1 Immunobiology Working Group, and
2 Biomolecular Structure and Function Working Group, Fox Chase Cancer Center, Philadelphia, PA 19111, USA
3 Department of Medicine, Division of Experimental Medicine, McGill University, Montréal, Québec H3A 1A3, Canada
4 PROCREA BioSciences, Inc., Montreal, Québec H4P 2R2, Canada
Correspondence to: D. L. Wiest
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Abstract |
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Keywords: pre-TCR, pT, structural model, thymocyte development, Vpre-T
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Introduction |
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Most receptor signals are initiated by ligand engagement, for which antibody usually serves as an effective surrogate; however, this does not appear to be the case for the pre-TCR complex. Indeed, the exodomains of pT and TCRß can be eliminated without adversely affecting the pre-TCR's ability to signal thymocyte development (2528). Moreover, antibody engagement of surface pre-TCR complexes actually inhibits development of thymocytes to the DP stage (2931). Two different models of ligand-independent pre-TCR signaling have been proposed: (i) the surface expression model postulates that pre-TCR signaling requires accumulation of completely assembled pre-TCR complexes within the plasma membrane, possibly because of their apposition with glycolipid-enriched microdomains which are important in T cell activation (25,3234), or (ii) the internal signaling model postulates that pre-TCR signals are initiated during assembly of pre-TCR complexes within the endoplasmic reticulum or during their transport to the cell surface (32,35). These models are currently unresolved.
One of the impediments to understanding how thymocyte development is controlled by the pre-TCR complex is the possibility that immature thymocytes express variant pre-TCR isoforms which have different functions. Such isoforms of the pre-B cell receptor (BCR) have been detected in B lymphoid precursors (8). The pre-BCR comprises Ig and Igß signaling subunits in association with a dimer of Igµ heavy chains, each of which is disulfide-linked to surrogate light chain (SLC), a dimeric complex containing a constant (C) domain-like
5 subunit and V domain-like Vpre-B subunit (36,37). An isoform of the pre-BCR which lacks Igµ is expressed on the surface of pro-B cells (38,39). It has not been established whether an equivalent isoform of the pre-TCR complex lacking TCRß is expressed on early thymocytes.
We report here that immature thymocytes (and thymic lymphoma cells) express a pre-TCR isoform whose TCRß chains are unrecognizable by antibody specific for either the Vß or Cß domains. Consequently, we termed this novel pre-TCR isoform the MB (masked ß)-pre-TCR. MB-pre-TCR complexes represent approximately half of the pre-TCR complexes expressed on the surface of both normal thymocytes and also most thymic lymphoma lines. However, one of the lymphoma lines (SL-12ß.12) is virtually devoid of the MB-pre-TCR isoform. The differential representation of MB-pre-TCR complexes in different lymphoma lines suggests that their expression may be controlled by some trans-acting factor(s). The masking of TCRß in MB-pre-TCR complexes depends upon association of TCRß with pT and correlates with extensive O-glycosylation of pT
; nevertheless, preventing O-glycosylation does not restore antibody access to the TCRß subunits of MB-pre-TCR complexes. Antibody access is restored if the vacant V region of pT
is filled in with a V domain from a TCR
subunit. Taken together, these data provide indirect evidence that antibody access to TCRß may be governed by the presence of an additional pre-TCR subunit (possibly the `Vpre-T' subunit proposed by others) that occupies the vacant V domain of pT
(7,23). The mechanistic basis for masking of the TCRß subunits of MB-pre-TCR complexes and its potential impact on pre-TCR function are discussed.
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Methods |
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Cell lines and antibodies
Scid.adh, SL-12 and SL-343 are spontaneous thymic lymphomas derived from mice with the scid mutation (Scid mice), and so they lack the clonotypic subunits of the TCR, and ß. The SL-4210 lymphoma also arose spontaneously in a Scid mouse; however, SL-4210 does express a TCRß subunit from the Vß16 family (M. Rhodes, unpublished observation). SL-12, SL-343 and SL-4210 were provided by Dr M. Bosma (Fox Chase Cancer Center, Philadelphia, PA) (41,42). SL-12ß.12 cells were generated by electroporating a Vß3 cDNA into the pre-TCR-deficient parental SL-12 cell line, as described (10). Cells were maintained in RPMI supplemented as described (43). The following mAb were used: anti-TCRß (H57-597) (44), anti-CD3
/
(145-2C11) (45), anti-CD3
(7D6) (46), anti-TCR
(H28-710) (47), anti-Vß3 (KJ-25) (48), anti-Vß8.1, 8.2, 8.3 (F23.1; PharMingen, San Diego, CA) (49), anti-2B4 TCR
(A2B4) (50), anti- influenza virus hemaglutinin (HA) (12CA5; Boehringer Mannheim, Indianapolis, IN), anti-TCR
(GL-3) (51) and anti-V
11.1, 11.2 (RR8-1) (52). The following polyclonal rabbit antibodies were used: anti-TCR
(551) (53), anti-CD3
(R9, provided by Dr L. Samelson, NIH, Bethesda, MD) (54) and anti-pT
(10). Anti-pT
antibodies were raised against a GST-fusion protein encompassing either the cytoplasmic domain (pT
-cyt) or the exodomain (pT
-exo) of pT
as described (10,53).
Retroviral gene transfer
The 2 retroviral packaging cell lines expressing the MFG-2B4 (Vß3) and MFG-D011.10 (Vß8) constructs were provided by Dr L. Spain (Holland Laboratory, American Red Cross, Rockville, MD) (55). The murine stem cell virus (MSCVneo, MSCVpuro and MSCVhygro) expression vectors were provided by Dr R. Hawley (American Red Cross, Rockville, MD). The LZRSpBMN-linker-IRES-EGFP (LZRS) retroviral vector was provided by Dr A. Kruisbeek (Netherlands Cancer Institute, Amsterdam, The Netherlands) with permission from Dr Garry Nolan (Stanford University, Stanford, CA) (56). The following cDNAs were obtained as inserts: the 2B4 TCRßHA fusion protein (provided by Dr Y. Takahama, University of Tsukuba, Tsukuba, Japan) was obtained as an insert from the pCAGGS vector and the 2B4 TCR
chain (provided by Dr J. Bonifacino, NICHD, NIH, Bethesda, MD) was obtained as an insert from the pCDM8 vector. The V
11pT
chimera was constructed by fusing the V
11J
region (first Ig-like domain) of the AD10 TCR
chain to a pT
cDNA lacking the leader sequence and the first five amino acids (primer sequences available upon request) (57). 2B4 TCRßHA, 2B4 TCR
and V
11pT
were shuttled through the pCR2.1 cloning vector (Invitrogen, San Diego, CA) into either the MSCV (for 2B4 TCRßHA and 2B4 TCR
) or LZRS (for V
11pT
) retroviral expression vectors listed above by PCR using standard methodology (58). Briefly, the 2B4ßHA and 2B4
inserts were amplified using oligonucleotide primers flanking the translational start and stop codons that were appended with linkers encoding the HpaI (5) and XhoI (3) restriction sites while the V
11pT
insert was amplified with linkers encoding the XhoI (5) and NotI (3) restriction sites. The amplified inserts were directionally subcloned into MSCVneo (2B4ßHA), MSCVpuro (2B4
) or LZRS (V
11pT
) using standard techniques. MSCVneo-2B4ßHA and MSCVpuro-2B4
were separately transfected into the
2 retroviral packaging line using lipofectamine (Gibco/BRL, Grand Island, NY) according to the manufacturer's instructions. Stable viral producing cell lines were selected in 500 µg/ml G418, 1 µg/ml puromycin or 250 µg/ml hygromycin. LZRS-V
11pT
was transfected into the
NX-E retroviral ecotropic viral packaging line (generated by Dr Gary Nolan, Stanford University) using lipofection (56). The Scid.adh or SL-343 thymic lymphoma lines were retrovirally infected by co-culture as follows: 3x105 lymphoma cells were combined with 106
2 retroviral producers in a final volume of 3 ml RPMI containing 4 µg/ml polybrene and incubated at 37°C for 48 h. For V
11pT
expression, TCRß-expressing SL-343 cells (SL-343ß.1) were retrovirally infected with virus-containing supernatants generated from transfected
NX-E cells. For the 2B4 constructs, the infected thymic lymphoma cell lines were harvested and grown in medium containing the appropriate antibiotic for selection. For the V
11pT
chimera, cells simultaneously expressing high levels of V
11 and GFP were isolated by cell sorting, following which surface expression of V
11-pT
was verified by flow cytometry.
Surface biotinylation, immunoprecipitation and electrophoresis
Biotin surface labeling was performed as previously described (53,59) after which cell viability was consistently >98%. Labeled cells were lysed in buffer containing 1% digitonin (Wako, Kyoto, Japan) and immunoprecipitated for 2 h with antibody pre-adsorbed to Protein ASepharose (Sigma, St Louis, MO). Eluted immune complexes were resolved by one-dimensional non-reducing or two-dimensional non-equilibrium pH gradient electrophoresis (NEPHGE)/SDSPAGE gels as described followed by transfer onto Immobilon PVDF membranes (Millipore, Bedford, MA). Surface-biotinylated proteins were visualized with horseradish peroxidase-conjugated streptavidin (HRPAv; Southern Biotechnology Associates, Birmingham, AL) as described (53).
Recapture assay
The recapture assay was performed as previously described (10). Briefly, immune complexes from primary immunoprecipitations were disrupted by boiling in 1% SDS. The SDS eluates were quenched with 10 volumes of 1% NP-40 lysis buffer and secondarily immunoprecipitated using anti-pT, anti-TCR
or anti-epitope tag antibody described above. The recaptured immune complexes were resolved either by one-dimensional or two-dimensional SDSPAGE gels and visualized as described above.
Glycosidase digestion
Recaptured pTß heterodimers were denatured by boiling in 1% SDS either containing 2-mercaptoethanol for NEPHGE analysis or without 2-mercaptoethanol for one-dimensional non-reducing SDSPAGE. The eluted glycoproteins, which were neutralized with 10% NP-40 and the appropriate stock digestion buffer (provided by the manufacturers listed below), were treated as follows: (i) mock treatment, digestion buffer only; (ii) for N-glycan removal, 2500 U peptide-N-glycosidase F (PNGaseF; New England Biolabs, Beverly, MA); and (iii) for N- and O-glycan removal, PNGaseF in combination with 20 U/ml sialidase from Vibrio cholerae (Oxford GlycoSciences, Wakefield, MA) and 4 mU of O-glycanase (Genzyme, Cambridge, MA). For all groups, samples were incubated for 1824 h at 37°C followed by quenching with the appropriate sample buffer.
Inhibition of protein O-glycosylation by treatment with benzyl-2-acetamido-2-deoxy--D-galactopyranoside (benzyl-
-galnac)
Cells were seeded at an initial concentration of 1x105 cells/ml and cultured for 48 h in the presence of benzyl--GalNac (Toronto Research Chemicals, Toronto, Canada) that was solubilized in DMSO and used at a final concentration of 2 mM (60). Mock-treated control cultures were incubated with an equivalent volume of DMSO (0.1% final). The effectiveness of benzyl-
-GalNAc treatment was verified by the binding of a fluorochrome conjugate of the lectin derived from Helix pomatia (HPAFITC; EY Laboratories, San Mateo, CA) as analyzed by flow cytometry (60).
Molecular modeling of the pTß heterodimer complexed with anti-TCRß antibody
The model of the pre-TCR complex was based on the crystal structure of the ßTCR heterodimer (PDB: 1NFD) solved at 2.8 Å resolution (61). To build the structure model, we replaced the TCR
chain with a model of pT
. This model was based on the pT
sequence (GenBank accession no. 967187) and a homologous sequence of known structure, which was identified using PSI-BLAST 2.0.8 (62). The pT
sequence was compared to a non-redundant protein sequence database available from GenBank. Four PSI-BLAST iterations were performed and a position-specific similarity matrix was created which was used to search the PDB sequence database. The E-value cut-off for inclusion of new sequences into the position-specific matrix was 0.0001. The sequence with the best E-value was used as the structure template for the pT
model. Residues 20129 of the pT
sequence were assigned the backbone coordinates of residues 110228 of the FAB CH chain of the mAb IgG2a (PDB: 1IGT), as defined by the PSI-BLAST alignment. Side-chains were built onto the pT
model backbone using SCWRL 2.1 (63), which uses a backbone-dependent rotamer library (64) to assign the side-chain
angles, followed by a combinatorial search of allowable rotamers to minimize steric clashes. In order to find the proper orientation between the pT
model and the TCRß chain, it was first necessary to align the constant domains of the template and the TCR structure, then apply this same rotation/translation to the model. The structure of the CH domain of IgG2a was aligned with the structure of the constant domain of the TCRß chain using MINAREA (65). This rotation and translation was then applied to the pT
model to dock it to the TCR structure with the
chains removed. The minimal O-glycan structure was predicted to be a tetrasaccharide having a NeuNAc
23Galß13(NeuNAc
26)GalNAc structure based on the known mode of action of O-glycosidase, which cleaves de-sialylated Galß13GalNAc residues from serine or threonine residues. The coordinates of the O-linked tetrasaccharides were generated with the computer program Sweet and the sugars were manually docked with MidasPlus in proportion onto the residues predicted as probable O-linked glycosylation sites (T24, S28, T75, S79 and T131), according to the NetOGlyc server (6668). The fifth sugar was placed close to the last residue of the model (S129) since T131 is not part of the model. Figure 7
(A) was produced with MolScript and Raster3D (69,70).
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Results |
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Our results suggested that either the novel isoform actually contains the TCRß subunit or, alternatively, that ß expression induces a distinct protein that is required for formation of the novel pre-TCR isoform. To distinguish between these possibilities, we transduced Scid.adh with an HA-tagged TCRß cDNA, allowing the expressed TCRß subunit to be directly identified in recapture assays using the anti-HA antibody. Using the anti-HA antibody we were able to recapture pT multimers from both the anti-TCRß precipitable pre-TCR complex as well as from the novel pre-TCR isoform, demonstrating that TCRß is in fact a component of the novel isoform despite its inaccessibility to antibody reactive with the C domain of TCRß (Fig. 2B
). Interestingly, the V domain of the TCRß subunits in the novel pre-TCR isoform was similarly inaccessible to antibody (Fig. 3A
). Indeed, neither the novel pre-TCR isoform containing Vß3 nor that containing Vß8 could be recognized by anti-Vß3 or anti-Vß8 antibody respectively (Fig. 3A
). Consequently, we term this novel pre-TCR isoform the MB (for masked ß)-pre-TCR, because the ß subunit of this complex is inaccessible to both antibody directed against the C domain and V domain of TCRß.
Since the Scid.adh lymphoma represents an early stage of thymocyte development, we reasoned that the masking of TCRß might be a consequence of the stage of development rather than a unique feature of the pre-TCR complex. More specifically, we wanted to determine if TCRß subunits associated with TCR would be masked similarly as when associated with pT
. This was assessed by retroviral transduction of a TCR
cDNA into Scid.adh cells already expressing TCRß. Because expression of TCR
prevents pT
incorporation into receptor complexes, the pre-TCR complexes expressed by the Scid.adh recipient cells were completely replaced by
ß TCR complexes (S. Trop et al., manuscript submitted). Immunoprecipitation of detergent lysates of surface-labeled
ßTCR+ Scid.adh cells with either anti-Cß or anti-Vß domains effectively pre-cleared all
ßTCR complexes, demonstrating that the masking of TCR Cß and Vß domains in MB-pre-TCR complexes is dependent upon pT
and therefore is a unique property of pre-TCR complexes (Fig. 3B
). Consequently, the key to understanding the mechanistic basis for masking of TCRß lay in focusing on those features of pT
that distinguish it from TCR
.
Conventional and MB-pre-TCR contain differentially O-glycosylated forms of pT
Since masking of the TCRß subunit depends upon its association with pT and correlates with the increased Mr of pT
ß heterodimers from MB-pre-TCR complexes, it was possible that ß chain masking was due to differential post-translational processing of pT
. The most likely post-translational modification of pT
would be oligosaccharide addition. Note that we employed the pre-TCR+ SL-343ß.1 thymic lymphoma line (generated by retroviral transduction of a TCRß cDNA into the TCRß-deficient parental line) to assess the role of glycans in masking the ß subunits of MB-pre-TCR complexes because SL-343ß.1 expresses higher surface levels of the pre-TCR, making this analysis more straightforward. pT
contains two Asp-X-Ser consensus motifs for N-glycan attachment (9); however, while removal of the N-linked glycans using PNGase F treatment did reduce the Mr of pT
ß heterodimers from both the anti-TCRß precipitable and MB-pre-TCR isoforms, their relative size difference remained unchanged (Fig. 4A
). Thus, differential processing or addition of N-linked glycans to pT
is not responsible for the increased Mr of the pT
ß dimers of the MB-pre-TCR complex. Similar results were obtained with TCR
-/ thymocytes (data not shown).
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Inhibition of O-glycosylation does not restore antibody access to TCRß
To test whether the extensive modification of pT with O-glycans were actually responsible for inhibiting antibody binding to TCRß, we prevented the addition of O-glycans using a competitive inhibitor, benzyl-
-GalNAc. If O-linked glycans were responsible for masking the ß chains of MB-pre-TCR complexes, then preventing their addition should restore the recognition of pT
ß heterodimers by anti-TCRß antibody. Detergent extracts of pre-TCR+ SL-343ß.1 cells that were cultured for 2 days with benzyl-
-GalNAc and surface-biotinylated were immunoprecipitated first with anti-TCRß, then with anti-CD3
antibody. pT
ß dimers recaptured from the immune complexes were digested with PNGaseF and resolved on two-dimensional gels (Fig. 5
). Interestingly, despite the fact that benzyl-
-GalNAc treatment prevented the addition of O-glycans to pT
(as evidenced by the change in migration), antibody specific for the TCR Cß domain remained unable to pre-clear all pre-TCR complexes (Fig. 5
, bottom panels). Moreover, benzyl-
-GalNAc treatment did not even result in a quantitative increase in the proportion of pT
ß heterodimers accessible to anti-TCRß antibody (compare mock- and BenzylGalNAc-treated samples; Fig. 5
). These results suggest that the O-linked glycans are not responsible for TCRß chain masking.
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The two most likely explanations for the ability of the V appendage to restore antibody access to associated TCRß subunits are that: (i) an additional pre-TCR subunit occupies the V region of pT
in about half of pre-TCR complexes and modulates accessibility of the ß subunit to antibody or (ii) TCRß may stochastically adopt alternative structural conformationsone accessible to anti-ß antibody and one inaccessiblewith the accessible conformation preferentially stabilized by the V
appendage on pT
. The latter possibility predicts that in different pre-TCR-expressing cell populations, the ratio of conventional (i.e. TCRß is accessible to antibody) to MB (i.e. TCRß is inaccessible)-pre-TCR complexes should be constant; however, regulation of ß accessibility by a trans-acting factor (i.e. an additional pre-TCR subunit) should cause this ratio to vary with the expression level of that additional subunit. To distinguish these possibilities we assessed the relative proportion of anti-ß accessible and inaccessible pre-TCR complexes in a panel of pre-TCR-expressing thymic lymphomas (Fig. 6B
). Interestingly, the ratio of anti-TCRß accessible (Fig. 6B
; lane 1) to inaccessible (Fig. 6B
; lane 3) pre-TCR complexes did vary among the cell lines examined, with the extreme example being SL-12ß.12 cells, which expressed almost exclusively conventional pre-TCR in which TCRß is accessible to anti-TCRß antibody (Fig. 6B
; bottom panel). Since the ratio of anti-ß accessible:inaccessible pre-TCR complexes varied among the lines examined and all of these lines express all of the known components of the pre-TCR complex, these data suggest that accessibility of TCRß to antibody is controlled by a trans-acting factor(s), possibly an additional pre-TCR subunit.
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Discussion |
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Prior to the current report, there was functional evidence suggesting that immature thymocytes might express an isoform(s) of the pre-TCR complex (which we define by the presence of pT) (74). Indeed, treatment of TCR
transgenic (V
2) RAG-deficient mice with anti-V
2 antibody slightly increased thymic cellularity and the number of DP thymocytes generated, neither of which was observed in similarly treated mice lacking pT
. Consequently, the effects of anti-TCR
antibody treatment were attributed to a pre-TCR isoform comprising pT
, TCR
and associated CD3 signaling components (74); however, neither the extent to which this complex is expressed nor its relevance during normal thymocyte development are clear at present. The MB-pre-TCR isoform is clearly distinct from the aforementioned isoform in that MB-pre-TCR complexes lack components of the
TCR and are expressed extensively, accounting for about half of the pT
ß-containing complexes on the surface of TCR
-/ thymocytes. The expression of pre-TCR isoforms on normal thymocytes has also been predicted by analogy with early B lineage precursors, which express a pro-BCR complex comprising surface SLC but not the Igµ heavy chain (38). The analogous `pro-TCR' complex in T lineage precursors would comprise pT
in association with CD3, but not with TCRß. While MB-pre-TCR complexes are not recognized by anti-TCRß antibody, they do contain TCRß subunits and so are distinct from the aforementioned `pro-TCR' complex. Moreover, we find no evidence for surface expression of pT
-CD3 complexes lacking ß in our lymphoma cell lines, even when using our sensitive recapture assay (Fig. 2A
and data not shown). However, we have found abundant pT
-CD3 complexes retained intracellularly within the endoplasmic reticulum or Golgi complex of thymic lymphoma cells that lack TCRß (data not shown). Consequently, if these pT
-CD3 complexes are playing a role in early thymocyte development, they presumably do so from an intracellular location.
Because masking of the V and C domains of TCRß occurred when TCRß was associated with pT, but not when TCRß was associated with TCR
, our investigation of the mechanistic basis for masking focused upon structural features of pT
that differed from those of TCR
. These features of pT
were: (i) the presence of consensus motifs for addition of O-linked glycans and (ii) the absence of a V-like domain (9).
First, we found that pT is modified by O-linked glycans and that the pT
molecules associated with masked ß chains are more extensively glycosylated. Nevertheless, preventing the addition of O-glycans to pT
using the competitive inhibitor benzyl-
-GalNAc did not alleviate the masking of TCRß subunits in MB-pre-TCR complexes (60). This suggested that while the extent of O-glycosylation did correlate with the masking of TCRß in the MB-pre-TCR, the O-glycosylation of pT
was not responsible for the masking. Then why might the extent of O-glycan addition correlate with masking of the TCRß subunit? Since masking of TCRß seems to be controlled by a cellular factor, one simple explanation could be that the cellular factor also modulates accessibility of the glycosylation enzymes to the O-glycan addition sites of pT
(see below). The inability of O-glycosylation of pT
to account for masking of TCRß in MB-pre-TCR complexes is supported by the structure and location of the O-glycans in a three-dimensional structural model of a pT
ß dimer that we generated (Fig. 7A
). The model was based on the crystal structure of the N15
ßTCR complexed with a Fab' fragment of H57-597 (anti-TCR Cß) (61) and was produced by replacing TCR
with a model of pT
whose consensus O-glycosylation motifs were appended with O-glycans (see Methods). The resulting three-dimensional model revealed that it was unlikely that O-glycans on pT
could interfere with antibody recognition of Cß or Vß domains both because of their small size and remote location. Moreover, steric clashes would prevent the O-glycans from rotating into physical proximity with the ß chain epitopes. Although O-glycosylation of pT
is not responsible for masking the TCRß subunits of MB-pre-TCR complexes, the O-glycans may play other important roles in pre-TCR function. Indeed, O-glycan biosynthesis is developmentally regulated by pre-TCR signals during thymocyte maturation (75). Moreover, recent studies have shown that O-glycans are important mediators of neutrophil migration and inflammation (76), peripheral T cell activation, T cellantigen-presenting cell interaction (77), and apoptosis during thymocyte development (7880). The involvement of O-glycoproteins in apoptosis of developing thymocytes is mediated by an intrathymic O-glycan-binding lectin termed galectin, which when co-engaged with the TCR induces extensive apoptosis of DP thymocytes, but not DN thymocytes (78,79). It is possible that differential O-glycosylation of pT
and TCR
may play some role in susceptibility to galectin-mediated apoptosis (80).
Our assessment of the other structural feature of pT that differed from TCR
(i.e. the absence of a V-like domain) did provide insight into the basis for masking of the TCRß subunits in MB-pre-TCR complexes. Indeed, filling in the `missing' V domain of pT
with a V domain from TCR
completely eliminated the masking of associated TCRß subunits. There are three potential explanations for this result. The first is that TCRß subunits paired with pT
can stochastically adopt two conformationsone of which is recognizable and one which is not recognizable by anti-TCRß antibody; and the recognizable conformation is favored when ß pairs with a two-domain partner subunit like TCR
or the V
pT
chimera. This interpretation predicts that the ratio of recognizable and masked conformations should be an inherent property of the ß subunit and should not vary with the cellular context; however, we find that the SL-12ß.12 thymic lymphoma expresses only surface pre-TCR in which TCRß is accessible to anti-ß antibody (Fig. 6B
), suggesting that accessibility of antibody to TCRß is regulated by a trans-acting factor(s). The second possibility is that a trans-acting factor such as a molecular chaperone could direct TCRß to assume either its antibody-accessible or masked conformation; however, despite the intensive study of chaperone function in protein folding no precedent exists for a chaperone directing two distinct conformations, both of which are competent to be transported to the cell surface. The final possibility, which best fits our experimental evidence, is that accessibility of the TCRß subunit is modulated by the presence of an additional subunit, possibly a Vpre-T-like molecule, that occupies the V region of pT
. This configuration, i.e. a surrogate
complex comprising a C-like pT
and a V-like Vpre-T, is analogous to the two-subunit SLC of the pre-BCR (7,8). Like the C-domain equivalent of the SLC,
5, which is disulfide linked to the Igµ chain, pT
is disulfide linked to TCRß (6,9,36). Moreover, the V domain equivalent of the pre-BCR, Vpre-B, is not covalently associated with either the
5 or Igµ subunit (36). If the putative V-like subunit of the pre-TCR similarly lacks covalent attachment to pT
or ß, this would explain its apparent absence from the immune complexes of our recapture assay, which retains only those molecules that are covalently coupled to pT
. In addition, the V-like subunit of the pre-TCR may not be efficiently visualized by surface biotinylation, requiring a distinct method for detection. This was true of the pT
subunit, which could not be visualized by iodination but could be visualized by biotinylation (6). A resolution to this issue will require further investigation. When taken together our data are most consistent with the interpretation that access of the TCRß subunit of MB-pre-TCR complexes to anti-TCRß antibody is controlled by an additional V-like subunit of the surrogate
chain of the pre-TCR complex.
If the V-like subunit is responsible for modulating antibody accessibility to the TCRß subunit, then how might it do so? The two most likely possibilities are: (i) pairing of the V-like molecule with TCRß may stabilize TCRß's conformation, facilitating recognition by antibody, or (ii) pairing of the V-like molecule with TCRß may interfere with antibody binding. The first possibilitythat accessible ß subunits are paired with the V-like subunit and inaccessible ß chains are notis consistent with the differential O-glycosylation of pT (Fig. 7B
, upper right). Indeed, the pT
subunits of pre-TCR complexes in which TCRß is accessible to antibody tend to have fewer O-glycans, which might result from the obscuring of the consensus O-glycan addition sites by the presence of the V-like subunit (Fig. 7A and B
, upper right). However, the possibility that pairing of the V-like molecule with the TCRß chain is required to maintain TCRß's conformation in a state compatible with antibody recognition appears to be inconsistent with crystallographic analysis of the TCRß subunit. X-ray crystals of TCRß as a monomer, as a dimer with TCR
and in association with superantigen reveal no significant differences in conformation, suggesting that the TCRß structure is quite rigid and not significantly influenced by the subunits with which it is paired (8183). Nevertheless, no crystal structure of TCRß in association with pT
exists, leaving open the possibility that pairing of TCRß with a partner subunit bearing only a C domain may influence TCRß conformation. It should also be noted that crystallographic analysis does not always reliably predict the actual structure of proteins in solution. For example, NMR spectroscopy revealed that the structure of the homodimer interface of the UmuD' protein, which regulates SOS mutagenesis, is not the structure deduced from crystallographic analysis (84,85). The other possible explanation for the effect of a V-like subunit on TCRß accessibility is that this additional subunit could interfere with antibody accessibility to the Vß and Cß epitopes (Fig. 7B
). This explanation requires no conformational changes in ß and so does agree with the crystallographic analysis; however, it is not readily apparent how this possibility explains the more extensive O-glycosylation of pT
subunits in the MB-pre-TCR complex. Nor is it clear how such a subunit could interfere with antibody binding to both the V and C domains of TCRß. Experimentation attempting to resolve these models is in progress.
While the physiologic relevance of the MB-pre-TCR and putative VpreT subunit is uncertain at present, it may serve to modulate the intensity of pre-TCR signals. Since supraphysiologic pre-TCR signaling, such as that induced by antibody engagement of surface pre-TCR complexes, arrests thymocyte development (2931), the V-like subunit might act to limit the intensity of pre-TCR signals by preventing lateral interactions between the pre-TCR complex and other structures on the surface of the thymocyte or thymic stroma. Conversely, since there appears to be no ligand for the pre-TCR complex, the V-like subunit might serve to amplify pre-TCR signals by facilitating lateral interactions between the pre-TCR and other surface molecules involved in signaling. Experiments are in progress to identify the developmental stages at which MB-pre-TCR complexes are expressed and to assess the relative efficiency with which conventional and MB-pre-TCR complexes promote thymocyte development.
In this report, we characterized a novel pre-TCR isoform, the MB-pre-TCR complex, in which the pT subunits are heavily modified with O-glycans and in which the V and C domains of the TCRß subunit are inaccessible to antibody binding. Moreover, we have presented evidence which suggests that antibody access to the ß subunit of MB-pre-TCR complexes is governed by the presence of a trans-acting factor, possibly an additional pre-TCR subunit that occupies the `missing' V region of pT
. Future studies will focus on identifying the putative V-like subunit as a first step towards understanding its requirements for assembly into the pre-TCR complex and its role in pre-TCR function. Answers to these questions will represent a substantial increase in our understanding of how thymocyte development is controlled by the pre-TCR complex.
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Acknowledgments |
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Abbreviations |
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BCR B cell receptor |
benzyl-![]() ![]() |
C constant |
DN double negative (CD4CD8) |
DP double positive (CD4+CD8+) |
HA influenza virus hemaglutinin |
HRPAv horseradish peroxidase-conjugated avidin |
LZRS LZRSpBMN-linker-IRES-EGFP |
MB masked ß |
MSCV murine stem cell virus |
NEPHGE non-equilibrium pH gradient electrophoresis |
PNGaseF peptide-N-glycosidase F |
pT![]() ![]() |
SLC surrogate light chain |
V variable |
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Notes |
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5 Present address: Purdue BioPharma, LP, 201 College Road East, Princeton, NJ 08540, USA
Received 2 May 2000, accepted 1 August 2000.
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References |
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