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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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Primary structure of human CD3-ε and its proposed intra-chain disulfide bonding pattern. *Cysteine residues in the extracellular domain. The transmembrane region is underlined in the sequence and  shaded in the schematic figure. A disulfide bond that stabilizes the Ig fold  involves cysteine residues 27 and 76 (57). Two remaining cysteines are located close to the membrane and may be available for the formation of interchain disulfide bonds
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Figure 5: Primary structure of human CD3-ε and its proposed intra-chain disulfide bonding pattern. *Cysteine residues in the extracellular domain. The transmembrane region is underlined in the sequence and shaded in the schematic figure. A disulfide bond that stabilizes the Ig fold involves cysteine residues 27 and 76 (57). Two remaining cysteines are located close to the membrane and may be available for the formation of interchain disulfide bonds

Mentions: The CD3-ε chain contains two cysteine residues presumably involved in stabilization of the Ig fold, and two more cysteines in the lumenal domain (2), one of which is located within a few residues of the lipid bilayer (Fig. 5). The occurrence of disulfide-linked ε homodimers has been reported in vivo (45, 46), but the monomer is the prevalent form found in living cells. We investigated the biochemical properties of CD3-ε translated in vitro under both reducing and oxidizing conditions. Immunoprecipitations were performed with the anti–CD3-ε antibody in the presence of 50 mM iodoacetamide to prevent the formation of disulfide bridges after lysis. As visualized by SDS-PAGE under nonreducing conditions, translation of CD3-ε performed under oxidizing conditions resulted in oligomeric structures that collapsed to the monomer when analyzed by SDS-PAGE under reducing conditions (Fig. 6 A). Moreover, the difference in mobility for the oxidized and reduced monomeric form of CD3-ε is clearly visible (Fig. 6 A, left). We noted that it is only the oxidized form of CD3-ε that coimmunoprecipitated with fully assembled TCR complexes. We do not know whether the more rapidly migrating, oxidized form of CD3-ε contains any remaining free sulfhydryl groups, but we consider this unlikely. Inclusion of DTT in the translation reaction abrogated oligomerization but led to a monomeric polypeptide with a slightly decreased mobility, as a consequence of alkylation by iodoacetamide upon lysis. We confirmed the homooligomeric nature of CD3-ε by two-dimensional gel electrophoresis (first dimension: isoelectric focusing under nonreducing conditions; second dimension: reducing SDS-PAGE; data not shown). We conclude that CD3-ε translated in vitro in the absence of related subunits tends to form mostly disulfide-linked oligomers, a process that appears to be redox dependent.


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

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

Primary structure of human CD3-ε and its proposed intra-chain disulfide bonding pattern. *Cysteine residues in the extracellular domain. The transmembrane region is underlined in the sequence and  shaded in the schematic figure. A disulfide bond that stabilizes the Ig fold  involves cysteine residues 27 and 76 (57). Two remaining cysteines are located close to the membrane and may be available for the formation of interchain disulfide bonds
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Primary structure of human CD3-ε and its proposed intra-chain disulfide bonding pattern. *Cysteine residues in the extracellular domain. The transmembrane region is underlined in the sequence and shaded in the schematic figure. A disulfide bond that stabilizes the Ig fold involves cysteine residues 27 and 76 (57). Two remaining cysteines are located close to the membrane and may be available for the formation of interchain disulfide bonds
Mentions: The CD3-ε chain contains two cysteine residues presumably involved in stabilization of the Ig fold, and two more cysteines in the lumenal domain (2), one of which is located within a few residues of the lipid bilayer (Fig. 5). The occurrence of disulfide-linked ε homodimers has been reported in vivo (45, 46), but the monomer is the prevalent form found in living cells. We investigated the biochemical properties of CD3-ε translated in vitro under both reducing and oxidizing conditions. Immunoprecipitations were performed with the anti–CD3-ε antibody in the presence of 50 mM iodoacetamide to prevent the formation of disulfide bridges after lysis. As visualized by SDS-PAGE under nonreducing conditions, translation of CD3-ε performed under oxidizing conditions resulted in oligomeric structures that collapsed to the monomer when analyzed by SDS-PAGE under reducing conditions (Fig. 6 A). Moreover, the difference in mobility for the oxidized and reduced monomeric form of CD3-ε is clearly visible (Fig. 6 A, left). We noted that it is only the oxidized form of CD3-ε that coimmunoprecipitated with fully assembled TCR complexes. We do not know whether the more rapidly migrating, oxidized form of CD3-ε contains any remaining free sulfhydryl groups, but we consider this unlikely. Inclusion of DTT in the translation reaction abrogated oligomerization but led to a monomeric polypeptide with a slightly decreased mobility, as a consequence of alkylation by iodoacetamide upon lysis. We confirmed the homooligomeric nature of CD3-ε by two-dimensional gel electrophoresis (first dimension: isoelectric focusing under nonreducing conditions; second dimension: reducing SDS-PAGE; data not shown). We conclude that CD3-ε translated in vitro in the absence of related subunits tends to form mostly disulfide-linked oligomers, a process that appears to be redox dependent.

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