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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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Proper N glycosylation of in vitro translated TCR subunits.  In vitro transcripts of the indicated TCR subunits were translated for 1 h  at 30°C. Immunoprecipitates obtained with the appropriate antibodies  were either mock digested or digested with EndoH, and subsequently analyzed by SDS-PAGE (12.5%) performed under reducing conditions. The  lower band in the anti–CD3-ε immunoprecipitates may constitute a degradation product of CD3-ε.
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Figure 2: Proper N glycosylation of in vitro translated TCR subunits. In vitro transcripts of the indicated TCR subunits were translated for 1 h at 30°C. Immunoprecipitates obtained with the appropriate antibodies were either mock digested or digested with EndoH, and subsequently analyzed by SDS-PAGE (12.5%) performed under reducing conditions. The lower band in the anti–CD3-ε immunoprecipitates may constitute a degradation product of CD3-ε.

Mentions: In living cells, TCR-α, -β, CD3-γ and δ, but not CD3-ε and the ζ subunit are co-translationally N-glycosylated in the lumen of the ER. To confirm proper glycosylation in vitro, we translated the mRNAs of all TCR subunits individually in the presence of microsomes, followed by recovery of the translation products by immunoprecipitation using the appropriate antibodies. Immunoprecipitates were digested with EndoH or mock digested to remove N-linked glycans (Fig. 2). The number of glycans attached could be estimated for in vitro translated TCR-α (four glycans), -β, CD3-γ and -δ (two glycans), and CD3-ε and -ζ (no glycans), all in agreement with in vivo data (2–5, 25).


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

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

Proper N glycosylation of in vitro translated TCR subunits.  In vitro transcripts of the indicated TCR subunits were translated for 1 h  at 30°C. Immunoprecipitates obtained with the appropriate antibodies  were either mock digested or digested with EndoH, and subsequently analyzed by SDS-PAGE (12.5%) performed under reducing conditions. The  lower band in the anti–CD3-ε immunoprecipitates may constitute a degradation product of CD3-ε.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198996&req=5

Figure 2: Proper N glycosylation of in vitro translated TCR subunits. In vitro transcripts of the indicated TCR subunits were translated for 1 h at 30°C. Immunoprecipitates obtained with the appropriate antibodies were either mock digested or digested with EndoH, and subsequently analyzed by SDS-PAGE (12.5%) performed under reducing conditions. The lower band in the anti–CD3-ε immunoprecipitates may constitute a degradation product of CD3-ε.
Mentions: In living cells, TCR-α, -β, CD3-γ and δ, but not CD3-ε and the ζ subunit are co-translationally N-glycosylated in the lumen of the ER. To confirm proper glycosylation in vitro, we translated the mRNAs of all TCR subunits individually in the presence of microsomes, followed by recovery of the translation products by immunoprecipitation using the appropriate antibodies. Immunoprecipitates were digested with EndoH or mock digested to remove N-linked glycans (Fig. 2). The number of glycans attached could be estimated for in vitro translated TCR-α (four glycans), -β, CD3-γ and -δ (two glycans), and CD3-ε and -ζ (no glycans), all in agreement with in vivo data (2–5, 25).

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