By
From the Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
The T cell receptor for antigen (TCR) is a multisubunit complex that consists of at least seven
polypeptides: the clonotypic, disulfide-linked /
heterodimer that is noncovalently associated with the invariant polypeptides of the CD3 complex (CD3-
, -
, -
) and
, a disulfide-linked
homodimer. We achieved the complete assembly of the human TCR in an in vitro transcription/translation system supplemented with dog pancreas microsomes by simultaneous translation of the messenger RNAs encoding the TCR-
, -
and CD3-
, -
, -
, and -
subunits.
CD3-
, one of the subunits that initiates the assembly of the TCR in living cells, forms misfolded, disulfide-linked homooligomers when translated alone. However, co-translation of one
of its first binding partners in the course of assembly, CD3-
or -
, led to the expression of
mainly monomeric and correctly folded
subunits, the only form we could detect as part of a properly assembled TCR complex. In the absence of these subunits, the ER-resident chaperone calnexin interacted with oligomeric, i.e. misfolded, structures of CD3-
in a glycan-independent manner. A glycan-dependent interaction between CD3-
and calnexin was mediated
by CD3-
and concerned only monomeric CD3-
complexed with CD3-
, but was dispensable for proper folding of CD3-
. We suggest that in addition to its signaling function, CD3-
serves as a monitor for proper subunit assembly of the TCR.
Most T lymphocytes express on their plasma membrane the TCR-CD3 complex. This multisubunit
receptor has served as a paradigm for the analysis of the
biogenesis of multimembrane proteins, and shown how, in
the absence of a properly assembled complex, the remaining subunits are purged from the endoplasmic reticulum (ER)1 (1).
At the cell surface, the clonotypic TCR- Besides fulfilling signaling functions, the CD3 subunits
and the The molecular determinants underlying the subunit-specific interactions that promote assembly and the degradation of single TCR-CD3 subunits in the ER have been the
subject of intensive research, but are still not fully understood. Experimental evidence supports an assembly model
based on salt bridges formed in the lipid bilayer between
charged residues within the transmembrane of the individual TCR subunits (12). The presence of these charged residues in the transmembrane domains of single TCR- The molecular chaperone calnexin (IP90, p88) is also involved in the assembly of TCR. Originally discovered in
association with partially assembled TCR complexes devoid
of The role of the CD3 subunits in TCR assembly has been
explored mostly in transfection-based expression systems.
The requirement for the simultaneous presence of all CD3
subunits for proper complex formation has been convincingly demonstrated. However, the contribution of the individual TCR subunits to folding, assembly, and quality control, such as the monitoring of proper quaternary structure, has received far less attention.
We have studied the assembly of the human TCR-CD3
complex with a particular focus on early events of this
process, using a cell-free translation system supplemented
with dog pancreas microsomes. Optimization of the translation conditions allow the simultaneous translation of nine
different messenger RNAs (mRNAs) with high efficiency.
If proper redox conditions are imposed on the ER-derived microsomes, we observe correct folding of translated polypeptides, which are assembled in a subunit-specific manner
into TCR-CD3 complexes indistinguishable from those
reported for living cells. We have used this in vitro system
to monitor the folding of CD3- DNA Constructs.
The cDNAs encoding the HA1.7 TCR- Antibodies and Reagents.
The following mouse mAbs were
used in this study: Tü36 (30) recognizes properly conformed HLA-
DR1 heterodimers, OKT4 (31, 32), which is reactive against properly conformed human CD4 and OKT3 (31), recognizes CD3
(both obtained from American Type Culture Collection, Rockville, MD). JOVI.1 and JOVI.3 were raised against human TCR- and -
subunits appear as a disulfide-linked heterodimer that constitutes the true ligand binding unit and determines the specificity of the receptor. To transduce extracellular signals into
the cytoplasm, the TCR associates noncovalently with a
number of accessory polypeptides, jointly referred to as the
CD3 complex. This complex consists of the evolutionarily
related CD3-
, -
, and -
subunits, all of which belong to
the Ig gene family (2), and a disulfide-linked homodimer
of the TCR-
subunit. The
chain, a member of the gene family that also includes the
chain of the high affinity IgE receptor, lies largely on the cytoplasmic side of the plasma
membrane and has an extracellular domain of only nine
residues (5). Although the occurrence of
,
,
,
,
2, and
2
2 structures has been reported in vivo
(6, 7), the exact stoichiometry of a completely assembled
TCR-CD3 complex remains to be determined accurately (8).
chain are also required for cell surface expression
of the TCR-
/
heterodimer (9, 10). The efficiency of
TCR assembly in the ER determines receptor density at
the cell surface of T cells; single subunits that fail to join a
complex are retained in the ER and subsequently degraded
(11), whereas partial complexes are targeted to lysosomal
compartments for destruction (10).
, -
,
and CD3-
subunits has been shown to play a key role in
rapid ER degradation (13). In addition, the role of extracellular domains in the assembly of the TCR subunits has
been well documented in several studies (14, 15). Furthermore, the
homodimer seems to monitor the quaternary
structure of the partial TCR-CD3 complex to ensure that
only functionally active complexes are displayed at the cell surface. It is seen in association only with completely assembled receptor complexes and, in the absence of
2 homodimers, surface expression of TCR is compromised (10).
The
2 homodimer may be associated with the TCR complex only peripherally, as it can apparently be exchanged
for
subunits that reside at the cell surface (16).
subunits and in association with MHC class I molecules (17, 18), calnexin and its close relative calreticulin facilitate protein folding in the ER (19). Although calnexin
can act exclusively as a lectin, binding to monoglucosylated
trimming intermediates of N-linked glycans attached to the
target polypeptide (20), the initially glycan-dependent calnexin-MHC class I interaction was maintained after the removal of the N-linked glycans (21). Furthermore, glycan-independent binding between calnexin and aggregates of the vesicular stomatitis virus G protein (VSV-G protein)
has been reported (22). Because calnexin associates with all
TCR subunits except TCR-
(17, 18, 23, 24), it has been
suggested that it promotes the assembly of
2 complexes. The half-life of newly synthesized TCR-
proteins
that fail to join a TCR complex and become a target of ER
degradation is significantly prolonged due to their interaction with calnexin (23). However, the extent to which calnexin is involved in the folding of the individual TCR subunits and the assembly of the TCR-CD3 complex is not
known.
in an early stage of TCR
assembly, a process influenced dramatically by the presence
of related CD3 subunits. Translation of CD3-
alone gives
rise mainly to misfolded, disulfide-linked homooligomeric structures, the formation of which was substantially suppressed when CD3-
or -
were translated in the same reaction. The molecular chaperone calnexin was found to bind
oligomeric forms of CD3-
in a glycan-independent manner.
When CD3-
, which carries two N-linked glycans, was
translated simultaneously, monomeric CD3-
interacted with
calnexin exclusively in a glycan-dependent manner. We propose a role for CD3-
in sensing the quarternary structure of the TCR-CD3 complex.
and -
chains were provided by Dr. M.J. Owen (Imperial Cancer
Research Fund, London, UK; reference 25). We obtained the
cDNAs encoding CD3-
, -
and -
(2) and TCR-
(5, 26)
from Dr. C. Terhorst (Beth Israel Hospital, Boston, MA). The
cDNAs encoding HLA-DR1-
(27) and -
chains (28) were a
gift from Dr. Trowsdale (Human Immunogenetics Group, Imperial Cancer Research Fund, London, UK). The sequences which
encode the cleavable signal peptide of the TCR-
, -
, CD3-
, -
,
-
, TCR-
, and HLA-DR1-
were exchanged for the signal peptide encoding sequence of the mouse class I heavy chain H2-Kb.
The CD4 encoding cDNA (29) was cloned into pSP64 (Promega Corp., Madison, WI) under the control of the SP6 promotor. All other cDNAs were cloned into pSP72 (Promega Corp.) under the
control of the T7 promotor.
(V
3C
1; 33) and TIA-2 recognizes preferentially human TCR-
homodimers (34; gift from Dr. P. Anderson, Dana-Farber Cancer
Institute, Boston, MA).
, -
and -
were provided by Dr. J. Coligan (National Institutes of Health, Bethesda, MD) and the antisera raised against the human TCR-
and -
chain (35) were a gift from Dr. O. Acuto (Institut Pasteur, Paris, France). The rabbit polyclonal antiserum against the COOH terminus of canine calnexin was purchased from StressGen Biotechnologies Corp. (Victoria, Canada).
Gel Electrophoresis. SDS-glycine electrophoresis (SDS-PAGE) was performed as described (37). SDS-glycine gels were fluorographed using DMSO-2,5-diphenyl oxazole (Sigma Chemical Co.) and exposed to XAR-5 films (Kodak, Rochester, NY).
In Vitro Transcription and In Vitro Translation.
In vitro transcriptions were performed using T7 or SP6 RNA polymerase
(Promega Corp.). RNA was equipped with a G5ppp5
N-cap structure either co- or posttranscriptionally according to the vendor's
instructions (Promega Corp.). RNA was stored in water at
80°C.
The optimal amount of RNA for translation was determined empirically for each stock of RNA prepared. Before its use in translations, the RNA was heated (80°C) for 2 min and subsequently
chilled on ice.
Immunoprecipitation and Endoglycosidase H Digestion. After translation, microsomes were pelleted by centrifugation (5415C centrifuge; Eppendorf North America, Inc., Madison, WI; 14,000 rpm, 10 min) and subsequently lysed for 30 min on ice in 1 ml NP-40 lysis buffer (0.5% NP-40, 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2) or digitonin (Wako, Kyoto, Japan) lysis mix (1% digitonin, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co.). Insoluble debris was removed by centrifugation (14,000 rpm, 10 min). Supernatants were precleared twice with either normal rabbit or mouse serum and formalin fixed Staphylococcus aureus and then immunoprecipitated with the indicated antibodies as described elsewhere (38). Digestions with endoglycosidase H (EndoH)f (New England Biolabs, Beverly, MA) were performed on immune complexes bound to S. aureus according to the vendor's instruction.
The translational performance of the mRNAs encoding the TCR subunits was optimized to ensure the likelihood of subsequent assembly of subunits translated simultaneously. We substituted the 5 untranslated sequence as well as the signal peptide encoding sequence of all cDNAs with the corresponding sequence of the murine MHC class I molecule Kb, the
in vitro transcript of which had proven to be translated with high efficiency (40). A comparison of the translation
products of both sets of mRNAs bearing either the endogenous or the Kb signal sequence shows that in all cases this
substitution led to an enhanced translation efficiency (Fig.
1). This holds true as well for the translation of CD3-
, the
nonmodified in vitro transcript that failed to be translated
(data not shown). Since sequence substitution as described
above did not affect the mobility of the translation products
in SDS-PAGE, we performed all subsequent experiments
with modified mRNAs.
Proper N-linked Glycosylation of TCR Subunits In Vitro.
In living cells, TCR-, -
, CD3-
and
, but not CD3-
and the
subunit are co-translationally N-glycosylated in
the lumen of the ER. To confirm proper glycosylation in
vitro, we translated the mRNAs of all TCR subunits individually in the presence of microsomes, followed by recovery of the translation products by immunoprecipitation using the appropriate antibodies. Immunoprecipitates were
digested with EndoH or mock digested to remove N-linked
glycans (Fig. 2). The number of glycans attached could be
estimated for in vitro translated TCR-
(four glycans), -
,
CD3-
and -
(two glycans), and CD3-
and -
(no glycans), all in agreement with in vivo data (2, 25).
Proper Formation of Intra- and Intermolecular Disulfide Bridges Depends on Redox Conditions and Is Essential for Correct Folding.
The formation of disulfide bridges occurs in the lumen of the ER both co- and posttranslationally, and requires an oxidizing environment. Disulfide bonds constrain
the tertiary structure and help maintain the conformation
of a polypeptide chain (41). In this study, we used conformation-specific mAbs to monitor disulfide bond-dependent folding. TCR- and CD4 translated in the presence of
the reducing agent DTT do not acquire their native conformation, demonstrated by their failure to bind to JOVI.1,
JOVI.3 (anti-TCR
mAbs) and OKT4 (anti-CD4 mAb),
respectively. However, the epitopes recognized by all three
antibodies were recovered upon inclusion of oxidized glutathion (GSSG) (Fig. 3 A) in a dose-dependent manner.
Similarly, disulfide bond-mediated homodimerization of
the subunit is dependent on proper redox conditions. In
the presence of 4 mM DTT,
homodimers could not be
detected with TIA-2 (anti-
mAb, reacting preferentially
with dimeric
) but were recovered upon addition of
GSSG (Fig. 3 B). We conclude that the proper formation
of intra- and intermolecular disulfide bonds in vitro requires an oxidizing environment and is essential for correct folding.
Because the complexity of polypeptides resulting from in vitro translation is limited and their specific radioactivity is high, specificity of immunoprecipitation must be rigorously established. All TCR subunits were therefore translated separately (Fig. 4 A, left), and the microsomes lysed in digitonin lysis buffer. Lysates of the different translations were pooled and subjected to immunoprecipitation using the antibodies as indicated (Fig. 4 A), or lysates of the individual translations were immunoprecipitated with a mixture of antibodies lacking only the antibody specific for the translated polypeptide (Fig. 4 A, middle).
All of the antibodies used precipitated exclusively the appropriate chain from a polypeptide mix (Fig. 4 A, middle) but failed to recover inappropriate polypeptides (Fig. 4 A, right). This result also demonstrates that assembly of the TCR does not occur artefactually after lysis, but requires the presence of all subunits in a shared membrane compartment (see below).
To examine subunit-specific assembly of the TCR in
vitro, we translated simultaneously all chains of the TCR as
well as three unrelated polypeptides; the human CD4 coreceptor and the and
chain of the human MHC class II
molecule HLA-DR1 (DR). The peptide of the influenza
hemagglutinin (HA306-318) was added to the reaction as a
ligand of DR to promote the formation of peptide-bound
SDS-stable MHC class II molecules, which migrate as SDS-stable heterodimer in SDS-PAGE (44). This control serves
to show the ability of the in vitro system to support the
proper assembly of this heterodimer, in accord with our
earlier studies (40). After translation, microsomes were solubilized in digitonin lysis buffer, subjected to immunoprecipitation using OKT4 (anti-CD4 mAb), Tü36 (anti-DR mAb), JOVI.1 (anti-TCR-
mAb), or OKT3 (anti-CD3
mAb). The resulting immunoprecipitates were analyzed by
SDS-PAGE under reducing conditions (Fig. 4 B, left). CD4
and DR were recovered only by their appropriate antibodies. A sizable proportion of DR displayed the SDS-stable phenotype, indicative of proper peptide loading and specific assembly. The immunoprecipitates obtained with JOVI.1
(anti-TCR-
) and OKT3 (anti-CD3) contained subunits
of the TCR exclusively and were devoid of co-translated
CD4 or DR.
To assess the formation of disulfide-linked subunits, complexes recovered with JOVI.1 and OKT3 were analyzed by
SDS-PAGE under nonreducing conditions (Fig. 4 B, middle). We observed the presence of /
heterodimers and
homodimers, as well as the absence of monomeric
,
,
and
subunits in complexes immunoprecipitated with
OKT3. To resolve TCR-
from TCR-
, immunoprecipitates from JOVI.1 and OKT3 were digested with EndoH
before separation by reducing SDS-PAGE (Fig. 4 B, right).
The methionine content (TCR-
, 4 methionines; TCR-
,
6 methionines) and relative intensity of both subunits recovered with OKT3 strongly suggest a 1:1 stoichiometry for
both chains, in accordance with in vivo data.
We conclude that the formation of TCR complexes as
monitored by coimmunoprecipitation of the relevant subunits occurs in a specific manner. The recovery of TCR-
with both JOVI.1 and OKT3 indicate the formation of
complete TCR complexes, since this subunit joins the TCR
during the last stage of assembly (10). Moreover, specific
assembly of multimolecular complexes (TCR versus DR) can
occur simultaneously in an in vitro translation system, as complexes containing inappropriate subunits were not observed.
The CD3- chain contains two cysteine
residues presumably involved in stabilization of the Ig fold,
and two more cysteines in the lumenal domain (2), one of
which is located within a few residues of the lipid bilayer
(Fig. 5). The occurrence of disulfide-linked
homodimers
has been reported in vivo (45, 46), but the monomer is the
prevalent form found in living cells. We investigated the
biochemical properties of CD3-
translated in vitro under
both reducing and oxidizing conditions. Immunoprecipitations were performed with the anti-CD3-
antibody in the
presence of 50 mM iodoacetamide to prevent the formation of disulfide bridges after lysis. As visualized by SDS-PAGE under nonreducing conditions, translation of CD3-
performed under oxidizing conditions resulted in oligomeric structures that collapsed to the monomer when analyzed by SDS-PAGE under reducing conditions (Fig. 6 A).
Moreover, the difference in mobility for the oxidized and
reduced monomeric form of CD3-
is clearly visible (Fig. 6
A, left). We noted that it is only the oxidized form of CD3-
that coimmunoprecipitated with fully assembled TCR complexes. We do not know whether the more rapidly migrating, oxidized form of CD3-
contains any remaining free
sulfhydryl groups, but we consider this unlikely. Inclusion of DTT in the translation reaction abrogated oligomerization but led to a monomeric polypeptide with a slightly decreased mobility, as a consequence of alkylation by iodoacetamide upon lysis. We confirmed the homooligomeric
nature of CD3-
by two-dimensional gel electrophoresis
(first dimension: isoelectric focusing under nonreducing conditions; second dimension: reducing SDS-PAGE; data not
shown). We conclude that CD3-
translated in vitro in the absence of related subunits tends to form mostly disulfide-linked oligomers, a process that appears to be redox dependent.
To investigate whether the quantity of CD3- expressed
in microsomes exerts any influence on the formation of
disulfide-linked
oligomers, we translated CD3-
together
with increasing amounts of an unrelated chain, the DR-
chain. Total microsome-associated material as well as immunoprecipitates of CD3-
were analyzed by SDS-PAGE
performed under both reducing and nonreducing conditions (Fig. 6 B). As the ratio between monomeric and disulfide-linked oligomeric CD3-
does not depend on the
amount of translated CD3-
(and DR-
), the formation of
homooligomers must be an inherent property of the CD3-
subunit.
We addressed the kinetics of this reaction (Fig. 6 C) by
terminating the translation of CD3- after 15 min by the
addition of cycloheximide. Aliquots of microsomes were
lysed in the presence of iodoacetamide at indicated times
after translation and subjected to immunoprecipitation with
anti-CD3-
antibodies. Analysis of immune complexes by
SDS-PAGE performed under nonreducing conditions reveals that dimeric CD3-
was already prevalent at the end
of the translation period, but higher order forms gradually
increased with time.
The propensity of CD3-
to form disulfide-linked oligomers was examined by translating CD3-
either alone or together with the other subunits
of the TCR. Total microsome-associated material as well as
immunoprecipitates obtained from lysed microsomes were analyzed by SDS-PAGE performed under both reducing
and nonreducing conditions (Fig. 7 A). Lysis in 0.5% NP-40 facilitates the visualization of CD3-
as it leads to the
disruption of all noncovalent interactions between the subunits of the TCR, but not those between CD3-
, -
, and
-
(6).
As shown above, CD3- translated on its own is found
primarily in oligomeric structures. However, its behavior
changes dramatically in the presence of TCR subunits, where
CD3-
is now predominantly monomeric. The role of
each individual subunit in "chaperoning" CD3-
was studied by translating
alone and pairwise with other subunits
of the TCR or with DR-
. Analysis of anti-CD3-
immunoprecipitates by nonreducing SDS-PAGE (Fig. 7 B)
shows that in the presence of TCR-
, -
, -
and DR-
the formation of
oligomers is not affected. However, co-translation of CD3-
and CD3-
drastically shifts the equilibrium towards the formation of
monomers. The strong
interaction between CD3-
and CD3-
or -
(which survives solubilization in 0.5% NP-40) may account for this
effect. As binding of both
and
to CD3-
appears to
compete with the formation of
homooligomers, the high
degree of homology between CD3-
, -
, and, to some degree, CD3-
may be particularly noteworthy and suggests
the presence of a common binding motif for complex formation. Since Ig-like domains are present not only in the
CD3 subunits, but also in the clonotypic
/
heterodimer
and in DR-
, the mere presence of Ig-like domains is
clearly not sufficient to confer the ability of blocking homooligomerization of CD3-
.
Calnexin, a molecular chaperone, binds preferentially to monoglucosylated trimming intermediates in the
ER (21). Interestingly it also interacts with free CD3- and
CD3-
complexed with CD3-
or -
, although CD3-
lacks N-linked glycans (24, 47). How calnexin binds to
CD3-
is not known. To study calnexin-CD3-
interactions and their importance for proper folding of CD3-
, we designed the following experiment: CD3-
alone,
and
, or
alone were translated in the absence and presence of
the glucosidase inhibitor N-7-oxadecyl-dNM (7-O-dec),
which blocks glucose trimming of N-linked glycans (48)
and consequently prevents a glycan-dependent interaction
between calnexin and glycoproteins. The change in mobility
for the glycosylated
subunit upon translation in the presence of 7-O-dec is due to glucose retention and is clearly visible on SDS-PAGE. After translation (1-h duration), microsomes were lysed in digitonin lysis buffer. Immunoprecipitates obtained with antibodies directed against calnexin,
CD3-
, and -
were analyzed by SDS-PAGE under nonreducing conditions (Fig. 8).
Translated alone, oligomeric subunits could be recovered with the anticalnexin antibody regardless of the absence or presence of 7-O-dec in the translation reaction.
However, the
subunit strongly interacted with calnexin
in a glycan-dependent manner, as this interaction appeared
to be 7-O-dec sensitive. Monomeric CD3-
was co-immunoprecipitated with calnexin in the presence of CD3-
.
Complete sensitivity of this interaction to 7-O-dec implies
a role of CD3-
as a bridging intermediate for CD3-
in this binding.
Notably, 7-O-dec did not exert any effect on the oligomerization of CD3- translated alone or together with
CD3-
. Therefore, for both glycan-dependent and -independent modes of binding, the interaction of calnexin with
the CD3-
does itself not influence the oligomerization of
CD3-
. Hence, we conclude that the folding of
is guided
by the formation of a complex with CD3-
and most likely
also with CD3-
, two subunits competing for binding to CD3-
at an early stage of TCR assembly.
Our understanding of the requirements for the assembly of the TCR stems largely from studies in which the individual subunits were transfected into suitable recipient cell lines, e.g., mutant T cell lines deficient in one of the six subunits or by transfections into nonlymphoid cells. The expression of all subunits of the TCR-CD3 complex is necessary and sufficient to accomplish its assembly in the ER and its subsequent expression at the cell surface (49, 50), without obvious involvement of lymphoid-specific factors.
The analysis of heterooligomerization of the TCR-CD3
complex in living cells is complicated by the presence of
widely different pool sizes of the participating subunits, and
by the egress of incompletely assembled receptors out of the
ER, a complication avoided in the present study. We sought
to reproduce the early stages of the TCR assembly in a
complete in vitro system, with particular emphasis on the
role of the CD3- subunit. We optimized the translation
efficiency of all mRNAs involved to allow the simultaneous translation of multiple mRNAs, without having to consider the confounding effects of the 5
untranslated sequence and signal peptides on the efficiency of translation
and membrane insertion. In this fashion, we successfully
translated nine different mRNAs simultaneously, and,
provided proper redox conditions were imposed on the
ER-derived microsomes, observed specific complex formation amongst the appropriate subunits produced in vitro. Specifically, the co-translation of class II HLA-DR-
and
-
subunits in the presence of an appropriate peptide
ligand, along with CD4 and all subunits of the TCR-CD3
complex, resulted in the formation of properly assembled
TCR-CD3 complexes, correctly folded CD4 molecules, as
well as peptide-loaded class II molecules, without cross-contamination of any of these complexes by inappropriate subunits. To our knowledge, no oligomeric membrane
proteins of greater complexity have been generated by in
vitro translation. The ability to obtain such complexes testifies to the robustness of these microsome-supplemented
in vitro systems. The actual concentrations of the TCR
subunits obtained by in vitro translation are difficult to estimate, but likely to be quite low, compared to the concentrations of the proteins in the lumen of the microsomes
used. The remarkably efficient assembly and folding of the
polypeptides analyzed thus suggests the possibility that
these processes might be limited to small numbers of "insertion sites", where conditions for folding and assembly are
favorable. Through association of the translocon with accessory proteins (N-oligosaccharyl transferase, peptidyl disulfide isomerase, calnexin), some specialization through accomplishing this goal within the ER could occur.
The sequence of subunit interactions that leads to a complete TCR-CD3 complex has been addressed for living cells
(7, 9). Given the complexity of this receptor, it is perhaps
not surprising that assembly occurs in a well ordered and
stepwise fashion; the formation of CD3-/
and
/
pairs
takes place early and depends on the specific interaction between their ectodomains (15). Both CD3-
and CD3-
compete for binding to CD3-
(51). The crucial sites in the
extracellular portion of CD3-
for its interaction with CD3-
are also conserved in the primary structure of CD3
, which
suggests that both CD3-
and -
share a common binding
motif in CD3-
(15). Next, pairs of CD3-
/
and
/
recruit TCR-
and -
, respectively. Upon formation of an
intrachain disulfide bridge between TCR-
and -
, trimeric complexes merge to a hexameric
-
complex,
into which the
homodimer is integrated as the last remaining subunit. It is noteworthy that the stoichiometry of
the CD3-TCR complex is still considered tentative (8).
When CD3- is translated alone, it tends to form disulfide-linked homooligomers; moreover, a substantial amount of
monomeric CD3-
is found in a reduced, i.e., incompletely, folded state. The quantity of CD3-
translated and
inserted into microsomes does not influence the ratio between monomeric (reduced and oxidized) and oligomeric
structures. In all likelihood, homooligomerization of CD3-
is an inherent property of the
subunit; it is fast and occurs
under physiological redox conditions. However, co-translation of TCR subunits under conditions that support complex formation leads to a dramatic change in the balance of
newly synthesized CD3-
subunits towards oxidized and
properly folded monomers, the only form of CD3-
recovered with the conformation-dependent mAb OKT3. The
mere co-translation of CD3-
or -
was sufficient to keep
CD3-
primarily in an oxidized and monomeric state, indicating that CD3-
and -
guide the folding of CD3-
at
the initial stage of TCR assembly.
Although the disulfide bonding status for individual cysteine residues in CD3- has not been established, we consider it likely that the Ig fold may facilitate formation of the
disulfides between the cysteines at position 27 and position
76, which stabilize this structure. The extent to which the
remaining extracellular cysteine residues (at position 97 and
100) are involved in disulfide bonding in the mature
TCR-CD3 complex is not known. Our data suggest that
when no DTT is added, some CD3-
may not acquire a
proper disulfide bonding status. We therefore cannot indicate which cysteine residues are involved in the formation
of these
oligomers.
The presence of disulfide-linked homodimers in a completely assembled and functional TCR complex has been reported for several human and murine T cell lines (45, 46).
However, the presence of these
homodimers was established either using conformation-independent anti-CD3-
reagents (46) or by examining immunoprecipitates obtained
with the conformation-specific antibody OKT3 after cell surface labeling under oxidative conditions (45). Although
we observe oligomerizaton of CD3-
as a major pathway
in the course of its biosynthesis in the absence of CD3-
and -
, we could not detect these structures as part of a
complete TCR-CD3 complex by immunoprecipitation with
the mAb OKT3. This antibody, the epitope of which has
not been mapped accurately, only recovered CD3 subunits in immunoprecipitations when these were translated simultaneously with TCR-
and -
under redox conditions that
allow proper folding (data not shown). Intracellular immunostaining of CD3-
transiently expressed in COS cells
could only be achieved with OKT3 when either CD3-
or
CD3-
were coexpressed (52).
Furthermore, using OKT3 we could only co-immunoprecipitate TCR- and -
as disulfide-linked heterodimers,
despite the presence of free TCR-
, -
, and CD3 pairs in
the lysate. Hence, OKT3 most likely recognizes a conformation-dependent epitope on CD3-
that becomes available when CD3-
or -
is present, and remains stable after
solubilization in detergent when the hexameric
-
structure has been formed. A conformational change of
murine CD3-
, dependent on the presence of murine CD3-
or -
, has been reported (53). Taken together, our data
strongly support the notion that CD3-
requires the presence of CD3-
or -
to fold correctly and to allow the assembly of the TCR-CD3 complex to proceed.
CD3-, a nonglycosylated polypeptide of 28 kD, is expressed in human T cells and transiently interacts with pairs
of CD3-
/
at an early stage of TCR assembly (54). Upon
maturation and egress of the nascent TCR from the ER, it
is released from the complex (54). CD3-
is commonly regarded as a T cell-specific molecular chaperone and hence,
is likely to be absent from the canine microsomes used in
this study. The function of CD3-
is unknown since the
expression of CD3-
is dispensable for the assembly and
cell surface expression of the TCR-CD3 complex.
We have also investigated the role of the molecular
chaperone calnexin in the folding of CD3-. Calnexin binds
in a prolonged fashion to misfolded proteins or single subunits that fail to join a complex, suggesting that it may participate in the ER quality control machinery that prevents
egress of incompletely folded or unassembled polypeptides
from the ER (24, 55). A characteristic of calnexin is its
preference for monoglucosylated trimming intermediates of N-linked glycoproteins (21). Calnexin can bind exclusively in a lectin-like fashion, i.e., independent of the folding state of the substrate (20), an observation that ties the
cycle of deglucosylation and reglucosylation to glycoprotein folding (56). Although the interaction between heavy
chains of MHC class I molecules and calnexin is only initiated by partially trimmed glycans, removal of glycans of
class I-calnexin complexes by treatment with EndoH does
not lead to the dissociation of this complex (21). Calnexin-dependent ER retention has been observed for the nonglycosylated CD3-
subunit transfected into COS cells, based
on colocalization of CD3-
with mutant forms of calnexin,
engineered with respect to their retention in the ER (24).
The mode of binding between calnexin and CD3-
therefore appears to deviate from that between calnexin and
other molecules.
When we translated CD3- in the absence of CD3-
and -
, we could only detect oligomeric CD3-
subunits in
a complex with calnexin. Theoretically, this interaction could
involve an unlabeled ER-resident protein that resides in
the microsomes used in the translation experiments and
would escape detection. Calnexin can interact in a glycan-independent manner with aggregates of the glycosylated
VSV-G protein (22). Possibly some structural attribute of
such VSV-G protein aggregates might be mimicked by the
CD3-
oligomer. It will be important to define more accurately and in molecular terms, how calnexin can engage in
glycan-independent interactions, and whether the outcome
of these interactions could, under certain circumstances,
lead to the resolution of the aggregates.
In conclusion, we have demonstrated that the oligomerization of CD3- is a pathway essentially not used when
CD3-
and/or CD3-
subunits are available. In this sense,
the CD3-
could serve as a subunit that monitors proper
oligomerization of the CD3 complex.
Address correspondence to Hidde L. Ploegh, Center for Cancer Research, E17-325, Massachusetts Institute of Technology, 40 Ames St., Cambridge, MA 02139. Phone: 617-253-0519; FAX: 617-253-9891; E-mail: ploegh{at}mit.edu
Received for publication 25 March 1997 and in revised form 2 June 1997.
Johannes B. Huppa was supported by fellowship of the Daimler-Benz-Stiftung (Ladenburg, Germany) and is currently a fellow of the Boehringer-Ingelheim-Fonds (Stuttgart, Germany). This work received support from the National Institutes of Health (1 PO1 AI 37833-01).We would like to thank Dr. Armin Rehm for continuous discussions and all present lab members for helpful suggestions. We thank Dr. O. Acuto, Dr. P. Anderson, Dr. J. Coligan, Dr. M. Owen, Dr. C. Terhorst, and Dr. J. Trowsdale for providing essential reagents.
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