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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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DOα chain variant constructs. Circled letters indicate the predicted NH2-terminal amino acid of the mature α chain.
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Figure 2: DOα chain variant constructs. Circled letters indicate the predicted NH2-terminal amino acid of the mature α chain.

Mentions: We used PCR techniques (36, 37) to construct four versions of the Vα portion of DOα chain gene using either the Vα11.2 (AV11S2) or Vα13.1 (AV13S1) leader peptide and having a predicted NH2terminal amino acid of either aspartic acid (D) or glutamic acid (E). These are designated DOαL11terD, DOαL11terE, DOαL13terD, and DO(L13terE (identical to wild-type DOα (Fig. 2). These were cloned in pTZ18R fused in frame to the mouse Cα gene using a XhoI site at the 5′-end of the Vα segment and an introduced AccIII site at the 5′-end of Cα (36), such that the final complete α chain was flanked by EcoRI sites. To express these variant DOα constructs in the 22.3α− hybridoma, the EcoRI fragments were isolated and cloned into the EcoRI site of the murine retrovirus–based vector, LXSN (38). Using methods modified from Miller et al. (38), DOα RNAs were packaged into retroviruses in the GP&env AM12 (39) and PA-317 (40) cell lines, and were introduced into the 22.3α− hybridoma by infection. Infectants with stably integrated DOα genes were selected and maintained with culture medium containing 1–1.2 mg/ml G418 (Geneticin; GIBCO BRL).


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)

DOα chain variant constructs. Circled letters indicate the predicted NH2-terminal amino acid of the mature α chain.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196340&req=5

Figure 2: DOα chain variant constructs. Circled letters indicate the predicted NH2-terminal amino acid of the mature α chain.
Mentions: We used PCR techniques (36, 37) to construct four versions of the Vα portion of DOα chain gene using either the Vα11.2 (AV11S2) or Vα13.1 (AV13S1) leader peptide and having a predicted NH2terminal amino acid of either aspartic acid (D) or glutamic acid (E). These are designated DOαL11terD, DOαL11terE, DOαL13terD, and DO(L13terE (identical to wild-type DOα (Fig. 2). These were cloned in pTZ18R fused in frame to the mouse Cα gene using a XhoI site at the 5′-end of the Vα segment and an introduced AccIII site at the 5′-end of Cα (36), such that the final complete α chain was flanked by EcoRI sites. To express these variant DOα constructs in the 22.3α− hybridoma, the EcoRI fragments were isolated and cloned into the EcoRI site of the murine retrovirus–based vector, LXSN (38). Using methods modified from Miller et al. (38), DOα RNAs were packaged into retroviruses in the GP&env AM12 (39) and PA-317 (40) cell lines, and were introduced into the 22.3α− hybridoma by infection. Infectants with stably integrated DOα genes were selected and maintained with culture medium containing 1–1.2 mg/ml G418 (Geneticin; GIBCO BRL).

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.

Show MeSH