Limits...
Murine transporter associated with antigen presentation (TAP) preferences influence class I-restricted T cell responses.

Yellen-Shaw AJ, Laughlin CE, Metrione RM, Eisenlohr LC - J. Exp. Med. (1997)

Bottom Line: The transporter associated with antigen presentation (TAP) complex shuttles cytosolic peptides into the exocytic compartment for association with nascent major histocompatibility complex class I molecules.However, when T cell recognition was limited, either by blocking CD8 coreceptor interactions or by decreasing the amount of transport substrate synthesized, significant COOH-terminal effects were revealed.Therefore, TAP specificity operates in the intact cell, appears to reflect previously defined rules with regard to the influence of the COOH-terminal residue, and can strongly influence T cell responses.

View Article: PubMed Central - PubMed

Affiliation: Kimmel Cancer Institute, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

ABSTRACT
The transporter associated with antigen presentation (TAP) complex shuttles cytosolic peptides into the exocytic compartment for association with nascent major histocompatibility complex class I molecules. Biochemical studies of murine and human TAP have established that substrate length and COOH-terminal residue identity are strong determinants of transport efficiency. However, the existence of these specificities in the intact cell and their influences on T cell responses have not been demonstrated. We have devised a method for studying TAP- mediated transport in intact cells, using T cell activation as a readout. The approach makes use of a panel of recombinant vaccinia viruses expressing peptides containing the Kd-restricted nonamer influenza nucleoprotein residues 147-155. The COOH terminus of each construct was appended with a dipeptide composed of an internal threonine residue followed by a varying amino acid. Synthetic peptide versions of these 11-mers exhibit vastly different transport capabilities in streptolysin O-permeabilized cells, in accordance with the predicted influence of the COOH-terminal residues. Presentation of the endogenously expressed version of each construct requires TAP-mediated transport and cooexpression with a vac-encoded exocytic COOH-terminal dipeptidase, angiotensin converting enzyme, to allow liberation of the minimal epitope. Recognition by epitope-specific CTLs therefore signifies TAP-mediated transport of a complete 11-mer within the target cell. Under normal assay conditions no influences of the COOH-terminal residue were revealed. However, when T cell recognition was limited, either by blocking CD8 coreceptor interactions or by decreasing the amount of transport substrate synthesized, significant COOH-terminal effects were revealed. Under such conditions, those peptides that transported poorly in biochemical assays were less efficiently presented. Therefore, TAP specificity operates in the intact cell, appears to reflect previously defined rules with regard to the influence of the COOH-terminal residue, and can strongly influence T cell responses.

Show MeSH

Related in: MedlinePlus

Kinetics of purified ACE modification of synthetic substrates.  10 ng synthetic peptide was coincubated with ACE (0.001 U) for the indicated times. Samples were then subjected to reverse-phase HPLC. Reaction rates were monitored by the disappearance of the peak corresponding to starting substrate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199128&req=5

Figure 4: Kinetics of purified ACE modification of synthetic substrates. 10 ng synthetic peptide was coincubated with ACE (0.001 U) for the indicated times. Samples were then subjected to reverse-phase HPLC. Reaction rates were monitored by the disappearance of the peak corresponding to starting substrate.

Mentions: It was necessary to ensure that differences in ACE-mediated substrate modification would not confound our analysis, perhaps masking true transport differences. Purified ACE was incubated with a starting concentration of 10 nM of synthetic peptide. The reaction was stopped at various time points and the reaction products were analyzed by reverse-phase HPLC. The kinetics of ACE-mediated peptide modification were determined by following the rate of disappearance of the peak representing starting substrate (Fig. 4). Of the substrates tested, only ACE-mediated presentation of TE differed significantly from the other substrates, being modified more slowly. Therefore, this construct was excluded from further analysis. Consistent with this, note that this construct exhibited slightly but repeatably lower levels of presentation under standard assay conditions (Fig. 2).


Murine transporter associated with antigen presentation (TAP) preferences influence class I-restricted T cell responses.

Yellen-Shaw AJ, Laughlin CE, Metrione RM, Eisenlohr LC - J. Exp. Med. (1997)

Kinetics of purified ACE modification of synthetic substrates.  10 ng synthetic peptide was coincubated with ACE (0.001 U) for the indicated times. Samples were then subjected to reverse-phase HPLC. Reaction rates were monitored by the disappearance of the peak corresponding to starting substrate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199128&req=5

Figure 4: Kinetics of purified ACE modification of synthetic substrates. 10 ng synthetic peptide was coincubated with ACE (0.001 U) for the indicated times. Samples were then subjected to reverse-phase HPLC. Reaction rates were monitored by the disappearance of the peak corresponding to starting substrate.
Mentions: It was necessary to ensure that differences in ACE-mediated substrate modification would not confound our analysis, perhaps masking true transport differences. Purified ACE was incubated with a starting concentration of 10 nM of synthetic peptide. The reaction was stopped at various time points and the reaction products were analyzed by reverse-phase HPLC. The kinetics of ACE-mediated peptide modification were determined by following the rate of disappearance of the peak representing starting substrate (Fig. 4). Of the substrates tested, only ACE-mediated presentation of TE differed significantly from the other substrates, being modified more slowly. Therefore, this construct was excluded from further analysis. Consistent with this, note that this construct exhibited slightly but repeatably lower levels of presentation under standard assay conditions (Fig. 2).

Bottom Line: The transporter associated with antigen presentation (TAP) complex shuttles cytosolic peptides into the exocytic compartment for association with nascent major histocompatibility complex class I molecules.However, when T cell recognition was limited, either by blocking CD8 coreceptor interactions or by decreasing the amount of transport substrate synthesized, significant COOH-terminal effects were revealed.Therefore, TAP specificity operates in the intact cell, appears to reflect previously defined rules with regard to the influence of the COOH-terminal residue, and can strongly influence T cell responses.

View Article: PubMed Central - PubMed

Affiliation: Kimmel Cancer Institute, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

ABSTRACT
The transporter associated with antigen presentation (TAP) complex shuttles cytosolic peptides into the exocytic compartment for association with nascent major histocompatibility complex class I molecules. Biochemical studies of murine and human TAP have established that substrate length and COOH-terminal residue identity are strong determinants of transport efficiency. However, the existence of these specificities in the intact cell and their influences on T cell responses have not been demonstrated. We have devised a method for studying TAP- mediated transport in intact cells, using T cell activation as a readout. The approach makes use of a panel of recombinant vaccinia viruses expressing peptides containing the Kd-restricted nonamer influenza nucleoprotein residues 147-155. The COOH terminus of each construct was appended with a dipeptide composed of an internal threonine residue followed by a varying amino acid. Synthetic peptide versions of these 11-mers exhibit vastly different transport capabilities in streptolysin O-permeabilized cells, in accordance with the predicted influence of the COOH-terminal residues. Presentation of the endogenously expressed version of each construct requires TAP-mediated transport and cooexpression with a vac-encoded exocytic COOH-terminal dipeptidase, angiotensin converting enzyme, to allow liberation of the minimal epitope. Recognition by epitope-specific CTLs therefore signifies TAP-mediated transport of a complete 11-mer within the target cell. Under normal assay conditions no influences of the COOH-terminal residue were revealed. However, when T cell recognition was limited, either by blocking CD8 coreceptor interactions or by decreasing the amount of transport substrate synthesized, significant COOH-terminal effects were revealed. Under such conditions, those peptides that transported poorly in biochemical assays were less efficiently presented. Therefore, TAP specificity operates in the intact cell, appears to reflect previously defined rules with regard to the influence of the COOH-terminal residue, and can strongly influence T cell responses.

Show MeSH
Related in: MedlinePlus