Abstract
The CD1 family consists of five proteins that are related to the peptide-presenting MHC class I family. T cells can recognize the presentation of both foreign and self-derived lipids on four CD1 family members. The identities of the self-lipids capable of stimulating autoreactive T cell responses remain elusive or controversial. Here, we employed mass spectrometry to analyze the lipid content of highly purified CD1c and CD1d protein samples. We report the identification of 11 novel self-lipids presented by CD1c and nine by CD1d. Rigorous controls provide strong evidence that the identified lipids were specifically loaded into the lipid-binding site of the CD1 molecules. The diverse but distinct population of lipids identified from each CD1 family member implies each present a different subset of self-lipids, and the enrichment of particular motifs indicates that the lipids that are presented by CD1 family members could be predicted. Finally, our results imply the CD1 system surveys the endoplasmic reticulum, Golgi apparatus, and/or secretory compartments, in addition to its well characterized surveillance of the endocytic and lysosomal compartments.
Highlights
The CD1 family is related to the MHC class I family in sequence and structure
Using CD1c and CD1d protein samples with undetectable levels of impurities, we investigated the self-lipids that were loaded into these samples, which had trafficked through the endoplasmic reticulum (ER), Golgi apparatus, and secretory compartments prior to their purification
SDS-PAGE, blue native (BN)-PAGE, and MS assessed the purity of the final protein samples, which suggested they were of high purity and contained undetectable levels of impurities
Summary
Protein Production, Purification, and Analyses—The protein constructs have been described previously [36]. After the eluate was incubated with the glutathione-Sepharose beads for 3 h at 4 °C on a roller shaker, the beads and their associated GST-tagged HRV 3C protease were removed from the eluate through 0.22-m filtration After this first round of protein purification, the CD1c-2M and CD1d-2M protein samples, with their Fc tags removed, were further purified through size-exclusion chromatography. The fractioned elutes that contained homogenous protein, as determined by non-reducing SDS-PAGE, were further purified through ion-exchange chromatography to reduce lipid contaminants. All of the fractions containing homogenous protein, as determined by non-reducing SDSPAGE, were pooled into a single aliquot and further purified through size-exclusion chromatography and ion-exchange chromatography as described above. Using this approach, a CD1d-2M-His sample was produced. The post-column splitter diverted ϳ10% of the LC flow to the ESI source of the mass spectrometer
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