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Influence of the NH2-terminal amino acid of the T cell receptor alpha chain on major histocompatibility complex (MHC) class II + peptide recognition.

Seibel JL, Wilson N, Kozono H, Marrack P, Kappler JW - J. Exp. Med. (1997)

Bottom Line: Although cross-linking of this TCR with an antibody to the TCR idiotype elicited vigorous T cell hybridoma activation, stimulation with its natural MHC + peptide ligand did not.The substitution also dramatically reduced the affinity of soluble alpha/beta-TCR heterodimers for soluble MHC + peptide molecules in a cell-free system, suggesting that it did not exert its effect simply by disrupting TCR interactions with accessory molecules on the hybridoma.These results demonstrate for the first time that amino acids which are not in the canonical TCR complementarity determining regions can be critical in determining how the TCR engages MHC + peptide.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, National Jewish Medical and Research Center, University of Colorado Health Sciences Center, Denver, Colorado 80206, USA.

ABSTRACT
The alpha/beta T cell receptor (TCR) recognizes peptide fragments bound in the groove of major histocompatibility complex (MHC) molecules. We modified the TCR alpha chain from a mouse T cell hybridoma and tested its ability to reconstitute TCR expression and function in an alpha chain-deficient variant of the hybridoma. The modified alpha chain differed from wild type only in its leader peptide and mature NH2-terminal amino acid. Reconstituted cell surface TCR complexes reacted normally with anti-TCR and anti-CD3 antibodies. Although cross-linking of this TCR with an antibody to the TCR idiotype elicited vigorous T cell hybridoma activation, stimulation with its natural MHC + peptide ligand did not. We demonstrated that this phenotype could be reproduced simply by substituting the glutamic acid (E) at the mature NH2 terminus of the wild type TCR alpha chain with aspartic acid (D). The substitution also dramatically reduced the affinity of soluble alpha/beta-TCR heterodimers for soluble MHC + peptide molecules in a cell-free system, suggesting that it did not exert its effect simply by disrupting TCR interactions with accessory molecules on the hybridoma. These results demonstrate for the first time that amino acids which are not in the canonical TCR complementarity determining regions can be critical in determining how the TCR engages MHC + peptide.

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Substitution of D for E at the DOα chain NH2 terminus  lowers the affinity of cell-free α/β-TCR for IAd-OVA. Various concentrations of IAd-OVA were injected in flow cells with immobilized DOα/β  TCRs bearing DOα chains with either D or E at the NH2 terminus as  described in the Materials and Methods. A flow cell with immobilized  free β chain from this receptor was used as a control for signal from protein in solution and buffer differences.
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Figure 7: Substitution of D for E at the DOα chain NH2 terminus lowers the affinity of cell-free α/β-TCR for IAd-OVA. Various concentrations of IAd-OVA were injected in flow cells with immobilized DOα/β TCRs bearing DOα chains with either D or E at the NH2 terminus as described in the Materials and Methods. A flow cell with immobilized free β chain from this receptor was used as a control for signal from protein in solution and buffer differences.

Mentions: Anti-Cβ mAb was used to immobilize DOα/β heterodimers containing either DOαterE or DOαterD chains in separate flow cells of a biosensor chip. A flow cell in which the free DOβ chain was immobilized was used as a negative control. Various concentrations of IAd-OVA were passed through the flow cells and the binding kinetics followed (Fig. 7). Obvious binding of IAd-OVA to a TCR containing DOαterE was seen at all class II concentrations. The interaction had a relatively slow on rate, ka = 1.60 × 103 M−1s−1, and a fast off rate, kd = 0.05s−1, resulting in a dissociation constant (KD) of 31 μM. These kinetic and thermodynamic constants are similar to those observed for other TCRs binding to MHC class II + antigen complexes (50). In contrast, IAd-OVA bound very poorly to a TCR containing DOαterD. Very weak specific binding detected only at the highest concentration of IAd-OVA. This suggested a dissociation constant >300 μM. The results showed that the NH2 terminal E to D substitution in DOα disrupted TCR binding to class II + peptide in the absence of accessory molecules. We concluded that N terminal E must play a direct role in the interaction of this TCR with IAd/OVA.


Influence of the NH2-terminal amino acid of the T cell receptor alpha chain on major histocompatibility complex (MHC) class II + peptide recognition.

Seibel JL, Wilson N, Kozono H, Marrack P, Kappler JW - J. Exp. Med. (1997)

Substitution of D for E at the DOα chain NH2 terminus  lowers the affinity of cell-free α/β-TCR for IAd-OVA. Various concentrations of IAd-OVA were injected in flow cells with immobilized DOα/β  TCRs bearing DOα chains with either D or E at the NH2 terminus as  described in the Materials and Methods. A flow cell with immobilized  free β chain from this receptor was used as a control for signal from protein in solution and buffer differences.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196340&req=5

Figure 7: Substitution of D for E at the DOα chain NH2 terminus lowers the affinity of cell-free α/β-TCR for IAd-OVA. Various concentrations of IAd-OVA were injected in flow cells with immobilized DOα/β TCRs bearing DOα chains with either D or E at the NH2 terminus as described in the Materials and Methods. A flow cell with immobilized free β chain from this receptor was used as a control for signal from protein in solution and buffer differences.
Mentions: Anti-Cβ mAb was used to immobilize DOα/β heterodimers containing either DOαterE or DOαterD chains in separate flow cells of a biosensor chip. A flow cell in which the free DOβ chain was immobilized was used as a negative control. Various concentrations of IAd-OVA were passed through the flow cells and the binding kinetics followed (Fig. 7). Obvious binding of IAd-OVA to a TCR containing DOαterE was seen at all class II concentrations. The interaction had a relatively slow on rate, ka = 1.60 × 103 M−1s−1, and a fast off rate, kd = 0.05s−1, resulting in a dissociation constant (KD) of 31 μM. These kinetic and thermodynamic constants are similar to those observed for other TCRs binding to MHC class II + antigen complexes (50). In contrast, IAd-OVA bound very poorly to a TCR containing DOαterD. Very weak specific binding detected only at the highest concentration of IAd-OVA. This suggested a dissociation constant >300 μM. The results showed that the NH2 terminal E to D substitution in DOα disrupted TCR binding to class II + peptide in the absence of accessory molecules. We concluded that N terminal E must play a direct role in the interaction of this TCR with IAd/OVA.

Bottom Line: Although cross-linking of this TCR with an antibody to the TCR idiotype elicited vigorous T cell hybridoma activation, stimulation with its natural MHC + peptide ligand did not.The substitution also dramatically reduced the affinity of soluble alpha/beta-TCR heterodimers for soluble MHC + peptide molecules in a cell-free system, suggesting that it did not exert its effect simply by disrupting TCR interactions with accessory molecules on the hybridoma.These results demonstrate for the first time that amino acids which are not in the canonical TCR complementarity determining regions can be critical in determining how the TCR engages MHC + peptide.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, National Jewish Medical and Research Center, University of Colorado Health Sciences Center, Denver, Colorado 80206, USA.

ABSTRACT
The alpha/beta T cell receptor (TCR) recognizes peptide fragments bound in the groove of major histocompatibility complex (MHC) molecules. We modified the TCR alpha chain from a mouse T cell hybridoma and tested its ability to reconstitute TCR expression and function in an alpha chain-deficient variant of the hybridoma. The modified alpha chain differed from wild type only in its leader peptide and mature NH2-terminal amino acid. Reconstituted cell surface TCR complexes reacted normally with anti-TCR and anti-CD3 antibodies. Although cross-linking of this TCR with an antibody to the TCR idiotype elicited vigorous T cell hybridoma activation, stimulation with its natural MHC + peptide ligand did not. We demonstrated that this phenotype could be reproduced simply by substituting the glutamic acid (E) at the mature NH2 terminus of the wild type TCR alpha chain with aspartic acid (D). The substitution also dramatically reduced the affinity of soluble alpha/beta-TCR heterodimers for soluble MHC + peptide molecules in a cell-free system, suggesting that it did not exert its effect simply by disrupting TCR interactions with accessory molecules on the hybridoma. These results demonstrate for the first time that amino acids which are not in the canonical TCR complementarity determining regions can be critical in determining how the TCR engages MHC + peptide.

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