Page 45 - The autoimmune hypothesis of narcolepsy and its unexplored clinical features M.S. Schinkelshoek
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Peptides
All peptides used were produced at the Peptide Synthesis Facility of the Department of Immunohematology and Blood Transfusions of the Leiden University Medical Center. Two Hcrt peptides, Hcrt56-68 and Hcrt87-99, and one 2009 H1N1 influenza A hemagglutinin (H1N1-HA) peptide, H1N1-HA275-287, were selected based on sequence similarity between these peptides and those described in the aforementioned retracted article (De la Herran-Arita et al., 2013, De la Herran- Aritaetal.,2014).AllpeptidesequencescanbefoundinSupplementaryTable3.1. For follow-up experiments, we used processed Hcrt peptides: two truncated peptides lacking the first 4 residues (including histidines on position 59 and 90 of Hcrt56-68 and Hcrt87-99, respectively), thereby increasing homology between H1N1-HA275-287 and the Hcrt peptides. Additionally, the histidine residue on position 59 of the first Hcrt peptide (Hcrt56-68) was replaced by an oxo-histidine or alanine residue rendering the two peptides more homologous to H1N1-HA275-287. Soluble complexes of HLA-DQ6 containing the peptides H1N1-HA275-287, Hcrt56- 68 and Hcrt87-99 were produced essentially as described previously for HLA-DQ2 (Henderson et al., 2007, Petersen et al., 2014). Briefly, the αβ-heterodimer of the HLA-DQ6 extracellular domain was expressed in Hi5 insect cells, with each peptide linked to the N-terminus of the HLA-DQ6 β-chain. The C-termini of the constructs contained an enterokinase cleavable Fos/Jun zippers, and, at the C terminus of the β-chain, a BirA biotinylation sequence followed by a His10-Tag. The complexes were purified via diafiltration, metal affinity, size exclusion and ion exchange chromatography. For crystallisation experiments the Fos/Jun zippers were removed by enterokinase cleavage and ion exchange chromatography.
Crystallisation, data collection, structure determination and refinement
Peptide-HLA-DQ6 complexes were crystallised at 20°C via the hanging drop vapor diffusion method using equal volumes of mother liquor and protein solution at 10 mg/ml in a buffer containing 10mM Tris (pH 8) and 150 mM NaCl. HLA-DQ6-Hcrt56-68 and HLA-DQ6-Hcrt87-99 were crystallised with mother liquor containing 16-20% PEG4000, 0.1M NaOAc pH4.5-5.0, and HLA-DQ6- HA275-287 was crystallised with 23% PEG4000, 0.2M NaI, 0.1M HEPES pH 7. Prior to data collection the crystals were cryoprotected in mother liquor supplemented with 20% glycerol, or 20% ethylene glycol in the case of HLA-DQ6- Hcrt87-99, and flash frozen in liquid N2. X-ray diffraction data was
H1N1 reactivity in CD4+ T cells
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