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.
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.
Kurlander
R J
RJ
Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA. rkurlander@nih.gov
Chao
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E
Fields
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Nataraj
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D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation / Q000235:genetics; D001426:Bacterial Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001692:Biological Transport / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000276:immunology / Q000378:metabolism; D019286:Endopeptidase K / Q000502:physiology; D018984:Epitopes, T-Lymphocyte / Q000378:metabolism*; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism; D006183:H-2 Antigens / Q000235:genetics / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology*; D008089:Listeria monocytogenes / Q000235:genetics / Q000276:immunology*; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014157:Transcription Factors / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D018984:Epitopes, T-Lymphocyte; D006183:H-2 Antigens; C078177:H2-M3 antigen; D015395:Histocompatibility Antigens Class I; D010446:Peptide Fragments; C073119:TAP1 protein, human; C486171:Tap1 protein, mouse; D014157:Transcription Factors; C074165:lemA protein, bacterial; D019286:Endopeptidase K