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The Adjacent Flanking Region Plays a Critical Role in Facilitating the Presentation of the Listeria monocytogenes Product lemA to H2 M3^wt-Restricted, Peptide-Specific Murine CD8 Cells

Roger J. Kurlander^1,*, Elizabeth Chao^*, Janet Fields^* and Chandrasekaran Nataraj

^* Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892; and Department of Medicine, Duke Medical Center, Durham, NC 27710

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 M3^wt. 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 M3^wt, 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 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.

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