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In vitro translation and assembly of a complete T cell receptor-CD3 complex.

Huppa JB, Ploegh HL - J. Exp. Med. (1997)

Bottom Line: The T cell receptor for antigen (TCR) is a multisubunit complex that consists of at least seven polypeptides: the clonotypic, disulfide-linked alpha/beta heterodimer that is noncovalently associated with the invariant polypeptides of the CD3 complex (CD3-gamma, -delta, -epsilon) and zeta, a disulfide-linked homodimer.A glycan-dependent interaction between CD3-epsilon and calnexin was mediated by CD3-gamma and concerned only monomeric CD3-epsilon complexed with CD3-gamma, but was dispensable for proper folding of CD3-epsilon.We suggest that in addition to its signaling function, CD3-epsilon serves as a monitor for proper subunit assembly of the TCR.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
The T cell receptor for antigen (TCR) is a multisubunit complex that consists of at least seven polypeptides: the clonotypic, disulfide-linked alpha/beta heterodimer that is noncovalently associated with the invariant polypeptides of the CD3 complex (CD3-gamma, -delta, -epsilon) and zeta, a disulfide-linked homodimer. We achieved the complete assembly of the human TCR in an in vitro transcription/translation system supplemented with dog pancreas microsomes by simultaneous translation of the messenger RNAs encoding the TCR-alpha, -beta and CD3-gamma, -delta, -epsilon, and -zeta subunits. CD3-epsilon, one of the subunits that initiates the assembly of the TCR in living cells, forms misfolded, disulfide-linked homooligomers when translated alone. However, co-translation of one of its first binding partners in the course of assembly, CD3-gamma or -delta, led to the expression of mainly monomeric and correctly folded epsilon subunits, the only form we could detect as part of a properly assembled TCR complex. In the absence of these subunits, the ER-resident chaperone calnexin interacted with oligomeric, i.e. misfolded, structures of CD3-epsilon in a glycan-independent manner. A glycan-dependent interaction between CD3-epsilon and calnexin was mediated by CD3-gamma and concerned only monomeric CD3-epsilon complexed with CD3-gamma, but was dispensable for proper folding of CD3-epsilon. We suggest that in addition to its signaling function, CD3-epsilon serves as a monitor for proper subunit assembly of the TCR.

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Co-translation of related TCR subunits prevents the formation  of disulfide-linked ε homooligomers. (A) CD3-ε was either translated  alone or along with the remaining subunits of the TCR–CD3 complex  for 1 h at 30°C. Microsomes were lysed in NP-40 lysis buffer in the presence of 50 mM iodoacetamide. Total microsome-associated material as  well as immunoprecipitates obtained with the anti–CD3-ε antiserum  were analyzed by SDS-PAGE (12.5%) performed as indicated under reducing and nonreducing conditions. (B) CD3-ε was translated alone and  pairwise with one subunit of the TCR or with HLA-DRβ as indicated,  for 1 h at 30°C. Microsomes were lysed in NP-40 lysis buffer in the presence of 50 mM iodoacetamide. Total microsome-associated material as well  as immunoprecipitates obtained with the anti–CD3-ε antiserum were analyzed by SDS-PAGE (12.5%) performed under reducing and nonreducing conditions as indicated.
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Figure 7: Co-translation of related TCR subunits prevents the formation of disulfide-linked ε homooligomers. (A) CD3-ε was either translated alone or along with the remaining subunits of the TCR–CD3 complex for 1 h at 30°C. Microsomes were lysed in NP-40 lysis buffer in the presence of 50 mM iodoacetamide. Total microsome-associated material as well as immunoprecipitates obtained with the anti–CD3-ε antiserum were analyzed by SDS-PAGE (12.5%) performed as indicated under reducing and nonreducing conditions. (B) CD3-ε was translated alone and pairwise with one subunit of the TCR or with HLA-DRβ as indicated, for 1 h at 30°C. Microsomes were lysed in NP-40 lysis buffer in the presence of 50 mM iodoacetamide. Total microsome-associated material as well as immunoprecipitates obtained with the anti–CD3-ε antiserum were analyzed by SDS-PAGE (12.5%) performed under reducing and nonreducing conditions as indicated.

Mentions: The propensity of CD3-ε to form disulfide-linked oligomers was examined by translating CD3-ε either alone or together with the other subunits of the TCR. Total microsome-associated material as well as immunoprecipitates obtained from lysed microsomes were analyzed by SDS-PAGE performed under both reducing and nonreducing conditions (Fig. 7 A). Lysis in 0.5% NP-40 facilitates the visualization of CD3-ε as it leads to the disruption of all noncovalent interactions between the subunits of the TCR, but not those between CD3-ε, -γ, and -δ (6).


In vitro translation and assembly of a complete T cell receptor-CD3 complex.

Huppa JB, Ploegh HL - J. Exp. Med. (1997)

Co-translation of related TCR subunits prevents the formation  of disulfide-linked ε homooligomers. (A) CD3-ε was either translated  alone or along with the remaining subunits of the TCR–CD3 complex  for 1 h at 30°C. Microsomes were lysed in NP-40 lysis buffer in the presence of 50 mM iodoacetamide. Total microsome-associated material as  well as immunoprecipitates obtained with the anti–CD3-ε antiserum  were analyzed by SDS-PAGE (12.5%) performed as indicated under reducing and nonreducing conditions. (B) CD3-ε was translated alone and  pairwise with one subunit of the TCR or with HLA-DRβ as indicated,  for 1 h at 30°C. Microsomes were lysed in NP-40 lysis buffer in the presence of 50 mM iodoacetamide. Total microsome-associated material as well  as immunoprecipitates obtained with the anti–CD3-ε antiserum were analyzed by SDS-PAGE (12.5%) performed under reducing and nonreducing conditions as indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198996&req=5

Figure 7: Co-translation of related TCR subunits prevents the formation of disulfide-linked ε homooligomers. (A) CD3-ε was either translated alone or along with the remaining subunits of the TCR–CD3 complex for 1 h at 30°C. Microsomes were lysed in NP-40 lysis buffer in the presence of 50 mM iodoacetamide. Total microsome-associated material as well as immunoprecipitates obtained with the anti–CD3-ε antiserum were analyzed by SDS-PAGE (12.5%) performed as indicated under reducing and nonreducing conditions. (B) CD3-ε was translated alone and pairwise with one subunit of the TCR or with HLA-DRβ as indicated, for 1 h at 30°C. Microsomes were lysed in NP-40 lysis buffer in the presence of 50 mM iodoacetamide. Total microsome-associated material as well as immunoprecipitates obtained with the anti–CD3-ε antiserum were analyzed by SDS-PAGE (12.5%) performed under reducing and nonreducing conditions as indicated.
Mentions: The propensity of CD3-ε to form disulfide-linked oligomers was examined by translating CD3-ε either alone or together with the other subunits of the TCR. Total microsome-associated material as well as immunoprecipitates obtained from lysed microsomes were analyzed by SDS-PAGE performed under both reducing and nonreducing conditions (Fig. 7 A). Lysis in 0.5% NP-40 facilitates the visualization of CD3-ε as it leads to the disruption of all noncovalent interactions between the subunits of the TCR, but not those between CD3-ε, -γ, and -δ (6).

Bottom Line: The T cell receptor for antigen (TCR) is a multisubunit complex that consists of at least seven polypeptides: the clonotypic, disulfide-linked alpha/beta heterodimer that is noncovalently associated with the invariant polypeptides of the CD3 complex (CD3-gamma, -delta, -epsilon) and zeta, a disulfide-linked homodimer.A glycan-dependent interaction between CD3-epsilon and calnexin was mediated by CD3-gamma and concerned only monomeric CD3-epsilon complexed with CD3-gamma, but was dispensable for proper folding of CD3-epsilon.We suggest that in addition to its signaling function, CD3-epsilon serves as a monitor for proper subunit assembly of the TCR.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
The T cell receptor for antigen (TCR) is a multisubunit complex that consists of at least seven polypeptides: the clonotypic, disulfide-linked alpha/beta heterodimer that is noncovalently associated with the invariant polypeptides of the CD3 complex (CD3-gamma, -delta, -epsilon) and zeta, a disulfide-linked homodimer. We achieved the complete assembly of the human TCR in an in vitro transcription/translation system supplemented with dog pancreas microsomes by simultaneous translation of the messenger RNAs encoding the TCR-alpha, -beta and CD3-gamma, -delta, -epsilon, and -zeta subunits. CD3-epsilon, one of the subunits that initiates the assembly of the TCR in living cells, forms misfolded, disulfide-linked homooligomers when translated alone. However, co-translation of one of its first binding partners in the course of assembly, CD3-gamma or -delta, led to the expression of mainly monomeric and correctly folded epsilon subunits, the only form we could detect as part of a properly assembled TCR complex. In the absence of these subunits, the ER-resident chaperone calnexin interacted with oligomeric, i.e. misfolded, structures of CD3-epsilon in a glycan-independent manner. A glycan-dependent interaction between CD3-epsilon and calnexin was mediated by CD3-gamma and concerned only monomeric CD3-epsilon complexed with CD3-gamma, but was dispensable for proper folding of CD3-epsilon. We suggest that in addition to its signaling function, CD3-epsilon serves as a monitor for proper subunit assembly of the TCR.

Show MeSH
Related in: MedlinePlus