The Adjacent Flanking Region Plays a Critical Role in Facilitating the Presentation of the Listeria monocytogenes Product lemA to H2 M3wt-Restricted, Peptide-Specific Murine CD8 Cells

• Published in: The Journal of immunology (1950) Vol. 163; no. 12; pp. 6741 - 6747
• Main Authors: Kurlander, Roger J, Chao, Elizabeth, Fields, Janet, Nataraj, Chandrasekaran
• Format: Journal Article
• Language: English
• Published: United States Am Assoc Immnol 15-12-1999
• Subjects: Amino Acid Sequence; Animals; Antigen Presentation - genetics; ATP-Binding Cassette Sub-Family B Member 2; ATP-Binding Cassette Transporters - immunology; ATP-Binding Cassette Transporters - metabolism; Bacterial Proteins - chemical synthesis; Bacterial Proteins - genetics; Bacterial Proteins - immunology; Bacterial Proteins - metabolism; Biological Transport - immunology; CD8-Positive T-Lymphocytes - immunology; CD8-Positive T-Lymphocytes - metabolism; Cell Line; Cytoplasm - immunology; Cytoplasm - metabolism; Endopeptidase K - physiology; Epitopes, T-Lymphocyte - metabolism; Fibroblasts - immunology; Fibroblasts - metabolism; H-2 Antigens - genetics; H-2 Antigens - immunology; Histocompatibility Antigens Class I - genetics; Histocompatibility Antigens Class I - immunology; Listeria monocytogenes - genetics; Listeria monocytogenes - immunology; Macrophages - immunology; Macrophages - metabolism; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutagenesis, Insertional; Peptide Fragments - genetics; Peptide Fragments - immunology; Peptide Fragments - metabolism; Transcription Factors - chemical synthesis; Transcription Factors - genetics; Transcription Factors - immunology; Transcription Factors - metabolism
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.163.12.6741

Mice infected with Listeria monocytogenes (LM) generate CD8 effectors specific for f-MIGWII, the amino terminus of the bacterial product lemA presented by the class Ib MHC molecule H2 M3wt. lemA has several distinctive properties: 1) it is readily presented as an exogenous Ag in the absence of bacterial infection; 2) it is processed by a TAP-independent pathway, which is sensitive to chloroquine, pepstatin, and brefeldin; and 3) the immunogenic portion of the molecule is extremely resistant to proteolytic degradation even by proteinase K. To assess the structural basis for these findings, we expressed a truncated variant (t-lemA) containing the amino-terminal hexapeptide and the subsequent 27 amino acids linked to a histidine tail in Escherichia coli, and purified the product by affinity chromatography. Purified t-lemA could be presented to f-MIGWII-specific effectors by macrophages and fibroblasts at 1-10 nM. Unlike f-MIGWII, which binds directly to H2 M3wt, t-lemA required processing by a chloroquine-, pepstatin-, and brefeldin-sensitive pathway. Brefeldin sensitivity often implies endogenous processing in the cytoplasm, but several lines of evidence suggest translocation to the cytoplasm and proteosomal degradation are not critical for t-lemA presentation. Unlike f-MIGWII, t-lemA was profoundly resistant to proteinase K, and, using 35S-labeled t-lemA, we could identify the region from position 1 to approximately 30 as the protease-resistant element. Thus, the hydrophobic peptide sequence following f-MIGWII can account for the unusual properties of lemA noted above. Analogous modification could be used to alter the properties of other peptide Ags presented by class I MHC products.