Functional analysis by site-directed mutagenesis of the complex polymorphism in rat transporter associated with...

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• The transporter associated with antigen processing (TAP) is crucial for presenting antigens to the immune system.
• TAP facilitates the transport of peptides from the cytosol to the endoplasmic reticulum for loading onto MHC class I molecules.
• Differences in TAP2 alleles in rats affect peptide transport, influencing the repertoire of peptides presented by MHC class I molecules.

Purpose of the Study:

• To identify specific residues in rat TAP2 responsible for differential peptide transport between alleles.
• To investigate the role of these residues in the transport of peptides with C-terminal arginine.

Main Methods:

• Segmental exchanges between TAP2 alleles.
• Site-directed mutagenesis to alter specific amino acid residues.
• Analysis of peptide transport activity of modified TAP2 constructs.

Main Results:

• Two adjacent clusters of five amino acids in the membrane domain of TAP2 were identified as critical for differential transport of arginine-terminated peptides.
• Transferring these clusters via mutagenesis conferred partial arginine transport ability to the restrictive allele.
• Each cluster independently contributed to the transport of peptides with charged C termini.

Conclusions:

• The TAP2-A allele's permissiveness for charged C-terminal peptides likely evolved in at least two distinct steps.
• These findings elucidate the molecular basis for differential peptide transport by TAP and its evolutionary implications.