Calnexin Associates with Monomeric and Oligomeric (Disulfide-linked) CD3delta Proteins in Murine T Lymphocytes*

Kelly P. KearseDagger

From the Department of Microbiology & Immunology, East Carolina University, School of Medicine, Greenville, North Carolina 27858-4353

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The antigen-binding receptor expressed on most T lymphocytes consists of disulfide-linked clonotypic alpha beta heterodimers noncovalently associated with monomeric CD3gamma ,delta ,epsilon proteins and disulfide-linked zeta zeta 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 CD3delta proteins exist as both monomeric and oligomeric (disulfide-linked) species that differentially assemble with CD3epsilon subunits in CD4+CD8+ thymocytes and splenic T cells. Interestingly, unlike previous results on glucose trimming and TCR assembly of CD3delta 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 CD3delta glycoproteins prior to their assembly with CD3epsilon subunits in CD4+CD8+ thymocytes. Finally, these studies show that calnexin associates with both monomeric and disulfide-linked CD3delta proteins in murine T cells. The data in the current report demonstrate that CD3delta 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 CD3delta proteins is a function of their oxidation state.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Most T lymphocytes express on their surfaces a multisubunit complex consisting of clonotypic alpha beta proteins associated with invariant CD3-gamma ,delta ,epsilon and zeta  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 delta epsilon and gamma epsilon proteins, (ii) assembly of alpha ,beta proteins with delta epsilon ,gamma epsilon pairs to form alpha delta epsilon and beta gamma epsilon intermediate complexes, (iii) joining of alpha delta epsilon and beta gamma epsilon chains followed by disulfide bonding of CD3-associated alpha ,beta proteins to yield incomplete alpha beta delta epsilon gamma epsilon complexes, and finally (iv) addition of disulfide-linked zeta zeta homodimers to form the complete alpha beta delta epsilon gamma epsilon zeta zeta 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 alpha beta delta epsilon gamma epsilon and complete alpha beta delta epsilon gamma epsilon zeta zeta 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 alpha beta delta epsilon gamma epsilon zeta zeta TCR (3), immature CD4+CD8+ (double-positive) thymocytes express both complete TCR and partial complexes of CD3delta epsilon , CD3gamma epsilon 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 CD3epsilon proteins exists as disulfide-linked dimers in human T lymphocytes (8), which assemble with TCRbeta subunits; CD3epsilon dimers were likewise observed to be present in murine thymocytes, although their assembly status was not evaluated (8). Disulfide-linked heterodimers of CD3gamma -epsilon proteins have also been described in REX variant human T cell lines, which fail to express TCRalpha or TCRalpha ,beta molecules (9). More recently, Huppa and Ploegh demonstrated that human CD3epsilon 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 CD3delta or CD3gamma molecules was sufficient to maintain CD3epsilon proteins in a principally monomeric phase, however, suggesting that CD3delta and CD3gamma may guide the folding of CD3epsilon 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 CD3delta proteins exist as both monomeric and disulfide-linked molecules that differentially assemble with CD3epsilon molecules in CD4+CD8+ thymocytes and splenic T cells. In addition, these data document that calnexin associates with both monomeric and oligomeric (disulfide-linked) CD3delta proteins in murine T lymphocytes.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 CD3epsilon proteins associated with CD3delta or CD3gamma chains (11); HMT 3.1, which recognizes CD3epsilon proteins irrespective of their assembly state (12); H28-710, specific for TCRalpha (13); and H57-597, specific for TCRbeta (14), all kindly provided by Dr. Ralph Kubo (Cytel, San Diego, CA). The following antisera were used: R9, specific for CD3delta proteins (15), kindly provided by Dr. Larry Samelson (National Institutes of Health, Bethesda, MD); 551, specific for TCRzeta 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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-CD3epsilon immunoprecipitates of radiolabled CD4+CD8+ thymocytes on two-dimensional nonreducing × reducing (NR × R) gels shows expected nondisulfide-linked monomeric CD3gamma ,delta and CD3epsilon proteins that migrate on and slightly above the diagonal, respectively (Fig. 1A, top panel), and disulfide-linked TCRalpha beta heterodimers and zeta zeta 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-CD3epsilon mAbs of distinct specificity, 145-2C11 and HMT3.1 (Fig. 1, A, top panel, and B, top panel, respectively) (see "Experimental Procedures"), and by CD3delta -specific Ab (Figs. 1B, bottom panel) but not anti-TCRbeta mAb (Fig. 1A, bottom panel) or anti-zeta antiserum (data not shown). Unlike CD4+CD8+ thymocytes, disulfide-linked 26-kDa proteins were not observed in anti-CD3epsilon precipitates of radiolabeled splenic T cells (Fig. 2, A and B) but were clearly present in anti-CD3delta 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 CD3epsilon and TCRbeta 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-CD3epsilon mAb (145-2C11) and anti-TCRbeta 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-CD3epsilon mAb (HMT3.1) and anti-CD3delta Ab (R9).


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Fig. 2.   Disulfide-linked 26-kDa proteins are not assembled with CD3epsilon 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-CD3epsilon 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-CD3epsilon mAb (145-2C11) and anti-CD3delta Ab (R9).

CD3delta 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 CD3delta proteins, CD3epsilon proteins, or both. To determine whether CD3delta ,epsilon proteins were assembled into disulfide-linked dimers in CD4+CD8+ thymocytes, anti-CD3delta precipitates of CD4+CD8+ thymocytes were analyzed on two-dimensional NR × R gels and immunoblotted with antiserum specific for CD3delta and CD3epsilon molecules. As shown in Fig. 3, CD3delta proteins existed as both monomeric and disulfide-linked molecules in CD4+CD8+ thymocytes (Fig. 3, top panel). In contrast, CD3epsilon proteins were present exclusively as nondisulfide-linked monomers (Fig. 3, bottom panel). Identical results were obtained in immunoblot experiments of anti-CD3epsilon precipitates of CD4+CD8+ thymocytes (data not shown). These results show that CD3delta proteins exist as both monomeric and disulfide-linked molecules in CD4+CD8+ thymocytes.


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Fig. 3.   CD3delta proteins are assembled into disulfide-linked dimers in murine T cells. Anti-CD3delta immunoprecipitates of digitonin lysates of CD4+CD8+ thymocytes were analyzed on two-dimensional NR × R SDS-PAGE gels and immunoblotted with anti-CD3delta or anti-CD3epsilon Ab as indicated. The positions of monomeric and disulfide-linked CD3delta proteins are indicated. Note that anti-CD3epsilon proteins existed exclusively as nondisulfide-linked, monomeric proteins in these studies.

CD3delta and CD3epsilon proteins are easily distinguished from each other in that CD3delta is post-translationally modified by the addition of three N-linked oligosaccharide chains unlike CD3epsilon , which does not contain N-glycans (1, 17). To confirm that CD3delta glycoproteins were disulfide-linked in murine T cells, digitonin lysates of radiolabeled CD4+CD8+ thymocytes were immunoprecipitated with anti-CD3delta Ab, CD3delta precipitates were boiled in SDS to release bound material, and CD3delta proteins were recaptured by precipitation with anti-CD3delta 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 CD3delta 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 CD3delta 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-CD3delta Ab (data not shown). These data are consistent with the assembly of CD3delta glycoproteins into disulfide-linked dimers that are composed of CD3delta proteins linked to itself (CD3delta -delta ) or to another molecule of similar size (CD3delta -x), which like CD3delta , must also contain N-glycans as the magnitude of decrease in molecular mass following deglycosylation is greater than would be expected for CD3delta associated with a nonglycosylated protein.


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Fig. 4.   A subset of newly synthesized CD3delta glycoproteins exists as disulfide-linked dimers in CD4+CD8+ thymocytes. Digitonin lysates of [35S]methionine-radiolabeled CD4+CD8+ thymocytes were immunoprecipitated with anti-CD3delta Ab, precipitates were boiled in 1% SDS to release bound material, and CD3delta proteins were specifically recaptured by precipitation with anti-CD3delta 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 CD3delta proteins are marked. An asterisk indicates a nonglycosylated molecule that nonspecifically coprecipitates with CD3delta proteins, believed to be actin; this molecule is not disulfide-linked to CD3delta proteins as determined by analysis on two-dimensional nonreducing × reducing gels (see Fig. 6).

Glc Trimming and Calnexin Assembly of CD3delta 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 TCRalpha ,beta and CD3delta ,gamma subunits (23-25). Previous studies on the processing of TCR glycoproteins in splenic T cells show that Glc residues are removed from newly synthesized CD3delta molecules prior to their assembly with other TCR subunits and that calnexin associates exclusively with unassembled, "free" CD3delta proteins containing incompletely trimmed glycan chains (24). To determine whether CD3delta 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-CD3delta Ab to purify total CD3delta proteins; alternatively, lysates were sequentially immunoprecipitated with anti-TCRbeta mAb to isolate CD3delta proteins assembled into complete alpha beta delta epsilon gamma epsilon zeta zeta and incomplete alpha beta delta epsilon gamma epsilon TCR complexes, followed by precipitation with anti-CD3epsilon mAb to capture CD3delta chains present in partial complexes of CD3delta epsilon components and finally precipitation with anti-CD3delta Ab to purify remaining unassembled, free CD3delta chains. Precipitates were boiled in SDS to release bound material, CD3delta proteins were specifically recaptured with anti-CD3delta 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), CD3delta proteins synthesized in CD4+CD8+ thymocytes existed in four major glycoforms (A-D), indicative of CD3delta 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, CD3delta proteins containing incompletely trimmed N-glycans in CD4+CD8+ thymocytes were present as both free, unassembled chains and as assembled molecules associated with CD3epsilon proteins (Fig. 5, A and B). In contrast, CD3delta proteins associated with TCRbeta 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 CD3delta glycoforms are similarly generated in immature CD4+CD8+ thymocytes and splenic T cells and that CD3delta 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 CD3delta glycoproteins in CD4+CD8+ thymocytes. A, digitonin lysates of [35S]methionine-radiolabeled CD4+CD8+ thymocytes were immunoprecipitated with anti-CD3delta Ab or anti-calnexin Ab or were sequentially immunoprecipitated with anti-TCRbeta mAb, followed by anti-CD3epsilon mAb, and finally were immunoprecipitated with anti-CD3delta Ab. Precipitated material was released by boiling in SDS and CD3delta proteins specifically recaptured by precipitation with anti-CD3delta Ab; recapture precipitates were digested with JB and EH glycosidases as indicated. The positions of CD3delta glycoforms (A-D) and Endo-H-sensitive, deglycosylated CD3delta proteins (CD3delta EHS) are marked. B, same as in A except that CD4+CD8+ thymocyte lysates were sequentially immunoprecipitated with anti-TCRbeta mAb, followed by immunoprecipitation with anti-CD3epsilon mAb and finally with anti-CD3delta Ab.

Next, the assembly of newly synthesized CD3delta proteins with the molecular chaperone calnexin was examined. Metabolically labeled CD3delta 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), CD3delta 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 CD3delta 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 CD3delta proteins were specifically recaptured with anti-CD3delta Ab. Analysis of recapture precipitates on two-dimensional NR × R SDS-PAGE gels showed that both monomeric and disulfide-linked CD3delta 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 CD3delta proteins associate with calnexin in murine T cells.


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Fig. 6.   Both monomeric and disulfide-linked CD3delta proteins associate with calnexin in CD4+CD8+ thymocytes. Digitonin lysates of [35S]methionine-radiolabeled CD4+CD8+ thymocytes were immunoprecipitated with anti-CD3delta Ab or anti-calnexin Ab; precipitates were boiled in SDS, CD3delta proteins were recaptured by precipitation with anti-CD3delta Ab, and recapture precipitates were analyzed on two-dimensional NR × R SDS-PAGE gels. The positions of monomeric and disulfide-linked CD3delta proteins are marked. An asterisk indicates a nondisulfide-linked molecule that nonspecifically coprecipitates with CD3delta proteins, believed to be actin.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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) CD3delta proteins exist as both monomeric and oligomeric (disulfide-linked) species in murine T cells; (ii) disulfide-linked CD3delta proteins differentially assemble with CD3epsilon and TCRbeta subunits in CD4+CD8+ thymocytes and splenic T cells; (iii) unlike CD3delta processing in splenic T cells, Glc residues are not invariably removed from CD3delta glycoproteins prior to their assembly with CD3epsilon chains in CD4+CD8+ thymocytes; and (iv) calnexin associates with both monomeric and disulfide-linked CD3delta proteins in murine T cells.

Previous studies by Jin et al. reported that a fraction of CD3epsilon proteins exists as disulfide-linked dimers in murine T cells, including thymocytes (8). The data in the current study show that CD3delta 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 CD3epsilon molecules in either cell type. The reason for these apparent discrepancies are unclear but may result from the fact that identification of disulfide-linked CD3epsilon proteins in previous studies relied on their detection by rabbit antiserum directed against murine CD3epsilon (8), which may detect unique epitopes not recognized by the anti-CD3epsilon mAbs used in our study. Regardless, the current study clearly demonstrates that CD3delta 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 CD3delta chains into homodimers containing CD3delta proteins disulfide-linked to itself or heterodimers of CD3delta proteins disulfide-bridged to another molecular of similar molecular mass and carbohydrate content.

Huppa and Ploegh have recently shown that human CD3epsilon 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 CD3delta 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 CD3delta proteins in CD4+CD8+ thymocytes and splenic T cells may represent CD3delta molecules that are synthesized in excess of other TCR subunits that rapidly dimerize and bind to calnexin. Curiously, however, disulfide-linked CD3delta proteins were not assembled with other TCR molecules in splenic T cells but were (noncovalently) associated with CD3epsilon 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 CD3delta proteins with CD3epsilon molecules in CD4+CD8+ thymocytes and splenic T cells. The molecular basis for the differential assembly of incompletely trimmed CD3delta chains with CD3epsilon proteins in CD4+CD8+ thymocytes and splenic T cells is unknown but may be influenced by the dissimilar stability of TCRalpha proteins in these two cell types (3, 17). We have previously noted that CD3delta proteins synthesized in T hybridoma cells under conditions of impaired glucosidase activity (which destabilizes TCRalpha molecules) show increased assembly with CD3epsilon subunits, similar to what is naturally observed in CD4+CD8+ thymocytes (3). Thus, it is possible that TCRalpha association with CD3delta or CD3epsilon proteins retards assembly of disulfide-linked CD3delta proteins with CD3epsilon molecules, although such alpha delta ,alpha epsilon intermediates remain to be directly demonstrated in primary murine T cells (3). In both CD4+CD8+ thymocytes and splenic T cells, dimeric CD3delta proteins were precluded from incorporation into complete TCR complexes as evidenced by their failure to coprecipitate with TCRbeta , zeta , and CD3gamma proteins (this study),2 indicating that quality control mechanisms exist in both cell types that control assembly of dimeric CD3delta proteins into higher ordered TCR complexes.

Finally, our results that newly synthesized CD3delta chains bearing incompletely trimmed oligosaccharides were assembled with CD3epsilon subunits in CD4+CD8+ thymocytes are in agreement with previous reports that Glc-containing CD3delta proteins are expressed on the surfaces of immature thymocytes in association with CD3epsilon molecules (5, 7). Importantly, however, the data in the current study provide the first assessment of the efficiency of Glc removal from newly synthesized CD3delta proteins in CD4+CD8+ thymocytes and show that CD3delta glycoforms are effectively generated in CD4+CD8+ thymocytes as in splenic T cells (24).

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed. Tel.: 252-816-2703; Fax: 252-816-3104; E-mail: kearse{at}brody.med.ecu.edu.

1 The abbreviations used are: TCR, T cell antigen receptor; ER, endoplasmic reticulum; mAb, monoclonal antibody; EH, endoglycosidase H; JB, jack bean mannosidase; PAGE, polyacrylamide gel electrophoresis; Ab, antibody.

2 K. P. Kearse, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Klausner, R. D., Lippincott-Schwartz, J., and Bonifacino, J. S. (1990) Annu. Rev. Cell Biol. 6, 403-431[CrossRef]
  2. Exley, M., Terhorst, C., and Wileman, T. (1991) Sem. Immunol. 3, 283-297[Medline] [Order article via Infotrieve]
  3. Kearse, K. P., Roberts, J. L., and Singer, A. (1995) Immunity 2, 391-399[Medline] [Order article via Infotrieve]
  4. Wiest, D. L., Kearse, K. P., Shores, E. W., and Singer, A. (1994) J. Exp. Med. 180, 1375-1382[Abstract]
  5. Wiest, D. L., Burgess, W. H., McKean, D., Kearse, K. P., and Singer, A. (1995) EMBO J. 14, 3425-3434[Abstract]
  6. Takase, K., Wakizaka, K., von Boehmer, H., Wada, I., Moriya, H., and Saito, T. (1997) J. Immunol. 159, 741-747[Abstract]
  7. Wiest, D. L., Bhandoola, A., Punt, J., Kreibich, G., McKean, D., and Singer, A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1884-1889[Abstract/Free Full Text]
  8. Jin, Y. J., Koyasu, S., Moingeon, P., Steinbrich, R., Tarr, G. E., and Reinherz, E. L. (1990) J. Biol. Chem. 265, 15850-15853[Abstract/Free Full Text]
  9. Sancho, J., Chatila, T., Wong, R. C. K., Hall, C., Blumberg, R., Alarcon, B., Geha, R. S., and Terhorst, C. (1989) J. Biol. Chem. 264, 20760-20769[Abstract/Free Full Text]
  10. Huppa, J. B., and Ploegh, H. L. (1997) J. Exp. Med. 186, 393-403[Abstract/Free Full Text]
  11. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E., and Bluestone, J. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1374-1378[Abstract]
  12. Born, W., Miles, C., White, J., O'Brien, R., Feed, J. H., Marrack, P., Kappler, J., and Kubo, R. T. (1987) Nature 330, 572-575[CrossRef][Medline] [Order article via Infotrieve]
  13. Becker, J. L. B., Near, R., Mudgett-Hunter, M., Margolies, M. M., Kubo, R. T., Kaye, J., and Hedrick, S. M. (1989) Cell 58, 911-921[Medline] [Order article via Infotrieve]
  14. Kubo, R. T., Born, J. W., Kappler, J., Marrack, P., and Pigeon, M. (1989) J. Immunol. 142, 2736-2742[Abstract/Free Full Text]
  15. Samelson, L. E., Weissman, A. M., Robey, F. A., Berkower, I., and Klausner, R. D. (1986) J. Immunol. 137, 3254-3258[Abstract/Free Full Text]
  16. Cenciarelli, C., Hou, D., Hsu, K. C., Rellahan, B. L., Weist, D. L., Smith, H. T., Fried, V. A., and Weissman, A. M. (1992) Science 257, 795-797[Medline] [Order article via Infotrieve]
  17. Kearse, K. P., Roberts, J. L., Munitz, T. I., Wiest, D. L., Nakayama, T., and Singer, A. (1994) EMBO J. 13, 4504-4514[Abstract]
  18. Moremen, K. W., Trimble, R. B., and Herscovics, A. (1994) Glycobiology 4, 113-125[Medline] [Order article via Infotrieve]
  19. Hammond, C., Braakman, I., and Helenius, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 913-917[Abstract]
  20. Bergeron, J. J. M., Brenner, M. B., Thomas, D. Y., and Williams, D. B. (1994) Trends Biochem. Sci. 19, 124-128[CrossRef][Medline] [Order article via Infotrieve]
  21. Spiro, R. G., Zhu, Q., Bhoyroo, V., and Soling, H.-D. (1996) J. Biol. Chem. 271, 11588-11594[Abstract/Free Full Text]
  22. Ware, F. E., Vassilakos, A., Peterson, P. A., Jackson, M. R., Lehrman, M. A., and Williams, D. B. (1995) J. Biol. Chem. 270, 4697-4704[Abstract/Free Full Text]
  23. Hochstenbach, F., David, V., Watkins, S., and Brenner, M. B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4734-4738[Abstract]
  24. van Leeuwen, J. E. M., and Kearse, K. P. (1996) J. Biol. Chem. 271, 9660-9665[Abstract/Free Full Text]
  25. van Leeuwen, J. E. M., and Kearse, K. P. (1996) J. Biol. Chem. 271, 25345-25349[Abstract/Free Full Text]
  26. Tarentino, A. L., and Maley, F. (1974) J. Biol. Chem. 249, 811-817[Abstract/Free Full Text]


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