Modification of the T Cell Antigen Receptor (TCR) Complex by
UDP-glucose:Glycoprotein Glucosyltransferase
TCR FOLDING IS FINALIZED CONVERGENT WITH FORMATION OF





COMPLEXES*
Terrence G.
Gardner and
Kelly P.
Kearse
From the Department of Microbiology and Immunology, East Carolina
University, School of Medicine, Greenville, North Carolina
27858-4354
 |
ABSTRACT |
Most T lymphocytes express on their surfaces a
multisubunit receptor complex, the T cell antigen receptor (TCR)
containing
,
,
,
,
, and
molecules, that has been
widely studied as a model system for protein quality control. Although
the parameters of TCR assembly are relatively well established, little
information exists regarding the stage(s) of TCR oligomerization where
folding of TCR proteins is completed. Here we evaluated the
modification of TCR glycoproteins by the endoplasmic reticulum folding
sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (GT) as a unique and sensitive indicator of how TCR subunits assembled into multisubunit complexes are perceived by the endoplasmic reticulum quality control system. These results demonstrate that all TCR subunits
containing N-glycans were modified by GT and that TCR proteins were differentially reglucosylated during their assembly with
partner TCR chains. Importantly, these data show that GT modification
of most TCR subunits persisted until assembly of CD3
chains and
formation of CD3-associated, disulfide-linked 
heterodimers.
These studies provide a novel evaluation of the folding status of TCR
glycoproteins during their assembly into multisubunit complexes and are
consistent with the concept that TCR folding is finalized convergent
with formation of 




complexes.
 |
INTRODUCTION |
The antigen receptor expressed on most T lymphocytes is the
multisubunit 
T cell receptor complex
(TCR),1 important for
recognition of major histocompatibility complex molecules containing
bound peptides (1). The 
TCR is composed of six distinct proteins:
clonotypic TCR
and -
molecules and invariant CD3
, -
, -
,
and -
chains (1). TCR assembly is initiated in the endoplasmic
reticulum (ER) and occurs via the ordered pairing of: (i) CD3
, -
,
and -
chains into partial complexes of 
and 
components;
(ii) association of clonotypic proteins with CD3 chains to form


and 

intermediate complexes; (iii) joining of


and 

molecules to create incomplete





complexes, within which disulfide linkage of
and
chains occurs; and finally, (iv) addition of 
homodimers to
form complete 






complexes (2, 3). In most T cell
types, intracellular transport and expression of TCR proteins is
tightly regulated by their assembly status. Unassembled and partially
assembled TCR proteins are retained within the ER and disposed of by
poorly understood mechanisms involving retrograde transport to the
cytosol and degradation by proteasomes (4-6). Incomplete
(




) and complete (






) TCR
complexes egress from the ER to the Golgi; however, incomplete TCR
complexes are sorted to lysosomes where they are degraded. Only
complete TCR complexes efficiently traffic to the cell surface (1).
Four TCR subunits are post-translationally modified by addition of
oligosaccharides TCR
(3 N-glycans), TCR
(4 N-glycans), CD3
(3 N-glycans), and CD3
(1 N-glycan) (1). N-Glycan chains on newly
translated proteins have the structure
Glc3Man9GlcNAc2 and are
sequentially processed by glucosidase I and II ER enzymes to form
monoglucosylated
Glc1Man9GlcNAc2 species,
important for interaction with the endogenous lectins calnexin and
calreticulin that function in the quality control system of protein
folding (7-10); the final, innermost Glc residue is removed by
glucosidase II (gII) before or after chaperone disassembly. Fully
trimmed (Glc0) proteins that persist in a malfolded state
are modified by UDP-glucose:glycoprotein glucosyltransferase (GT),
which transfers a single Glc residue, (re)creating monoglucosylated
(Glc1) species that can (re)enter the calnexin,
calreticulin assembly pathway (9). GT is proposed to be a major sensor
of protein folding in the ER (11-13) and will only add back Glc
residues removed by gII if a glycoprotein has not yet acquired its
proper tertiary structure (14). The deglucosylation/reglucosylation
cycle continues until correct conformation is achieved (9, 14).
GT modification of incompletely folded proteins involves interaction
with both polypeptide and glycan determinants, including recognition of
hydrophobic amino acids and interestingly, the innermost GlcNAc residue
of the glycan chain (the site of attachment of oligosaccharide to
protein) (15). Both recognition elements must be covalently linked to
effectively catalyze Glc transfer (15) and accessible to GT
modification, which for certain glycoproteins may be concealed by
molecular chaperone association in vivo, particularly under
conditions of extreme ER stress (14). The size of the glycan chain,
e.g. the oligomannose core, is also important for the
efficiency of reglucosylation; Man8-9GlcNAc2
glycans are reglucosylated much more effectively than shorter
Man5-7GlcNAc2 glycans (12, 16). As recently
demonstrated in mutant BW cell types synthesizing truncated
Glc3Man5GlcNAc2
N-glycans, TCR
molecules having shortened
oligosaccharides were reglucosylated much less efficiently than TCR
molecules having normal size glycans, which was correlated with TCR
instability (16).
To evaluate the folding status of TCR glycoproteins as a function of
their assembly into multisubunit complexes in the ER, we studied the GT
modification of TCR proteins in 2B4 T hybridoma cells. These studies
show that all TCR subunits bearing N-glycan chains were
modified by GT and that TCR proteins were differentially reglucosylated
during their assembly into multisubunit complexes. Furthermore, these
data demonstrate that reglucosylation of most TCR subunits was
extinguished following CD3
assembly and formation of
CD3-associated disulfide-linked 
heterodimers, indicating that
TCR folding is finalized convergent with formation of





complexes.
 |
EXPERIMENTAL PROCEDURES |
Cells and Reagents--
2B4 T hybridoma cells and the
TCR
-deficient 2B4 variant 21.2.2 were maintained by weekly passage
in RPMI 1640 medium containing 5% fetal calf serum at 37 °C in 5%
CO2 (17-19). The following monoclonal antibodies (mAb)
were used in this study: H57-597, specific for TCR
proteins (20);
A2B4 specific for 2B4 TCR
proteins (18); 145-2C11 specific for
CD3
, 
molecules (21); HMT3.2, which recognizes both murine
CD3
and CD3
proteins (22); the following antiserum was used: R9,
specific for CD3
molecules (23). Deoxymannojirimycin (dmj) was
purchased from Roche Molecular Biochemicals and was used at a final
concentration of 75 µg/ml.
[3H]Galactose Labeling and Biotinylation of
Proteins--
Metabolic pulse-labeling with
[3H]galactose was performed as described previously (16).
Briefly, cells were incubated in glucose-free RPMI 1640 medium (Life
Technologies, Inc.) containing 10% dialyzed fetal calf serum, 5 mM sodium pyruvate (Life Technologies, Inc.), and 1 mM cycloheximide (chx) for 3 min at 37 °C in 5%
CO2; cells were centrifuged and resuspended in similar
medium containing 0.5 mCi/ml ([6-3H]galactose) (ICN,
Irvine, CA) and labeled for 15-45 min at 37 °C in 5%
CO2. Effectiveness of chx treatment in blocking new protein synthesis was verified by parallel experiments using
[35S]methionine (data not shown). In experiments using
dmj, cells were cultured overnight in medium containing 75 µg/ml dmj
at 37 °C in 5% CO2; cell viability was identical in
medium- and dmj-treated cultures (data not shown). Biotinylation of
cell surface proteins was performed as described previously (24).
Cell Lysis, Immunoprecipitation, Gel Electrophoresis, and
Immunoblotting--
Cells were solubilized in 1% digitonin (Wako,
Kyoto, Japan) lysis buffer (20 mM Tris, 150 mM
NaCl, plus protease inhibitors) at 1 × 108 cells/ml
for 20 min at 4 °C. Cell lysates were clarified by centrifugation to
remove insoluble material and immunoprecipitated with the appropriate antibodies preabsorbed to protein A-Sepharose beads as described previously (16). Sequential immunoprecipitation, one- and
two-dimensional SDS-PAGE gel electrophoresis, and immunoblotting were
performed according to previously published methods (16, 25).
 |
RESULTS |
Recently we examined the reglucosylation of unassembled TCR
and
-
proteins in BW thymoma cells using [3H]galactose as
a radioactive tracer of Glc residues (16). Here we extended these
studies in 2B4 T hybridoma cells to approximate at which stage(s) of
TCR complex formation folding of individual TCR glycoproteins is
completed, with the rationale that GT modification (reglucosylation)
will cease upon attainment of proper conformation. As shown in Fig.
1, [3H]galactose may be
incorporated into N-linked oligosaccharides on glycoproteins
via three major pathways: (i) conversion into UDP-[3H]galactose, the sugar donor for
galactosyltransferase enzymes that transfer galactose residues to
mature, complex-type oligosaccharides in the trans-Golgi;
(ii) epimerization of UDP-[3H]galactose to
UDP-[3H]glucose, the sugar donor for GT that transfers
Glc residues to high mannose glycans on incompletely folded
glycoproteins in the ER; and (iii) conversion of
UDP-[3H]glucose into
dolichol-phospho[3H]glucose, which is incorporated into
nascent Glc3Man9GlcNAc2 glycans
that are cotranslationally added to newly synthesized polypeptides in
the ER (Fig. 1) (16, 26-28). In the current study, cycloheximide was
included in all experiments to inhibit incorporation of
[3H]glucose into newly translated proteins, thereby
restricting radiolabeling to galactosylation and reglucosylation routes
(Fig. 1) (16). 2B4 T hybridoma cells were used, which have served as a
model cell type for TCR assembly in numerous studies (1).

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Fig. 1.
Radiolabeling of glycoproteins with
[3H]galactose. Diagram illustrating the routes of
incorporation of [3H]galactose into glycoproteins
containing N-linked oligosaccharides and sensitivity of
labeling to chx and dmj. +, incorporation via this route; , no
incorporation via this route. See text for details.
|
|
TCR Subunits Bearing N-Glycans Are Substrates for GT--
As shown
in Fig. 2A, multiple TCR
subunits were detected in anti-CD3
precipitates of
[3H]galactose-labeled 2B4 T cells, including CD3
and
-
glycoproteins and clonotypic TCR
and -
proteins (Fig.
2A); as expected, nonglycosylated CD3
and TCR
molecules were not visualized (Fig. 2A). Because anti-CD3
precipitates contain a mixture of TCR components at various stages of
their assembly superimposed upon one another, sequential precipitation
techniques were used to separate more completely assembled TCR proteins
(capable of becoming galactosylated in the Golgi) from partially
assembled and unassembled TCR subunits (retained in the ER) (25). As
demonstrated, when supernatants from anti-CD3
precipitates were
sequentially precipitated with anti-TCR
mAb, radiolabeled TCR
proteins were detected (Fig. 2A), representing unassembled
TCR
proteins modified via the reglucosylation pathway. Consistent
with incorporation of [3H]glucose into glycan chains on
ER-localized TCR
proteins, the radioactive signal on reglucosylated
TCR
proteins was sensitive to digestion with endoglycosidase H (EH),
specific for immature oligosaccharides (data not shown). The vast
majority of radiolabeled CD3
glycoproteins associated with CD3
were not simultaneously assembled with TCR
but existed in partial
complexes of CD3
components (Fig. 2A, anti-TCR
anti-CD3
precipitates, respectively), indicating that most
radiolabeled CD3
chains associated with CD3
were modified by GT.
More than half of the CD3
-associated CD3
chains were assembled
with TCR
(Fig. 2A), with remaining CD3
proteins
existing in partial 
complexes containing reglucosylated N-glycans; unassembled CD3
glycoproteins were also
modified by GT as shown by sequential immunoprecipitation of
anti-CD3
precipitates with anti-CD3
Ab to capture "free,"
unassembled CD3
chains (Fig. 2A). Reglucosylation of
partially assembled and free CD3
and -
glycoproteins was verified
in 21.2.2 cells (Fig. 2B), a TCR
-deficient 2B4 variant
that cannot assemble CD3 chains into a form capable of ER exit (29).
Similar to our results in parental 2B4 cells, significantly more
radiolabeled CD3
proteins were associated with CD3
than CD3
proteins in 21.2.2 cells (Fig. 2B). In agreement with
previous studies showing that nascent glycoproteins undergo multiple
cycles of deglucosylation and reglucosylation in the ER (9, 16), Glc
residues did not persist on CD3
-associated
proteins in 21.2.2 cells (Fig. 2C), and CD3
proteins were effectively radiolabeled during a secondary pulse period with
[3H]galactose (Fig. 2C). Taken together, these
results demonstrate that glycosylated TCR subunits were substrates for
GT, including invariant CD3
and -
molecules and clonotypic TCR
proteins. In addition, these data show that both "free" and
assembled CD3
and -
proteins contained reglucosylated
glycans.

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Fig. 2.
Reglucosylation of TCR glycoproteins.
A, digitonin lysates of 2B4 T cells labeled with
[3H]galactose for 30 min were sequentially
immunoprecipitated with anti-CD3 mAb (145-2C11), followed by
anti-TCR mAb (H57-597); alternatively, lysates were sequentially
precipitated with anti-TCR mAb (H57-597), followed by anti-CD3
mAb (145-2C11); or with anti-CD3 mAb (145-2C11), followed by
anti-CD3 Ab (R9). B, 21.2.2 cells were radiolabeled with
[3H]galactose for 30 min and solubilized in 1% Nonidet
P-40, and lysates were sequentially immunoprecipitated with 145-2C11
anti-CD3 mAb, followed by HMT3.2 anti-CD3 mAb, which also
recognizes murine CD3 molecules. C, 21.2.2 cells were
radiolabeled with [3H]galactose for 15 min in the
presence of chx, chased in medium without radiolabel in the presence of
chx for 30 min, and then radiolabeled a second time with
[3H]galactose for 15 min in the presence of
cycloheximide. Nonidet P-40 lysates were precipitated with 145-2C11 mAb
and analyzed as in B.
|
|
Reglucosylation and Disulfide Linkage of Clonotypic TCR
and -
Proteins--
To determine the contribution of reglucosylation in
[3H]galactose radiolabeling of TCR proteins, studies were
performed using the mannosidase inhibitor dmj, which precludes
conversion of immature, high mannose glycans to mature (galactosylated)
glycans in the Golgi (30); thus, only reglucosylated glycoproteins are
visualized in such experiments (Fig. 1). As shown in Fig.
3A, markedly fewer radiolabeled TCR proteins were present in anti-TCR
(A2B4)
immunoprecipitates of dmj-treated cells relative to media-treated cells
with TCR
and CD3
and -
chains being completely absent and only
TCR
proteins detected (Fig. 3A). Consistent with
restriction of radiolabeling to immature N-glycans, TCR
proteins in dmj lysates migrated with increased mobility compared with
TCR
proteins from control lysates (Fig. 3A) and unlike
control TCR
molecules, disappeared completely following EH digestion
(Fig. 3A). Note that increased mobility of EH-digested
TCR
-associated TCR
proteins in control lysates results from the
fact that several N-glycans on TCR
proteins remain in the
immature high mannose form, even on surface-expressed molecules, which
is also true for CD3
proteins (31, 32 and see below). Interestingly,
increased amounts of reglucosylated (unassembled) TCR
proteins
existed in dmj lysates relative to control lysates (Fig.
3A), which was accompanied by augmented survival of newly synthesized TCR
proteins.2
These results were specific in that the half-life and reglucosylation of unassembled TCR
molecules were relatively unaffected; similar results were observed in BW thymoma cells (data not shown). Biochemical analysis of surface-labeled molecules showed that the vast majority of
TCR glycoproteins expressed on dmj-treated cells contained immature,
EH-sensitive oligosaccharides (Fig. 3B), demonstrating the
effectiveness of dmj in blocking maturation of
N-oligosaccharides in these studies and showing that dmj
treatment did not perturb TCR assembly. Taken together, these results
show that most radiolabeled TCR
and CD3
and -
glycoproteins
associated with 2B4 TCR
represent galactosylated species and not
reglucosylated TCR molecules. We conclude that TCR
proteins
assembled into TCR
and -
heterodimers are ineffectively modified
by GT (and thus no longer perceived by the ER quality control system as
incompletely folded) and relatedly, that reglucosylation of TCR
and
CD3
and -
proteins is terminated following their association with
TCR
molecules. In addition, these data show that inhibition of
mannosidase activity resulted in enhanced reglucosylation of
unassembled TCR
proteins.

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Fig. 3.
GT modification of TCR glycoproteins
associated with TCR . A, 2B4 T
cells were cultured in medium or deoxymannojirimycin (dmj) as indicated
and labeled with [3H]galactose in the presence of
cycloheximide for 45 min. The presence of dmj was maintained in
appropriate groups throughout the entire experiment. Digitonin lysates
of equivalent numbers of cells were sequentially immunoprecipitated
with A2B4 anti-TCR mAb, followed by H57-597 anti-TCR mAb, and
precipitates were treated /+ EH glycosidase as indicated. The
positions of TCR proteins are marked. B, media- and
dmj-treated 2B4 T cells were surface-labeled by biotinylation and
solubilized in 1% digitonin; digitonin lysates of equivalent numbers
of cells were precipitated with 145-2C11 anti-CD3 mAb and digested
with EH as indicated. Precipitates were analyzed on one-dimensional
13% SDS-PAGE gels under reducing conditions, and labeled proteins were
visualized by chemiluminescence. The positions of TCR proteins are
marked. R subscript denotes Endo H-resistant proteins;
S subscript denotes Endo H-sensitive proteins.
|
|
Reglucosylated TCR
and -
Proteins Are Assembled with CD3
Subunits--
Assembly of TCR
and -
proteins into
disulfide-linked heterodimers is preceded by the association of
monomeric TCR
and TCR
proteins with CD3 components in the form of


and 

intermediates, which join to form





complexes (3). Thus, we next wished to determine
whether CD3-associated TCR
and -
proteins were modified by GT. As
shown in Fig. 4, both monomeric and
dimeric radiolabeled TCR
and -
proteins were detected in
association with CD3 chains in lysates of
[3H]galactose-labeled 2B4 T cells (Fig. 4). Because
monomeric TCR
and -
proteins are restricted to the ER (1, 3),
these data indicate that reglucosylated (incompletely folded) TCR
and -
proteins exist in association with CD3 in 2B4 T cells, most
likely as CD3
and CD3
intermediates because our previous results
showed that few, if any, reglucosylated TCR
proteins were associated with TCR
chains. To determine the contribution of GT modification to
[3H]galactose radiolabeling of CD3-associated TCR
and
-
proteins, dmj treatment was utilized to restrict radiolabeling to
the reglucosylation pathway as before. In agreement with our previous
findings that CD3
and -
glycoproteins assembled into partial

, 
complexes were modified by GT, CD3
and -
chains
were effectively labeled in dmj-treated cells (Fig.
5A). As noted earlier,
reglucosylation of unassembled TCR
chains, captured in sequential
precipitates with anti-TCR
mAb, was enhanced in dmj-treated cells
relative to media-treated cells (Fig. 5A). Importantly,
these data show that reduced amounts of TCR
and -
proteins were
associated with CD3 chains in dmj-treated cells compared with
media-treated cells (Fig. 5A), which was expected as our
previous results showed that a significant portion of radiolabeled
TCR
and -
proteins were assembled into disulfide-linked
heterodimers modified by galactosylation. Analysis on two-dimensional
nonreducing × reducing (NR × R) gels showed that relatively
few radiolabeled TCR
and -
dimers were detected in anti-CD3
precipitates of dmj-treated cells (Fig. 5B), unlike CD3
chains, which were readily visible (Fig. 5B). Interestingly, radiolabeled TCR
proteins existed as both disulfide-linked and monomeric species whereas the vast majority of TCR
molecules were
present as non-disulfide-linked monomers (Fig. 5B).
Formation of TCR
and -
heterodimers was not precluded in
dmj-treated cells as shown by immunoblotting of CD3 precipitates with
anti-TCR
mAb (Fig. 6); dimeric TCR
proteins in media-treated groups existed as two species: an upper band
representing mature (EH-resistant) proteins and a lower band migrating
parallel with non-disulfide-linked TCR
monomers containing immature
(EH-sensitive) glycans (Fig. 6). As demonstrated, only immature TCR
proteins were present in lysates of dmj-treated cells (Fig. 6). These
results corroborate our previous findings that TCR
subunits
associated with TCR
proteins were ineffectively modified by GT and
that reglucosylated (incompletely folded) CD3-associated monomeric
TCR
and -
proteins exist in normal (untreated) cells. Taken
together, these studies demonstrate that reglucosylation of most TCR
components is extinguished following the CD3
and -
assembly and
formation of disulfide-linked TCR
and -
heterodimers, indicating
that TCR folding is finalized convergent with formation of





complexes.

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Fig. 4.
Disulfide linkage status of CD3-associated
TCR and - proteins in
[3H]galactose-labeled 2B4 T cells. Digitonin lysates
of 2B4 T cells labeled with [3H]galactose for 45 min were
sequentially immunoprecipitated with anti-CD3 mAb (145-2C11),
followed by anti-TCR mAb (H57-597), and analyzed on 13% SDS-PAGE
gels under nonreducing conditions or on two-dimensional
nonreducing × reducing (NR × R) gels. The positions of TCR
proteins are marked; TCR D D, dimeric
 proteins; TCR M and TCR M, monomeric
and proteins, respectively. Note that the spot below
D in the two-dimensional NR × R samples represents
a smudge on the gel that is enhanced by photography and is not a
radiolabeled protein.
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Fig. 5.
Reglucosylated TCR
and - proteins are assembled with CD3
components in 2B4 T cells. A, 2B4 T cells were cultured in
medium or dmj as indicated and labeled with [3H]galactose
for 45 min. Digitonin lysates of equivalent numbers of cells were
sequentially immunoprecipitated with 145-2C11 anti-CD3 mAb, followed
by H57-597 anti-TCR mAb. The positions of TCR proteins are marked.
B, anti-CD3 precipitates of dmj-treated
[3H]galactose-labeled 2B4 cells shown in A
were analyzed on two-dimensional nonreducing x reducing (NR × R)
gels. The positions of TCR proteins are marked.
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Fig. 6.
Dimerization of TCR
proteins in media- and dmj-treated 2B4 T cells. Aliquots of
the same cells used for [3H]galactose labeling in Fig. 5
were lysed in digitonin and immunoprecipitated with 145-2C11
anti-CD3 mAb. Lysates of equivalent numbers of cells were analyzed
in media and dmj groups. Precipitates were analyzed on two-dimensional
NR × R gels or digested with Endo H and analyzed on
one-dimensional gels under reducing conditions and immunoblotted with
anti-TCR mAb. The positions of immature (EH-sensitive) and mature
(EH-resistant) TCR proteins are indicated; D, dimeric
proteins; M, monomeric proteins.
|
|
 |
DISCUSSION |
The current report has examined the modification of TCR
glycoproteins by the ER folding sensor enzyme GT and provides the first
example where GT modification of a multisubunit protein complex has
been studied. The data in the current report significantly extend
previous studies on TCR processing in splenic T lymphocytes, which
showed that significant Glc trimming of newly synthesized CD3
and
TCR
glycoproteins takes place prior to association with partner TCR
chains (33), based on their comigration with calnexin-associated glycoforms following digestion with jack bean
-mannosidase. Indeed, the current study utilizes a sensitive radiolabeling method which specifically identifies TCR subunits containing monoglucosylated N-glycans generated via the reglucosylation pathway. The
current report establishes that all TCR subunits containing
N-glycans are substrates for GT and evaluated
reglucosylation as a function of TCR assembly, previously examined only
on unassembled TCR
and -
proteins expressed in BW thymoma cells,
which do not efficiently assemble TCR complexes due to deficient CD3
synthesis (16).
The results in this study suggest a scheme in which reglucosylation of
invariant CD3
and -
subunits persists until their association
with clonotypic TCR
and -
chains, and GT modification of TCR
proteins is terminated following the assembly of 




complexes and formation of disulfide-linked TCR
and -
heterodimers.
Interestingly, unlike CD3-associated TCR
molecules, which were
ineffectively modified by GT following disulfide linkage with TCR
proteins, reglucosylated TCR
molecules existed as both
CD3-associated monomers and dimers. Although the exact significance of
these findings remain to be determined, these data suggest that folding of TCR
may be one of the final steps of ER quality control that precedes TCR egress to the Golgi. It is conceivable that folding of the
TCR complex occurs concomitant with the ordered assembly of TCR
subunits and that GT recognition motifs become progressively "masked" as TCR oligomerization proceeds, similar to ER retention and lysosomal targeting information contained within the polypeptide sequences of certain TCR subunits (1, 34-36). Consistent with this
idea, the results in the current study provide evidence that most TCR
glycoproteins are no longer perceived by the ER quality control system
as incompletely folded following the assembly of 




TCR
complexes, which, interestingly, is the stage at which TCR complexes
become competent for ER exit. It is possible that reglucosylation of
higher ordered TCR complexes ceases due to relocalization from the ER
to the Golgi complex; however, we favor the idea that assembly,
folding, and intracellular transport of TCR proteins are closely
coupled events, similar to what has been observed for other multimeric
immune protein complexes, i.e. major histocompatibility complex
molecules (37-39). Indeed, previous studies have demonstrated that
protein reglucosylation is not static but proceeds in a rapid, cyclic
fashion in concert with Glc removal by glucosidase II enzymes (9,
16).
Interestingly, we found that reglucosylation of "free" TCR
proteins was increased under conditions of mannosidase blockade, which
was specific in that GT modification of TCR
proteins was relatively
unaffected.2 Because the efficiency of GT modification is
inversely correlated with N-glycan chain length (12, 16),
and dmj inhibits the activity of certain ER mannosidase enzymes (40,
41), it is reasonable that increased reglucosylation of unassembled
TCR
proteins in dmj-treated cells results from persistence of Man residues on N-glycan chains. However, it was also noted that
the stability of newly synthesized TCR
molecules was enhanced under these conditions, similar to what has been described for CD3
proteins by Weissman and colleagues (6). Thus, the relationship between
increased reglucosylation and increased survival of TCR proteins under
conditions of mannosidase blockade remains to be determined. Moreover,
despite the fact that GT modification of certain TCR subunits was
enhanced by prevention of Man removal, relatively few reglucosylated
TCR proteins assembled into higher ordered TCR complexes were detected
under these conditions, indicating that GT modification (folding) of
TCR subunits is tightly regulated.
Finally, it is unknown to what extent specific N-glycans on
TCR proteins containing multiple oligosaccharides may be differentially modified by GT enzymes. Recent studies by Dessen et al. (42) demonstrate that N-acetylglucosamine residues interact with
neighboring amino acids of proteins in native conformations, which may
be one of the major mechanisms by which GT modification of newly synthesized proteins is regulated (14, 43). The data in the current
report suggest that determinants that signify malfolded molecules may
persist on TCR
proteins compared with other TCR subunits, an idea
that is consistent with previous findings that TCR
survival is
uniquely sensitive to perturbations in the ER quality control system
(16, 27, 44). Identification of polypeptide and N-glycan
domains important for GT recognition of TCR glycoproteins should
provide valuable information regarding the molecular basis of GT
modification and the regulation of quality control mechanisms that
monitor the presence of unassembled and incompletely folded TCR
proteins in the ER.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Velislava Karaivanova and Tom
McConnell for critical reading of the manuscript. We are also grateful
to Dr. Ralph Kubo for the gift of HMT3.2 Ab and Dr. Larry Samelson for the gift of R9 Ab.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant R29 AI42104 (to K. P. K.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 252-816-2703;
Fax: 252-816-3104; E-mail: kearse{at}brody.med.ecu.edu.
2
K. P. Kearse, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
TCR, T cell
antigen receptor;
ER, endoplasmic reticulum;
GT, UDP-glucose:glycoprotein glucosyltransferase;
mAb, monoclonal antibody;
dmj, deoxymannojirimycin;
chx, cycloheximide;
PAGE, polyacrylamide gel
electrophoresis;
EH, endoglycosidase H.
 |
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