(Received for publication, December 11, 1995; and in revised form, February 6, 1996)
From the
Most T lymphocytes express on their surfaces an oligomeric
protein complex consisting of clonotypic polypeptides
associated with invariant CD3-
and
chains,
designated the T cell antigen receptor (TCR) complex. Assembly and
intracellular transport of nascent TCR proteins is believed to be
assisted by their interaction with the molecular chaperone calnexin,
which for certain molecules functions as a lectin for monoglucosylated
glycans. However, as most of our knowledge about calnexin-TCR protein
associations has been obtained under conditions of limited TCR
assembly, the role of calnexin in the formation of nascent TCR
complexes is unclear. Here, we studied the role of glucose (Glc)
trimming and calnexin association in the oligomerization of TCR
and CD3
glycoproteins in murine splenic T lymphocytes, a model
cell type for efficient assembly of complete TCR complexes. We show
that removal of Glc residues from both CD3
proteins and TCR
proteins occurred prior to their association with any other TCR
components and that calnexin specifically interacted with unassembled
TCR
and CD3
proteins containing incompletely trimmed
oligosaccharides. Interestingly, we found that removal of Glc residues
from glycan chains was necessary for efficient association of calnexin
with TCR
glycoproteins but not with CD3
glycoproteins. These
studies define Glc trimming and calnexin association as initial
molecular events in the translation of CD3
and TCR
proteins,
occurring coincident with or immediately after their translocation into
the endoplasmic reticulum and preceding the ordered pairing of TCR
chains. In addition, these data document that calnexin assembly with
CD3
and TCR
glycoproteins involves both glycan-dependent and
glycan-independent mechanisms.
Assembly of the T cell antigen receptor (TCR) ()complex occurs within the endoplasmic reticulum (ER) and
proceeds in a highly ordered manner by: (i) formation of noncovalently
associated pairs of
and
proteins, (ii)
association of individual clonotypic
,
polypeptides with
and
pairs to form intermediate
and
protein complexes, (iii) rapid pairing of
and
chains and disulfide-bonding of
CD3-associated
proteins to yield incomplete
TCR complexes, and finally (iv) addition
of
homodimers to form complete, fully assembled
TCR
complexes(1, 2, 3, 4) . Egress of
TCR proteins from the ER is directly related to their assembly status;
most unassembled TCR polypeptides and partial TCR complexes are
retained within the ER. Only TCR proteins assembled into incomplete
TCR complexes or complete
TCR complexes effectively
transit from the ER to the Golgi system(1) .
The molecular
chaperone calnexin is a nonglycosylated resident ER transmembrane
protein that associates with numerous oligomeric protein complexes
within the ER, including and
integrins(5) , major histocompatibility class
I(6, 7) and class II molecules(8) , and the
antigen receptors expressed on T and B
lymphocytes(7, 9, 10) . Regarding the TCR
complex, four individual TCR proteins have been shown to associate with
calnexin, including clonotypic TCR
and TCR
polypeptides (7, 10) and CD3
and CD3
chains(7, 9, 11) . Association between
calnexin and
proteins has never been observed(12) . While
convincing evidence exists for association of calnexin with individual,
unassembled TCR proteins, much less is known about the assembly of
calnexin with incompletely assembled TCR complexes containing multiple
TCR subunits. Indeed, the assembly status of most nascent TCR proteins
associated with calnexin has not been rigorously
evaluated(7, 9) . Recently, Wiest and co-workers
reported that calnexin is expressed on the surfaces of immature
thymocytes in association with CD3
and CD3
pairs (13, 14) . Expression of such clonotype-independent
complexes appears to be developmentally regulated as they were not
detected on the surfaces of mature T cells(14) . Unlike mature
T cells, immature thymocytes do not efficiently assemble CD3 components
into complete
TCR
complexes(15) , leading to the suggestion that calnexin
association with partial TCR complexes is exaggerated in cell types
that are deficient in formation of complete TCR complexes (14) .
A growing body of evidence signifies that removal of
glucose (Glc) residues from nascent oligosaccharide chains is important
for initial association of glycoproteins with
calnexin(10, 16, 17) . Removal of Glc
residues from immature GlcMan
GlcNAc
species (Glc, glucose; Man, mannose; GlcNAc, N-acetyl
glucosamine) is accomplished by the sequential action of ER glucosidase
I and glucosidase II enzymes, which remove the outermost and two
innermost Glc residues, respectively(18) , a process referred
to as Glc trimming. Recent studies by several laboratories indicate
that calnexin recognizes glycan chains on nascent glycoproteins bearing
monoglucosylated (Glc
Man
GlcNAc
)
saccharides(10, 16, 17, 18, 19) ,
indicating that both glucosidase I and glucosidase II activities are
necessary for creation of glycan substrates for calnexin
binding(20) . 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 (21) and the CD3
subunit of the TCR complex(11) .
The role of Glc trimming and calnexin association in the
oligomerization of TCR proteins within the ER is poorly understood.
Indeed, it is unknown at which stage(s) of TCR assembly Glc residues
are removed from oligosaccharide chains on nascent TCR glycoproteins in
any cell type. Regarding the role of calnexin in TCR assembly, it has
been suggested that calnexin interacts with individual newly
synthesized TCR proteins to facilitate their folding within the ER and
to prevent their escape to the Golgi
compartment(7, 9, 10, 12) .
Moreover, since calnexin has been shown to associate with all TCR
components except , it has also been proposed that calnexin
functions in the oligomerization of TCR proteins into incomplete
TCR complexes(9, 12) .
To determine at which stage(s) of TCR assembly Glc trimming and
calnexin association occurs and to see if calnexin does, in fact,
participate in the oligomerization of TCR subunits within the ER, we
studied the role of Glc trimming and calnexin association in the
assembly of TCR
and CD3
glycoproteins into TCR complexes in
murine splenic T cells. Our studies show that removal of Glc residues
from nascent glycan chains on both CD3
and TCR
proteins
occurs prior to their association with other TCR proteins and that
calnexin associates exclusively with unassembled CD3
and TCR
proteins containing incompletely trimmed glycans. Moreover, we
demonstrate that Glc trimming is required for effective interaction of
calnexin with TCR
proteins, but not with CD3
proteins, and
report that removal of oligosaccharides does not affect the stability
of existing protein complexes of calnexin and TCR
, CD3
proteins. The implications of these findings on our current knowledge
of TCR assembly are discussed.
Figure 1:
CD3 glycoforms reflect
differential processing of individual glycan chains on CD3
molecules. Digitonin lysates of metabolically pulse-labeled splenic T
cells were immunoprecipitated with anti-CD3
Ab and analyzed on
SDS-PAGE gels under reducing conditions. The positions of radiolabeled
,
,
,
, and
proteins are indicated (left-hand side). In the experiments shown on the right, CD3
proteins were precipitated from lysates of
splenic T cells treated with medium (MED) or castanospermine (CAS); precipitates were boiled in 1% SDS to release
precipitated material, and CD3
proteins recaptured with
anti-CD3
Abs; recaptured CD3
chains were either mock-treated
or digested with jack bean mannosidase (JB) or Endo H (EH). The positions of CD3
glycoforms (A-D) and Endo H-sensitive CD3
glycoproteins (CD3
EH
) are
indicated.
Figure 2:
Calnexin associates specifically with
unassembled CD3 proteins containing incompletely trimmed glycan
chains. A, digitonin lysates of radiolabeled splenic T cells
were immunoprecipitated with anti-CD3
Ab (left-hand side)
or with anti-calnexin Ab (right-hand side). Precipitated
material was released by boiling in SDS, CD3
proteins recaptured
by precipitation with anti-CD3
Ab, and precipitates digested with
glycosidases as indicated. The positions of CD3
glycoforms (A-D) and Endo H sensitive CD3
glycan chains (CD3
EH
) are marked. B, digitonin
lysates of splenic T cells metabolically pulse-labeled for 30 min were
immunoprecipitated with anti-CD3
Ab, or sequentially precipitated
with anti-TCR
Ab, followed by anti-CD3
mAb, and finally with
anti-CD3
Ab. Material was released from precipitates by boiling in
SDS, CD3
proteins were recaptured by precipitation with
anti-CD3
Ab, and precipitates digested with glycosidases as
indicated. The positions of CD3
glycoforms (A-D)
and Endo H-sensitive CD3
glycoproteins (CD3
EH
) are marked.
To determine if
calnexin remained associated with CD3 proteins following their
assembly with other TCR proteins and to ascertain at which stage(s) of
TCR assembly Glc residues are completely removed from CD3
proteins, a series of sequential immunoprecipitation studies was
performed to examine the Glc trimming status of CD3
proteins
containing incompletely trimmed and fully trimmed glycan chains. For
these experiments, digitonin lysates of radiolabeled splenic T cells
were precipitated with anti-CD3
antiserum to capture total
CD3
proteins, or were sequentially immunoprecipitated with: (i)
anti-TCR
mAb to isolate CD3
proteins existing in complete
TCR and incomplete
TCR complexes, followed by (ii)
anti-CD3
Ab to capture CD3
proteins existing in intermediate
and partial
protein complexes, and finally
(iii) anti-CD3
Ab to isolate free, unassembled CD3
proteins.
CD3
glycoproteins were recaptured from precipitates and their Glc
trimming status evaluated by JB digestion. As previously established,
four distinct glycoforms of CD3
chains existed in splenic T cell
lysates (Fig. 2B, left-hand side, A-D). Clearly, both incompletely trimmed and fully
trimmed CD3
glycoforms were present in the pool of unassembled
CD3
proteins (Fig. 2B, right-hand side, A-D). In contrast, CD3
proteins associated with
CD3
and TCR
molecules contained glycan chains that were
devoid of Glc residues as they were completely susceptible to JB
digestion (Fig. 2B, middle, D). Chase
studies showed that Glc residues were completely removed from CD3
glycoproteins within several hours of their synthesis in splenic T
cells, which correlated with their assembly into TCR complexes and
transport to the Golgi system (data not shown).
Taken together,
these results demonstrate that calnexin associates with unassembled
CD3 proteins containing incompletely trimmed oligosaccharide
chains. Moreover, since CD3
proteins assembled with CD3
containing complexes contained fully trimmed glycan chains, these
studies indicate that Glc trimming of CD3
glycoproteins occurs
prior to their assembly with other TCR proteins.
To confirm these
results, we wished to assess the Glc trimming status of CD3
proteins at an earlier time point prior to their association with
CD3
and TCR
proteins. For these studies, splenic T cells were
pulse-labeled for only 5 min and chased for 10 and 20 min. Sequential
precipitations of assembled and unassembled CD3
chains and
release/recapture procedures were performed as described in Fig. 2B. As expected, the vast majority of CD3
proteins existing at the end of the short pulse period were not
associated with CD3
chains, but existed as free, unassembled
CD3
chains (Fig. 3, top). During the chase period,
nascent CD3
proteins assembled with CD3
chains (Fig. 3, middle), and successively with TCR
molecules (Fig. 3, bottom). These results show that the
first detectable CD3
proteins assembled with other TCR proteins in
our studies contained fully trimmed glycan chains. Note that most
nascent CD3
proteins synthesized during a 5 min pulse period were
susceptible to JB digestion (Fig. 3, top, A-D), indicating that processing of nascent CD3
proteins by ER glucosidase enzymes occurs coincident with or
immediately after their translation and insertion into the ER
lumen(28, 29) .
Figure 3:
Glucose trimming and assembly of newly
synthesized CD3 glycoproteins in splenic T cells. Same as in Fig. 2B, except that cells were pulsed for only 5 min
and chased for the time period indicated. The positions of CD3
glycoproteins and CD3
glycoforms (A-D) are
marked.
Figure 4:
Calnexin associates with unassembled
TCR proteins containing incompletely trimmed oligosaccharides.
Digitonin lysates of radiolabeled splenic T cells were
immunoprecipitated with anti-calnexin Ab, or sequentially precipitated
with anti-CD3
mAb, followed by anti-V
11 mAb. Precipitated
material was released by boiling in SDS, TCR
proteins recaptured
by precipitation with anti-TCR
mAb, and samples digested with
glycosidases as indicated. Precipitates were analyzed on 13% SDS-PAGE
gels under reducing conditions. The positions of TCR
glycoforms (A and B) and Endo H-sensitive TCR
glycoproteins (TCR
EH
) are marked. Note that higher
percentage acrylamide gels reveal additional TCR
glycoforms that
are compressed between the A and B glycoforms displayed here (data not
shown); because resolution of other TCR
glycoforms was not optimal
on such gels, however, material was analyzed on 13% gels for these
studies.
Figure 5:
Glc
trimming is required for effective association of TCR proteins,
but not CD3
proteins, with calnexin. A, splenic T cells
were treated with medium (MED) or castanospermine (CAS), metabolically pulse-labeled, lysed in digitonin, and
precipitated with anti-calnexin Ab. The presence of Cas was maintained
throughout the pulse period. Calnexin precipitates were boiled in SDS,
TCR proteins recaptured, and precipitates were digested with Endo H.
The relative amount of TCR proteins present in Endo H-digested samples
was quantitated by densitometry scanning, with the amount of
calnexin-associated TCR proteins in medium groups set at 1.0. Multiple
exposures of autoradiographs were scanned to ensure linearity. B, digitonin lysates of Cas-treated splenic T cells were
incubated with three rounds each of protein A-Sepharose (control), 145-2C11 anti-CD3
mAb, HMT3.1 anti-CD3
mAb, or anti-CD3
Ab. Material was then sequentially precipitated
with anti-calnexin Ab, precipitates boiled in SDS, and CD3
proteins recaptured with anti-CD3
Ab. For quantitative purposes,
precipitates were digested with Endo H. The position of digested
CD3
proteins (CD3
EH
) is
indicated.
We reasoned that CD3 proteins
might associate directly with calnexin molecules in Cas-treated splenic
T cells, or indirectly, via pairing with CD3
proteins(11) . To examine this issue, we evaluated the ability
of various anti-CD3
mAbs to preclear calnexin-associated CD3
proteins from splenic T cell lysates. As demonstrated in Fig. 5B, precipitation of Cas-treated splenic T cell
lysates with anti-CD3
Abs effectively removed calnexin-associated
CD3
chains, as expected (Fig. 5B, compare lanes 1 and 4). In contrast, precipitation with two
different anti-CD3
mAbs failed to preclear calnexin-associated
CD3
proteins from lysates of Cas-treated splenic T cells (Fig. 5B, compare lanes 1, 2, and 3). Thus, we conclude that CD3
proteins associate
directly with calnexin molecules in Cas-treated splenic T cells. While
these experiments do not formally exclude the possibility that
anti-CD3
mAbs do not effectively recognize calnexin-associated
protein complexes in Cas-treated splenic T cells, it should
be noted that these mAbs are capable of precipitating
calnexin-associated CD3
proteins formed in immature T
cells(14) .
Finally, we wished to evaluate whether N-linked glycan chains on TCR and CD3
glycoproteins
were required for maintaining stable interaction with calnexin.
Calnexin precipitates of radiolabeled splenic T cell lysates were
either mock-treated or digested with Endo H, and association of TCR
glycoproteins with calnexin before and after deglycosylation was
compared. As demonstrated, equivalent amounts of TCR
and CD3
proteins were recaptured from mock-treated and Endo H-digested
anti-calnexin precipitates (Fig. 6), showing that once formed,
complexes of calnexin and individual CD3
and TCR
proteins do
not require oligosaccharide chains to maintain their association.
Figure 6:
Oligosaccharide chains are not necessary
to maintain stable association of calnexin with TCR and CD3
glycoproteins. Digitonin lysates of radiolabeled splenic T cells were
immunoprecipitated with anti-calnexin Ab; precipitates were resuspended
in Endo H digestion buffer in the presence or absence of Endo H and
incubated at 37 °C for 14 h. Following digestion, precipitates were
washed in PBS; supernatants containing material released by Endo H
digestion were removed, pellets (precipitates) were boiled in SDS, and
TCR proteins recaptured by precipitation with anti-TCR specific Abs.
The positions of CD3
and TCR
proteins are indicated. Note
that negligible amounts of TCR
and CD3
proteins were
recovered from supernatants of mock-treated and Endo H-digested
calnexin precipitates (data not shown).
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 assist in the
oligomerization of nascent TCR proteins within the ER and to play a
role in regulating the transport of assembled TCR complexes from the ER
to the Golgi compartment. In the current study, we evaluated the role
of Glc trimming and calnexin association in the oligomerization of
CD3 and TCR
proteins in splenic T lymphocytes. Our results
show that: (i) removal of Glc residues from nascent CD3
proteins
and TCR
proteins occurs prior to their association with partner
TCR chains within the ER; (ii) calnexin associates specifically with
unassembled CD3
proteins and unassembled TCR
proteins
containing incompletely trimmed glycan chains; (iii) Glc trimming is
required for effective association of TCR
proteins, but not
CD3
proteins, with calnexin; and (iv) oligosaccharide chains are
important in the initial assembly of TCR
-calnexin protein
complexes, but that once formed, are not required to maintain their
association. Taken together, these data effectively rule out the
postulate that calnexin functions as a scaffold for assembly of nascent
TCR complexes and show that calnexin assembly with TCR glycoproteins
involves both glycan-dependent and glycan-independent mechanisms.
Recent studies suggest that association of nascent glycoproteins
with calnexin proceeds in a two-step fashion involving initial binding
of monoglucosylated glycans by calnexin, followed by protein-protein
interactions which stabilize these
associations(19, 30) . In agreement with this model,
we found that inhibition of glucose trimming markedly impaired
association of nascent TCR proteins with calnexin in splenic T
cells and, similar to what has been reported for major
histocompatibility class I proteins (19) and major
histocompatibility class II proteins(31) , removal of
oligosaccharide chains from existing calnexin-TCR
complexes did
not affect the stability of their interaction. Interestingly, the
transmembrane domain of all of these proteins has been implicated in
maintaining their association with calnexin(31, 32) ,
suggesting that calnexin interactions may be stabilized within the
lipid bilayer of the ER. The finding that blockade of Glc trimming
severely limits the assembly of nascent TCR
proteins with calnexin
in splenic T cells is in agreement with previous studies showing that
association of nascent TCR
proteins with calnexin is impaired in
glucosidase II-deficient BW PHAR2.7 thymoma cells and in Cas-treated
wild type BW thymoma cells(10) . Importantly, the current study
extends these findings by showing that, unlike calnexin association
with TCR
proteins, assembly of calnexin with CD3
proteins was
only modestly affected by inhibition of Glc trimming. Thus, CD3
represents the second TCR component that has been described to interact
with calnexin in a glycan-independent manner, with CD3
being the
first(11) . Because CD3
and CD3
proteins are
structurally homologous(1) , it is reasonable to speculate that
CD3
and CD3
molecules share a common region that mediates
their association with calnexin. It remains to be determined why some
CD3
chains synthesized in Cas-treated splenic T cells failed to
associate with calnexin in our studies. The presence of
monoglucosylated glycans on CD3
proteins, although not absolutely
required, may increase the efficiency with which CD3
proteins
interact with calnexin. Alternatively, as persistence of Glc residues
on oligosaccharide chains has been observed to result in increased
association of CD3
chains with CD3
molecules(4) , it
is conceivable that enhanced assembly of CD3
chains into
pairs in Cas-treated splenic T cells precludes their
association with calnexin.
The role of calnexin in the
oligomerization of TCR proteins has been controversial as most studies
on calnexin association with TCR proteins have been performed in cell
types that are markedly impaired or completely deficient in the
assembly of complete TCR
complexes(10, 14) . Studies on calnexin interaction
with TCR proteins are further complicated by the fact that multiple TCR
proteins coprecipitate with calnexin, which may or may not be assembled
with each other into oligomeric TCR complexes(7, 9) .
To overcome these problems, we performed studies in murine splenic T
lymphocytes, which efficiently assemble complete
TCR complexes (15) and, most importantly, implemented a
precipitation/recapture/glycosidase digestion protocol that allowed us
to rigorously identify the Glc trimming status of TCR
and CD3
proteins associated with calnexin and partner TCR chains. Indeed, our
results demonstrate that calnexin associates exclusively with
unassembled CD3
and TCR
proteins containing incompletely
trimmed oligosaccharide chains in splenic T cells and show that
processing of oligosaccharides on CD3
and TCR
glycoproteins
by ER glucosidase enzymes precedes their assembly into intermediate
and incomplete
TCR
complexes(4) . Thus, we believe that calnexin assists in the
folding of newly synthesized, individual TCR proteins within the ER but
do not think that calnexin plays a significant role in the
oligomerization of multiple TCR protein subunits. It remains possible
that calnexin release occurs coincident with assembly of individual TCR
proteins with partner chains and that such assembly facilitates
displacement of calnexin; importantly, however, our data show that
calnexin does not remain associated with TCR
and CD3
proteins
assembled into multisubunit TCR complexes in splenic T cells. Finally,
it is noteworthy to mention that our results on calnexin association
with unassembled TCR components in splenic T cells parallel recent
findings on the assembly of IgM complexes in B cells, which showed that
calnexin associated with free IgM heavy chains but not with IgM chains
assembled with Ig
proteins(33) .
In summary, the
current report has evaluated the role of Glc trimming and calnexin
association in the assembly of CD3 and TCR
proteins into TCR
complexes in splenic T cells. These studies define Glc trimming and
calnexin association as initial molecular events in the assembly
cascade of TCR glycoproteins that precede the ordered pairing of TCR
chains within the ER.