endo H and
analyzed on SDS-PAGE gels under reducing conditions. The positions of
H-2K
proteins and
-microglobulin proteins
are indicated. K
= K
proteins resistant to digestion with Endo H; K
= K
proteins sensitive to digestion with
Endo H.
These data show that formation of nascent H-2K complexes
proceeds with similar efficiency in BW WT and BW PHAR2.7 cells and that
H-2K
complexes are effectively transported from the ER to
the Golgi compartment in both cell types, two findings which are in
agreement with our results regarding surface expression of H-2K
proteins on BW WT and BW PHAR2.7 cells (Fig. 1, A and B).
Figure 4:
Association of nascent H-2K
proteins with calnexin in BW WT and BW PHAR2.7 cells. A,
digitonin lysates of radiolabeled BW WT and BW PHAR2.7 cells were
immunoprecipitated with anti-calnexin Ab or anti-H-2K
mAb(37-6-5), and precipitates were analyzed on two-dimensional
NEPHGE/SDS-PAGE gels under reducing conditions. The position of
radiolabeled H-2K
proteins is indicated. B,
radiolabeled lysates of untreated and cas-treated BW WT cells were
immunoprecipitated with anti-calnexin Ab, and precipitates were
analyzed as in A. The position of H-2K
proteins is
indicated.
Figure 5:
Effect of total glucosidase blockade on
formation of nascent H-2K complexes in BW WT and BW PHAR2.7
cells. Untreated and cas-treated BW WT and BW PHAR2.7 cells were
metabolically pulse-labeled for 30 min with
[
S]methionine, chased for 45 min, solubilized in
1% Nonidet P-40, and lysates were immunoprecipitated with
anti-H-2K
-specific mAb(36-7-5). The presence of inhibitor
was maintained throughout the experiment. Precipitates were either
mock-treated or digested with glycosidases and analyzed on
one-dimensional SDS-PAGE gels under reducing conditions. Note that
protein synthesis was identical in untreated and cas-treated cell
groups ((10) ; data not shown). The positions of H-2K
proteins and
-microglobulin proteins are
indicated. In the experiment shown in A, precipitates were
digested with PNGase F which cleaves both immature and mature
oligosaccharides (K
=
deglycosylated K
polypeptides); in the experiment presented
in B, precipitates were digested with Endo H, which cleaves
only immature oligosaccharides (K
= Endo H-sensitive K
protein).
We conclude from these
data that removal of Glc residues from core glycan chains and calnexin
association is required for effective formation of H-2K complexes in BW WT cells but not BW PHAR2.7 cells. Impaired
formation of nascent H-2K
complexes in cas-treated BW WT
cells provides a molecular basis for reduced surface expression of
H-2K
proteins on BW WT cells cultured overnight in cas (Fig. 2). Moreover, since cas impaired formation of nascent
H-2K
complexes in BW WT cells and other T cell types,
including thymocytes and T hybridoma cells, (
)but not BW
PHAR2.7 cells, we further conclude that unique assembly mechanisms
exist in BW PHAR2.7 cells for assembly of nascent H-2Kk complexes in
the absence of Glc trimming and calnexin association.
Figure 6:
Increased Bip expression in BW PHAR2.7
cells relative to BW WT cells. A, Nonidet P-40 lysates of
equivalent numbers of BW WT and BW PHAR2.7 cells were precipitated with
anti-calnexin Abs (top) or anti-Bip Abs (bottom) and
blotted with the same Ab used for precipitation. The positions of
calnexin and Bip proteins are indicated; * denotes an unknown 44-kDa
protein which is reported to react with the anti-Bip Ab used in these
experiments (Stressgen product sheet). Lane 1, BW WT cells; lane 2, BW PHAR2.7 cells; lane 3, Ab + lysis
buffer. Identical results were obtained when whole lysates of BW WT and
BW PHAR2.7 were analyzed (data not shown). B, radiolabeled
digitonin lysates of BW WT and BW PHAR2.7 cells were immunoprecipitated
with anti-Bip Ab and precipitates were analyzed on two-dimensional
NEPHGE/SDS-PAGE gels under reducing conditions. The positions of Bip (arrowhead) and H-2K proteins (arrow) are
indicated.
The quality control system of the ER ensures that properly
folded, fully assembled protein complexes are expressed on the cell
surface. The molecular chaperone calnexin is believed to play a key
role in maintaining the fidelity of class I complexes expressed on the
cell surface by functioning in the assembly of nascent class I
HC--peptide complexes within the ER and regulating
their transport to the Golgi compartment. The data in the current study
show that: (i) removal of Glc residues from core glycans is required
for efficient interaction of nascent H-2K
proteins with
calnexin; (ii) calnexin association is not essential for stable
expression of H-2K
complexes; (iii) expression of Bip
chaperones is markedly increased in glucosidase II-deficient BW PHAR2.7
cells relative to BW WT cells; and (iv) H-2K
proteins were
associated with Bip proteins in BW PHAR2.7 cells but not BW WT cells.
Recent evidence indicates that initial association of oligomeric
glycoproteins with calnexin requires recognition of monoglucosylated
GlcMan
GlcNAc
species on
glycoproteins(12, 14) . In agreement with this idea,
we found that nascent H-2K
chains synthesized in BW WT
thymoma cells, but not in cas-treated BW WT cells or in glucosidase
II-deficient BW PHAR2.7 cells, associated with calnexin. Degradation of
murine and human MHC class I proteins under conditions of impaired
glucosidase activity has been reported(23, 24) . As
the Abs used in our studies effectively recognize
-assembled H-2K
chains, but not
unassembled H-2K
chains, it remains to be determined if
reduced formation of H-2K
complexes in cas-treated BW WT
cells results from rapid degradation of nascent H-2K
HC or
the failure of H-2K
HC to assemble effectively with
molecules. Regardless, our data clearly show that Glc
trimming and calnexin association are not essential for stable
expression of H-2K
complexes as H-2K
proteins
are effectively expressed in glucosidase II-deficient BW PHAR2.7
thymoma cells.
Maintenance of class I expression is important for
immune surveillance function within the body. The data in the current
study show that alternative folding pathways exist within the ER that
sustain expression of class I protein complexes under conditions where
Glc trimming and calnexin association are impaired. Unlike H-2K molecules, H-D
proteins were not stably expressed on
BW PHAR2.7 cells, indicating that assembly mechanisms exist within the
ER of BW PHAR2.7 cells that function effectively for the
oligomerization of H-2K proteins but not H-2D proteins. Regarding this
issue, we have considered the possibility that stable expression of
H-2K
proteins in BW PHAR2.7 cells results from mutations
introduced during their selection procedure (18) that allow
H-2K
polypeptides made in BW PHAR 2.7 cells to uniquely
assemble with
proteins and processed peptide in the
absence of Glc trimming and calnexin association. While the current
studies do not exclude this possibility, preliminary sequencing data
suggest that H-2 K
molecules expressed in BW WT and BW
PHAR2.7 cells are identical. (
)
The molecular basis for
proficient expression of H-2K complexes in BW PHAR2.7 cells
is unclear, but as suggested in this report may involve interaction
with Bip proteins. Unlike human class I molecules which stably interact
with Bip proteins(9, 25) , association with murine
class I molecules with Bip is controversial as Bip associates weakly
with endogenous mouse class I proteins (9) and fails to
interact with mouse gene products expressed in human cell
lines(25) . Indeed, no association between Bip and H-2K
proteins in BW WT thymoma cells was observed in our studies (Fig. 5B). It is well documented that Bip expression
increases during periods of ER stress and is stimulated by treatment
with compounds that interfere with glycan addition or processing of
oligosaccharide side chains(26, 27) . The current
report shows that expression of Bip proteins is markedly elevated in
glucosidase II-deficient BW PHAR2.7 cells relative to BW WT cells,
suggesting that persistence of diglucosylated glycans on nascent
glycoproteins results in the accumulation of numerous malfolded
proteins within the ER, which, in turn, results in increased Bip
synthesis via feedback regulatory mechanisms(28) . Thus, it is
conceivable that quantitatively increased levels of Bip proteins in BW
PHAR2.7 cells represent the molecular basis for effective expression of
H-2K
complexes in BW PHAR2.7 cells. As we were unable to
determine if H-2K
proteins associated with Bip in BW
PHAR2.7 cells represent intermediates in the assembly of
H-2K
-
-peptide complexes or end-stage
H-2K
proteins targeted for degradation, the fate of
Bip-associated H-2K
proteins in BW PHAR2.7 cells is
unclear. Alternatively, it is reasonable to speculate that stable
expression of H-2K
proteins in BW PHAR2.7 cells results
from their association with an unknown chaperone protein(s) that, like
Bip, functions independently of glycan processing and is up-regulated
during periods of ER stress. We were unsuccessful in our attempts to
sustain expression of H-2K
complexes in BW WT cells by
extended culture in cas, despite the fact that Bip expression was
increased under these conditions (data not shown). Whether these
findings reflect difficulties in approximating ER stress conditions in
BW WT and BW PHAR2.7 cells by drug treatment or denote true differences
between the ER environments of BW WT and BW PHAR2.7 cells remains to be
determined. Finally, it should be noted that Scott and Dawson recently
reported expression of human MHC class I proteins in a
calnexin-deficient cell line(29) , showing that calnexin is not
absolutely required for the transport and surface expression of human
MHC class I molecules.