This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.In the United States, a small percentage of persons infected with the spirochete Borrelia burgdorferi (Bb) develop a chronic arthritis that is refractory to antibiotic treatment. This condition can last for months to years despite apparent eradication of the spirochete. Antibiotic treatment refractory Lyme arthritis is associated with the MHC class II alleles HLA-DR*0401 and HLA-DR*0101, which suggests that the host response to Bb is important in disease progression. The goal of this project is to determine which Bb protein-derived peptides are presented by the disease susceptibility MHC class II alleles. We have established HLA-DR*0401 and *0101 homozygous cell lines from Lyme arthritis patients for use as antigen-presenting cells in cell culture. These cells will be incubated with recombinant Bb proteins or Bb whole cell sonicates. HLA-DR-peptide conjugates are immunopurified using anti-HLA-DR antibodies. Peptides are acid eluted, separated by centrifugal ultrafiltration, and purified by C18 solid-phase exraction. Purified peptides are identified by LC-MS/MS analysis followed by database searching.As a test of our methods, have purified peptides from HLA-DR*0401 cells incubated in media alone. LC-MS/MS analysis identified numerous self-derived peptides as well as some serum-derived peptides. These results indicate that our methods are working well and we have begun to study Bb proteins. We have expressed recombinant Bb outer surface protein A (OspA) which is labeled with 13C6-lysine. Carbon-13 was chosen as a label since it has a minimal effect on liquid chromatographic retention time. Lysine was selected because the OspA sequence contains numerous lysine residues well distributed throughout the protein. Each incorporated lysine residue adds exactly 6 Daltons to the mass of the peptide. When labeled and unlabeled OspA-MBP are mixed together and added to antigen-presenting cells, the processed peptides can be readily identified by mass spectrometry as pairs separated by intervals of exactly 6 Da. These pairs are then subjected to sequence analysis during automated LCMS data acquisition. We have now progressed to the analysis of patient-derived samples.
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