From the Department of Microbiology & Immunology, East Carolina
University, School of Medicine,
Greenville, North Carolina 27858-4353
The antigen-binding receptor expressed on most T
lymphocytes consists of disulfide-linked clonotypic 
heterodimers
noncovalently associated with monomeric CD3
,
,
proteins and
disulfide-linked 
homodimers, collectively referred to as the T
cell antigen receptor (TCR) complex. Here, we examined and compared the
disulfide linkage status of newly synthesized TCR proteins in murine
CD4+CD8+ thymocytes and splenic T cells.
These studies demonstrate that CD3
proteins exist as both monomeric
and oligomeric (disulfide-linked) species that differentially assemble
with CD3
subunits in CD4+CD8+ thymocytes and
splenic T cells. Interestingly, unlike previous results on glucose
trimming and TCR assembly of CD3
proteins in splenic T cells (Van
Leeuwen, J. E. M., and K. P. Kearse (1996) J. Biol. Chem. 271, 9660-9665), we found that glucose residues were
not invariably removed from CD3
glycoproteins prior to their assembly with CD3
subunits in CD4+CD8+
thymocytes. Finally, these studies show that calnexin associates with
both monomeric and disulfide-linked CD3
proteins in murine T cells.
The data in the current report demonstrate that CD3
proteins exist
as both monomeric and disulfide-linked molecules in murine T cells that
differentially associate with partner TCR chains in
CD4+CD8+ thymocytes and splenic T cells. These
results are consistent with the concept that folding and assembly of
CD3
proteins is a function of their oxidation state.
 |
INTRODUCTION |
Most T lymphocytes express on their surfaces a multisubunit
complex consisting of clonotypic 
proteins associated with
invariant CD3-
,
,
and
chains, designated the T cell antigen
receptor (TCR)1 complex
(1-3). Assembly of the TCR occurs in the endoplasmic reticulum (ER)
and proceeds in a highly ordered manner involving: (i) formation of
noncovalently associated paris of 
and 
proteins, (ii)
assembly of
,
proteins with 
,
pairs to form 

and 

intermediate complexes, (iii) joining of 

and


chains followed by disulfide bonding of CD3-associated
,
proteins to yield incomplete 




complexes, and
finally (iv) addition of disulfide-linked 
homodimers to form the
complete 






TCR complex (1-3).
In general, the surface expression of TCR proteins is tightly
associated with their assembly status (1, 3). Individual, unassembled
TCR proteins and partially assembled TCR complexes containing two or
three TCR subunits do not effectively exit the ER. Both incomplete





and complete 






TCR egress
from the ER to the Golgi complex; however, only complete TCR complexes are efficiently transported to the cell surface (1-3). Unlike mature
CD4+ and CD8+ (single positive) T cells, which
fundamentally express complete 






TCR (3),
immature CD4+CD8+ (double-positive) thymocytes
express both complete TCR and partial complexes of CD3
, CD3
proteins associated with calnexin (4-6), referred to as clonotypic
independent complexes (4, 5). The molecular basis for clonotypic
independent complex expression is unknown but is postulated to result
from inefficient ER retention mechanisms in
CD4+CD8+ thymocytes that do not persist in
mature T cells (5, 7).
Previous studies by Jin et al. reported that a small
subfraction of CD3
proteins exists as disulfide-linked dimers in
human T lymphocytes (8), which assemble with TCR
subunits; CD3
dimers were likewise observed to be present in murine thymocytes, although their assembly status was not evaluated (8). Disulfide-linked heterodimers of CD3
-
proteins have also been described in REX variant human T cell lines, which fail to express TCR
or TCR
,
molecules (9). More recently, Huppa and Ploegh demonstrated that human
CD3
molecules translated in vitro in the absence of other
TCR proteins have a tendency to form disulfide-linked homooligomers, which assemble with the molecular chaperone calnexin (10).
Cotranslation of CD3
or CD3
molecules was sufficient to maintain
CD3
proteins in a principally monomeric phase, however, suggesting
that CD3
and CD3
may guide the folding of CD3
proteins during
the initial stages of their biosynthesis (10). In the current study we
evaluated the disulfide linkage status of newly synthesized TCR
proteins in murine CD4+CD8+ thymocytes and
splenic T cells. These studies show that newly synthesized CD3
proteins exist as both monomeric and disulfide-linked molecules that
differentially assemble with CD3
molecules in CD4+CD8+ thymocytes and splenic T cells. In
addition, these data document that calnexin associates with both
monomeric and oligomeric (disulfide-linked) CD3
proteins in murine T
lymphocytes.
 |
EXPERIMENTAL PROCEDURES |
Animals, Cell Preparation, and Reagents--
C57BL/6 (B6) mice
were obtained from the Jackson Laboratory (Bar Harbor, ME).
CD4+CD8+ thymocytes were isolated by their
adherence to plastic plates coated with anti-CD8 mAb (83-12-5) and were
typically >95% CD4+CD8+ as described (3).
Splenic T cells were purified by incubation of single cell suspensions
of spleen cells on tissue culture plates coated with rabbit anti-mouse
immunoglobulin, (Organo-Technika-Cappel, Malvern, PA) for 60 min at
37 °C, followed by isolation of nonadherent cells. The resultant
cell populations were typically 80-85% CD3+ as determined
by flow cytometry analysis.
Antibodies--
The following mAb were used in this study:
145-2C11, specific for CD3
proteins associated with CD3
or
CD3
chains (11); HMT 3.1, which recognizes CD3
proteins
irrespective of their assembly state (12); H28-710, specific for
TCR
(13); and H57-597, specific for TCR
(14), all kindly provided
by Dr. Ralph Kubo (Cytel, San Diego, CA). The following antisera were used: R9, specific for CD3
proteins (15), kindly provided by Dr.
Larry Samelson (National Institutes of Health, Bethesda, MD); 551, specific for TCR
proteins (16), kindly provided by Dr. Allan
Weissman (National Institutes of Health, Bethesda, MD); SPA-860
anti-calnexin (Stressgen Biotechnologies, Victoria, BC, Canada) and
PA3-900 anti-calreticulin (Affinity BioReagents, Neshanic Station,
NJ).
Metabolic Labeling and Immunoprecipitation--
Metabolic pulse
labeling with [35S]methionine was performed as described
previously (3). Briefly, cells were pulse-labeled in
methionine-deficient medium (Biofluids, Rockville, MD) containing 1 mCi/ml [35S]methionine (Trans 35S-label; ICN,
Irvine, CA) for 30 min at 37 °C. Cells were lysed by solubilization
in 1% digitonin (Calbiochem, La Jolla, CA) lysis buffer (20 mM Tris, 150 mM NaCl, 10 mM
iodoacetamide, plus protease inhibitors) at 1 × 108
cells/ml for 20 min at 4 °C; lysates were clarified by
centrifugation to remove insoluble material and immunoprecipitated with
the appropriate antibodies preabsorbed to protein A-Sepharose beads
(Amersham Pharmacia Biotech). Sequential immunoprecipitation and
immunoprecipitation/release/recapture procedures were performed as
previously detailed (3).
Glycosidase Digestion and Gel Electrophoresis--
Digestion
with endoglycosidase H (EH) was performed by resuspending precipitates
in glycosidase digestion buffer (75 mM sodium phosphate, pH
6.1, 75 mM EDTA, 0.1% Nonidet P-40) containing 10 milliunits of EH (Genzyme, Cambridge, MA) for 16 h at 37 °C;
digestion with jack bean mannosidase (JB) (Oxford Glycosystems,
Rosedal, NY) was performed according to the manufacturer's
instructions. One- and two-dimensional SDS-PAGE was performed as
described previously (17).
 |
RESULTS |
Disulfide-linked 26-kDa Proteins Differentially Associate with TCR
Subunits in CD4+CD8+ Thymocytes and Splenic T
Cells--
As shown in Fig.
1A, analysis of anti-CD3
immunoprecipitates of radiolabled CD4+CD8+
thymocytes on two-dimensional nonreducing × reducing (NR × R) gels shows expected nondisulfide-linked monomeric CD3
,
and
CD3
proteins that migrate on and slightly above the diagonal,
respectively (Fig. 1A, top panel), and
disulfide-linked TCR
heterodimers and 
homodimers, which
migrate below the diagonal (Fig. 1A, top panel).
Interestingly, a dimeric species of unknown identity was also present
in such precipitates, migrating at approximately 26 kDa following
reduction of disulfide-linked bonds (Fig. 1A, top
panel, arrow). Disulfide-linked 26-kDa proteins were
isolated from CD4+CD8+ thymocyte lysates using
two different anti-CD3
mAbs of distinct specificity, 145-2C11 and
HMT3.1 (Fig. 1, A, top panel, and B, top panel, respectively) (see "Experimental
Procedures"), and by CD3
-specific Ab (Figs. 1B,
bottom panel) but not anti-TCR
mAb (Fig. 1A,
bottom panel) or anti-
antiserum (data not shown). Unlike
CD4+CD8+ thymocytes, disulfide-linked 26-kDa
proteins were not observed in anti-CD3
precipitates of radiolabeled
splenic T cells (Fig. 2, A and
B) but were clearly present in anti-CD3
precipitates of
splenic T cells (Fig. 2B, bottom panel).
Disulfide linkage of 26-kDa proteins did not result from artificial
formation of disulfide bonds during cell lysis because identical
results were observed when cells were solubilized in lysis buffer
containing excess (75 mM) iodoacetamide (data not shown).
Taken together, these data demonstrate that disulfide-linked 26-kDa
proteins differentially associate with CD3
and TCR
subunits in
CD4+CD8+ thymocytes and splenic T cells.

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Fig. 1.
Disulfide-linked 26-kDa proteins
coprecipitate with TCR proteins in CD4+CD8+
thymocytes. A, CD4+CD8+
thymocytes were radiolabeled with [35S]methionine for 30 min and solubilized in 1% digitonin, and lyates were
immunoprecipitated with anti-CD3 mAb (145-2C11) and anti-TCR mAb
(H57-597). Precipitates were analyzed on two-dimensional NR × R
SDS-PAGE gels. The positions of TCR proteins and disulfide-linked
26-kDa proteins (arrow) are indicated. B, same as
in A except that CD4+CD8+ thymocyte
lysates were immunoprecipitated with anti-CD3 mAb (HMT3.1) and
anti-CD3 Ab (R9).
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Fig. 2.
Disulfide-linked 26-kDa proteins are not
assembled with CD3 molecules in splenic T cells. A,
CD4+CD8+ thymocytes and splenic T cells were
radiolabeled with [35S]methionine for 30 min and
solubilized in 1% digitonin, and lyates were immunoprecipitated with
anti-CD3 mAb (145-2C11) mAb. Precipitates were analyzed on
two-dimensional NR × R SDS-PAGE gels. The positions of TCR
proteins and disulfide-linked 26-kDa proteins (arrow) are
indicated. B, same as in A, except that splenic T
cell lysates were immunoprecipitated with anti-CD3 mAb (145-2C11)
and anti-CD3 Ab (R9).
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|
CD3
Proteins Are Assembled into Disulfide-linked Dimers in
Murine T Cells--
Because their molecular mass is similar to that of
CD3 components, we reasoned that disulfide-linked 26-kDa proteins might represent newly synthesized CD3
proteins, CD3
proteins, or both. To determine whether CD3
,
proteins were assembled into
disulfide-linked dimers in CD4+CD8+ thymocytes,
anti-CD3
precipitates of CD4+CD8+ thymocytes
were analyzed on two-dimensional NR × R gels and immunoblotted with antiserum specific for CD3
and CD3
molecules. As shown in
Fig. 3, CD3
proteins existed as both
monomeric and disulfide-linked molecules in
CD4+CD8+ thymocytes (Fig. 3, top
panel). In contrast, CD3
proteins were present exclusively as
nondisulfide-linked monomers (Fig. 3, bottom panel).
Identical results were obtained in immunoblot experiments of
anti-CD3
precipitates of CD4+CD8+ thymocytes
(data not shown). These results show that CD3
proteins exist as both
monomeric and disulfide-linked molecules in
CD4+CD8+ thymocytes.

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Fig. 3.
CD3 proteins are assembled into
disulfide-linked dimers in murine T cells. Anti-CD3
immunoprecipitates of digitonin lysates of
CD4+CD8+ thymocytes were analyzed on
two-dimensional NR × R SDS-PAGE gels and immunoblotted with
anti-CD3 or anti-CD3 Ab as indicated. The positions of monomeric
and disulfide-linked CD3 proteins are indicated. Note that
anti-CD3 proteins existed exclusively as nondisulfide-linked,
monomeric proteins in these studies.
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CD3
and CD3
proteins are easily distinguished from each other in
that CD3
is post-translationally modified by the addition of three
N-linked oligosaccharide chains unlike CD3
, which does not contain N-glycans (1, 17). To confirm that CD3
glycoproteins were disulfide-linked in murine T cells, digitonin
lysates of radiolabeled CD4+CD8+ thymocytes
were immunoprecipitated with anti-CD3
Ab, CD3
precipitates were
boiled in SDS to release bound material, and CD3
proteins were
recaptured by precipitation with anti-CD3
Ab. Precipitates were
digested with Endo H glycosidase (specific for cleavage of immature
N-linked glycans) and analyzed on one-dimensional SDS-PAGE gels under nonreducing conditions. Most CD3
proteins radiolabeled during a 30-min pulse period migrated as monomeric 26-kDa proteins (Fig. 4, first lane), which
fell to 17 kDa following removal of N-linked glycan chains,
as expected (Fig. 4, second lane). Importantly, these data
show that remaining CD3
molecules existed as disulfide-linked proteins that migrated at approximately 52 kDa in mock treated samples
(Fig. 4, first lane) and at 34 kDa following glycosidase digestion (Fig. 4, second lane); these results were
confirmed by immunoblotting experiments using anti-CD3
Ab (data not
shown). These data are consistent with the assembly of CD3
glycoproteins into disulfide-linked dimers that are composed of CD3
proteins linked to itself (CD3
-
) or to another molecule of
similar size (CD3
-x), which like CD3
, must also contain
N-glycans as the magnitude of decrease in molecular mass
following deglycosylation is greater than would be expected for CD3
associated with a nonglycosylated protein.

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Fig. 4.
A subset of newly synthesized CD3
glycoproteins exists as disulfide-linked dimers in
CD4+CD8+ thymocytes. Digitonin lysates of
[35S]methionine-radiolabeled
CD4+CD8+ thymocytes were immunoprecipitated
with anti-CD3 Ab, precipitates were boiled in 1% SDS to release
bound material, and CD3 proteins were specifically recaptured by
precipitation with anti-CD3 Ab. Recapture precipitates were digested
with EH glycosidases as indicated and analyzed on 13% SDS-PAGE gels
under nonreducing conditions. The positions of monomeric and
disulfide-linked CD3 proteins are marked. An asterisk
indicates a nonglycosylated molecule that nonspecifically
coprecipitates with CD3 proteins, believed to be actin; this
molecule is not disulfide-linked to CD3 proteins as determined by
analysis on two-dimensional nonreducing × reducing gels (see Fig.
6).
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Glc Trimming and Calnexin Assembly of CD3
Molecules in
CD4+CD8+ Thymocytes--
Immature
N-glycan chains on newly synthesized glycoproteins having
the structure Glc3Man9GlcNAc2 are
initially processed by the sequential action of glucosidase I and
glucosidase II enzymes in the ER, creating monoglucosylated
Glc1Man9GlcNAc2 glycans important for interaction with the lectin-like chaperone calnexin (18-22). Calnexin is proposed to function in the quality control of folding and
assembly of numerous newly synthesized glycoproteins, including TCR
,
and CD3
,
subunits (23-25). Previous studies on the
processing of TCR glycoproteins in splenic T cells show that Glc
residues are removed from newly synthesized CD3
molecules prior to
their assembly with other TCR subunits and that calnexin associates exclusively with unassembled, "free" CD3
proteins containing incompletely trimmed glycan chains (24). To determine whether CD3
proteins were similarly processed in CD4+CD8+
thymocytes, cells were pulse-labeled with [35S]methionine
for 30 min and solubilized in 1% digitonin, and lysates were
immunoprecipitated with anti-CD3
Ab to purify total CD3
proteins;
alternatively, lysates were sequentially immunoprecipitated with
anti-TCR
mAb to isolate CD3
proteins assembled into complete 






and incomplete 




TCR
complexes, followed by precipitation with anti-CD3
mAb to capture
CD3
chains present in partial complexes of CD3
components and
finally precipitation with anti-CD3
Ab to purify remaining
unassembled, free CD3
chains. Precipitates were boiled in SDS to
release bound material, CD3
proteins were specifically recaptured
with anti-CD3
Ab, and recapture precipitates were digested with JB
and EH glycosidases. JB digestion is useful for evaluating the Glc
trimming status of newly synthesized glycoproteins because it removes
eight mannoses from fully trimmed N-glycan chains devoid of
Glc residues (Man8-9GlcNAc2) but only five mannoses from incompletely trimmed N-glycans containing one
to three Glc saccharides
(Glc1-3Man8-9GlcNAc2) (19, 24).
In contrast, EH removes all but a single GlcNAc from
N-glycan chains irrespective of their Glc content (26).
Similar to what was previously observed in splenic T cells (24), CD3
proteins synthesized in CD4+CD8+ thymocytes
existed in four major glycoforms (A-D), indicative of CD3
proteins
containing three (A), two (B), one (C), and zero (D) incompletely
trimmed glycan chains, respectively (Fig.
5A, left-hand
side). Interestingly, however, unlike splenic T cells, CD3
proteins containing incompletely trimmed N-glycans in
CD4+CD8+ thymocytes were present as both free,
unassembled chains and as assembled molecules associated with CD3
proteins (Fig. 5, A and B). In contrast, CD3
proteins associated with TCR
were totally devoid of Glc residues as
shown by their complete sensitivity to JB digestion (Fig. 5,
A and B). Taken together, these data show that
CD3
glycoforms are similarly generated in immature CD4+CD8+ thymocytes and splenic T cells and
that CD3
proteins containing incompletely trimmed
N-glycans exist as both free and assembled TCR subunits in
CD4+CD8+ thymocytes.

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Fig. 5.
Glc trimming and TCR assembly of CD3
glycoproteins in CD4+CD8+ thymocytes.
A, digitonin lysates of
[35S]methionine-radiolabeled
CD4+CD8+ thymocytes were immunoprecipitated
with anti-CD3 Ab or anti-calnexin Ab or were sequentially
immunoprecipitated with anti-TCR mAb, followed by anti-CD3 mAb,
and finally were immunoprecipitated with anti-CD3 Ab. Precipitated
material was released by boiling in SDS and CD3 proteins
specifically recaptured by precipitation with anti-CD3 Ab; recapture
precipitates were digested with JB and EH glycosidases as indicated.
The positions of CD3 glycoforms (A-D) and
Endo-H-sensitive, deglycosylated CD3 proteins (CD3
EHS) are marked. B, same as in A
except that CD4+CD8+ thymocyte lysates were
sequentially immunoprecipitated with anti-TCR mAb, followed by
immunoprecipitation with anti-CD3 mAb and finally with anti-CD3
Ab.
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Next, the assembly of newly synthesized CD3
proteins with the
molecular chaperone calnexin was examined. Metabolically labeled CD3
proteins coprecipitated with calnexin in
CD4+CD8+ thymocytes that, as expected,
contained incompletely trimmed glycan chains that were partially
resistant to JB digestion (Fig. 5A, middle
lanes). As similarly noted for other T cell types (25), CD3
proteins synthesized in CD4+CD8+ thymocytes did
not associate with the calnexin-related molecule, calreticulin (data
not shown). To determine the disulfide linkage status of CD3
proteins associated with calnexin, digitonin lysates of radiolabeled
CD4+CD8+ thymocytes were immunoprecipitated
with anti-calnexin Ab, precipitates were boiled in 1% SDS to release
bound material, and CD3
proteins were specifically recaptured with
anti-CD3
Ab. Analysis of recapture precipitates on two-dimensional
NR × R SDS-PAGE gels showed that both monomeric and
disulfide-linked CD3
proteins were assembled with calnexin in
CD4+CD8+ thymocytes (Fig.
6); similar results were observed in
splenic T cells (data not shown). These studies show that both
monomeric and disulfide-linked CD3
proteins associate with calnexin
in murine T cells.

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Fig. 6.
Both monomeric and disulfide-linked CD3
proteins associate with calnexin in CD4+CD8+
thymocytes. Digitonin lysates of
[35S]methionine-radiolabeled
CD4+CD8+ thymocytes were immunoprecipitated
with anti-CD3 Ab or anti-calnexin Ab; precipitates were boiled in
SDS, CD3 proteins were recaptured by precipitation with anti-CD3
Ab, and recapture precipitates were analyzed on two-dimensional NR × R SDS-PAGE gels. The positions of monomeric and disulfide-linked
CD3 proteins are marked. An asterisk indicates a
nondisulfide-linked molecule that nonspecifically coprecipitates with
CD3 proteins, believed to be actin.
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 |
DISCUSSION |
In the current report we evaluated the disulfide linkage status of
newly synthesized TCR proteins in CD4+CD8+
thymocytes and splenic T cells. These studies show that: (i) CD3
proteins exist as both monomeric and oligomeric (disulfide-linked) species in murine T cells; (ii) disulfide-linked CD3
proteins differentially assemble with CD3
and TCR
subunits in
CD4+CD8+ thymocytes and splenic T cells; (iii)
unlike CD3
processing in splenic T cells, Glc residues are not
invariably removed from CD3
glycoproteins prior to their assembly
with CD3
chains in CD4+CD8+ thymocytes; and
(iv) calnexin associates with both monomeric and disulfide-linked
CD3
proteins in murine T cells.
Previous studies by Jin et al. reported that a fraction of
CD3
proteins exists as disulfide-linked dimers in murine T cells, including thymocytes (8). The data in the current study show that
CD3
proteins were present as disulfide-linked dimers in both
CD4+CD8+ thymocytes and splenic T cells, but no
evidence was found for disulfide linkage of CD3
molecules in either
cell type. The reason for these apparent discrepancies are unclear but
may result from the fact that identification of disulfide-linked CD3
proteins in previous studies relied on their detection by rabbit
antiserum directed against murine CD3
(8), which may detect unique
epitopes not recognized by the anti-CD3
mAbs used in our study.
Regardless, the current study clearly demonstrates that CD3
glycoproteins were assembled into disulfide-linked dimers in murine T
lymphocytes using several different approaches, including
immunoblotting, immunoprecipitation/release/recapture experiments, and
glycosidase digestion studies. Our results are consistent with the
assembly of CD3
chains into homodimers containing CD3
proteins
disulfide-linked to itself or heterodimers of CD3
proteins
disulfide-bridged to another molecular of similar molecular mass and
carbohydrate content.
Huppa and Ploegh have recently shown that human CD3
molecules
translated in vitro in the absence of other TCR proteins
have a tendency to form disulfide-linked homooligomers, which assemble with calnexin (10). The data in the current report importantly extend
these studies by showing that murine CD3
proteins synthesized in
intact cells in the presence of other TCR proteins may also exist as
disulfide-linked molecules that can assemble with calnexin. Conceivably, disulfide-linked CD3
proteins in
CD4+CD8+ thymocytes and splenic T cells may
represent CD3
molecules that are synthesized in excess of other TCR
subunits that rapidly dimerize and bind to calnexin. Curiously,
however, disulfide-linked CD3
proteins were not assembled with other
TCR molecules in splenic T cells but were (noncovalently) associated
with CD3
proteins in CD4+CD8+ thymocytes.
Although the significance of these findings is currently unclear, it is
interesting to note that these results parallel the differential
assembly of Glc-containing CD3
proteins with CD3
molecules in
CD4+CD8+ thymocytes and splenic T cells. The
molecular basis for the differential assembly of incompletely trimmed
CD3
chains with CD3
proteins in CD4+CD8+
thymocytes and splenic T cells is unknown but may be influenced by the
dissimilar stability of TCR
proteins in these two cell types (3,
17). We have previously noted that CD3
proteins synthesized in T
hybridoma cells under conditions of impaired glucosidase activity
(which destabilizes TCR
molecules) show increased assembly with
CD3
subunits, similar to what is naturally observed in
CD4+CD8+ thymocytes (3). Thus, it is possible
that TCR
association with CD3
or CD3
proteins retards assembly
of disulfide-linked CD3
proteins with CD3
molecules, although
such 
,
intermediates remain to be directly demonstrated in
primary murine T cells (3). In both CD4+CD8+
thymocytes and splenic T cells, dimeric CD3
proteins were precluded from incorporation into complete TCR complexes as evidenced by their
failure to coprecipitate with TCR
,
, and CD3
proteins (this
study),2 indicating that
quality control mechanisms exist in both cell types that control
assembly of dimeric CD3
proteins into higher ordered TCR
complexes.
Finally, our results that newly synthesized CD3
chains bearing
incompletely trimmed oligosaccharides were assembled with CD3
subunits in CD4+CD8+ thymocytes are in
agreement with previous reports that Glc-containing CD3
proteins are
expressed on the surfaces of immature thymocytes in association with
CD3
molecules (5, 7). Importantly, however, the data in the current
study provide the first assessment of the efficiency of Glc removal
from newly synthesized CD3
proteins in
CD4+CD8+ thymocytes and show that CD3
glycoforms are effectively generated in
CD4+CD8+ thymocytes as in splenic T cells
(24).
I thank Drs. Ralph Kubo, Larry Samelson, and
Allan Weissman for generosity in providing anti-TCR antibodies and Drs.
Mark Mannie and Tom McConnell for critical reading of the
manuscript.