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Effects of complementarity determining region mutations on the affinity of an alpha/beta T cell receptor: measuring the energy associated with CD4/CD8 repertoire skewing.

Manning TC, Parke EA, Teyton L, Kranz DM - J. Exp. Med. (1999)

Bottom Line: We report that several mutants at positions lying within CDR3alpha and CDR2beta showed increased affinities for pMHC compared with the wild-type receptor.Two mutants (S27F and S51P), which each promote skewing toward a CD8(+) phenotype, exhibited significantly reduced affinity for pMHC-I, consistent with a quantitative-instructional model of CD4/CD8 lineage commitment.Together, the results (a) demonstrate that engineering higher affinity TCRs is feasible, and (b) provide TCR-pMHC energy values associated with CD4/CD8 repertoire skewing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.

ABSTRACT
It has been proposed that the generally low affinities of T cell receptors (TCRs) for their peptide-major histocompatibility complex (pMHC) ligands (Kd approximately 10(-4) to 10(-7) M) are the result of biological selection rather than an intrinsic affinity limitation imposed by the TCR framework. Using a soluble version of the 2C TCR, we have used complementarity determining region (CDR)-directed mutagenesis to investigate whether the affinity of this receptor for its allogeneic pMHC ligand can be improved upon. We report that several mutants at positions lying within CDR3alpha and CDR2beta showed increased affinities for pMHC compared with the wild-type receptor. Additionally, we have investigated whether Valpha mutations that have been implicated in the phenomenon of CD8(+) repertoire skewing achieve this skewing by means of generalized increases in affinity for MHC-I molecules. Two mutants (S27F and S51P), which each promote skewing toward a CD8(+) phenotype, exhibited significantly reduced affinity for pMHC-I, consistent with a quantitative-instructional model of CD4/CD8 lineage commitment. This model predicts that CD8 is downregulated on thymocytes that have TCR-ligand interactions above a minimal energy threshold. Together, the results (a) demonstrate that engineering higher affinity TCRs is feasible, and (b) provide TCR-pMHC energy values associated with CD4/CD8 repertoire skewing.

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Mass spectrometry of scTCR CDR mutants. Purified wild-type (WT) and mutant TCR proteins were dialyzed into 30% acetonitrile,  0.1% formic acid and subjected to mass spectrometry. Sensitivity of measurement was generally ±4 daltons (i.e., 0.01%). Observed mass differences  attributable to the amino acid substitutions are shown. Expected mass differences (in daltons) were as follows: S51P, +10; S27A, −16; S27F, +60.
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Figure 2: Mass spectrometry of scTCR CDR mutants. Purified wild-type (WT) and mutant TCR proteins were dialyzed into 30% acetonitrile, 0.1% formic acid and subjected to mass spectrometry. Sensitivity of measurement was generally ±4 daltons (i.e., 0.01%). Observed mass differences attributable to the amino acid substitutions are shown. Expected mass differences (in daltons) were as follows: S51P, +10; S27A, −16; S27F, +60.

Mentions: The Vα3.1/Vβ8.2 TCR from CTL clone 2C has been produced as a single-chain thioredoxin fusion protein in E. coli for use in binding and mutagenesis studies (7, 24, 25, 27). Various single, double, and triple mutants involving six residues (Glu56β, Thr55β, Ser76β, Ser102α, Ser27α, and Ser51α) were generated using a two-step PCR–based approach. Mutants were subjected to automated DNA sequencing to confirm mutations. TCRs were expressed in E. coli and purified by metal affinity and size exclusion chromatography to ≥95% purity. Electrospray mass spectrometry was used to confirm the expected mass difference relative to wild-type for several of the mutant TCRs (Fig. 2). The wild-type scTCR is evident as a single peak of mass 40,538 ± 4 daltons (0.01% accuracy), whereas the various mutant TCRs exhibit mass differences equivalent to those predicted based on their amino acid differences. This sensitive method is able to confirm the mass difference attributable to the loss of as little as a single oxygen atom in an ∼40,000-dalton protein, as demonstrated with the αS27A mutant (Fig. 2).


Effects of complementarity determining region mutations on the affinity of an alpha/beta T cell receptor: measuring the energy associated with CD4/CD8 repertoire skewing.

Manning TC, Parke EA, Teyton L, Kranz DM - J. Exp. Med. (1999)

Mass spectrometry of scTCR CDR mutants. Purified wild-type (WT) and mutant TCR proteins were dialyzed into 30% acetonitrile,  0.1% formic acid and subjected to mass spectrometry. Sensitivity of measurement was generally ±4 daltons (i.e., 0.01%). Observed mass differences  attributable to the amino acid substitutions are shown. Expected mass differences (in daltons) were as follows: S51P, +10; S27A, −16; S27F, +60.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192906&req=5

Figure 2: Mass spectrometry of scTCR CDR mutants. Purified wild-type (WT) and mutant TCR proteins were dialyzed into 30% acetonitrile, 0.1% formic acid and subjected to mass spectrometry. Sensitivity of measurement was generally ±4 daltons (i.e., 0.01%). Observed mass differences attributable to the amino acid substitutions are shown. Expected mass differences (in daltons) were as follows: S51P, +10; S27A, −16; S27F, +60.
Mentions: The Vα3.1/Vβ8.2 TCR from CTL clone 2C has been produced as a single-chain thioredoxin fusion protein in E. coli for use in binding and mutagenesis studies (7, 24, 25, 27). Various single, double, and triple mutants involving six residues (Glu56β, Thr55β, Ser76β, Ser102α, Ser27α, and Ser51α) were generated using a two-step PCR–based approach. Mutants were subjected to automated DNA sequencing to confirm mutations. TCRs were expressed in E. coli and purified by metal affinity and size exclusion chromatography to ≥95% purity. Electrospray mass spectrometry was used to confirm the expected mass difference relative to wild-type for several of the mutant TCRs (Fig. 2). The wild-type scTCR is evident as a single peak of mass 40,538 ± 4 daltons (0.01% accuracy), whereas the various mutant TCRs exhibit mass differences equivalent to those predicted based on their amino acid differences. This sensitive method is able to confirm the mass difference attributable to the loss of as little as a single oxygen atom in an ∼40,000-dalton protein, as demonstrated with the αS27A mutant (Fig. 2).

Bottom Line: We report that several mutants at positions lying within CDR3alpha and CDR2beta showed increased affinities for pMHC compared with the wild-type receptor.Two mutants (S27F and S51P), which each promote skewing toward a CD8(+) phenotype, exhibited significantly reduced affinity for pMHC-I, consistent with a quantitative-instructional model of CD4/CD8 lineage commitment.Together, the results (a) demonstrate that engineering higher affinity TCRs is feasible, and (b) provide TCR-pMHC energy values associated with CD4/CD8 repertoire skewing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.

ABSTRACT
It has been proposed that the generally low affinities of T cell receptors (TCRs) for their peptide-major histocompatibility complex (pMHC) ligands (Kd approximately 10(-4) to 10(-7) M) are the result of biological selection rather than an intrinsic affinity limitation imposed by the TCR framework. Using a soluble version of the 2C TCR, we have used complementarity determining region (CDR)-directed mutagenesis to investigate whether the affinity of this receptor for its allogeneic pMHC ligand can be improved upon. We report that several mutants at positions lying within CDR3alpha and CDR2beta showed increased affinities for pMHC compared with the wild-type receptor. Additionally, we have investigated whether Valpha mutations that have been implicated in the phenomenon of CD8(+) repertoire skewing achieve this skewing by means of generalized increases in affinity for MHC-I molecules. Two mutants (S27F and S51P), which each promote skewing toward a CD8(+) phenotype, exhibited significantly reduced affinity for pMHC-I, consistent with a quantitative-instructional model of CD4/CD8 lineage commitment. This model predicts that CD8 is downregulated on thymocytes that have TCR-ligand interactions above a minimal energy threshold. Together, the results (a) demonstrate that engineering higher affinity TCRs is feasible, and (b) provide TCR-pMHC energy values associated with CD4/CD8 repertoire skewing.

Show MeSH