Figure 2
Characterization of human HLA-DQ2.5-derived recombinant T-cell receptor ligand (RTL) molecules. (a) Size exclusion chromatography (superdex-75) of purified and refolded “empty” RTL800 and RTL801 (αC44S mutation), and gliadin-61-71 covalently tethered RTL802 and 803. Inset, Samples of purified RTLs were boiled for 5 min in Laemmli sample buffer±the reducing agent β-mercaptoethanol (β-ME), and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12%). Non-reduced RTLs (−β-ME) have a smaller apparent molecular weight than reduced RTLs (+β-ME), indicating the presence of a disulfide bond. First and last lanes show the molecular weight standards albumin (66 kDa), ovalbumin (45 D), carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (21.5 kDa). Note that the molecules with the αC44S mutation (RTL801 and 803) have dramatically reduced levels of intermolecular disulfide linked aggregates compared with RTLs 800 and 802 with the wild-type sequence. (b) DQ2.5-derived RTLs are recognized by DQ2.5-specific monoclonal antibody (mAb) 2.12.E11. Five μg DQ2-derived RTL800 and 802, DR4-derived RTL363 as a negative control,40 or full-length recombinant DQ2/CLIP as a positive control were blotted in duplicate onto nitrocellulose membranes. Membranes were blocked with 10% fetal calf serum in phosphate-buffered saline overnight and then incubated with anti-DQ mAbs SPV-L3 (undetermined epitope),41 2.12.E11 (DQB1*0201, 0202 and 0203-specific) 42 and SFR-20α5 (undetermined epitope) 43 for 1 h. Blots were washed 2 × and then secondary antibodies goat-anti-rat horseradish peroxidase (HRP) (SFR-20α5) and rabbit-anti-mouse HRP antibodies were used for the detection. Immunoglobulin G were used as the control. All three antibodies recognize full-length DQ2/CLIP, only mAb 2.12.E11 recognized DQ2-derived RTL800 and RTL802, and none of these antibodies could detect DR4-derived RTL363. Our data are consistent with SPV-L3 and SFR-20α5 recognizing the α2- or β2-domains of full-length DQ2 and mAb 2.12.E11 recognizing a unique epitope present in the DQ2.5-derived RTLs that minimally consists of β1-domain residues G45, E46, and F47.42 (c) Circular dichroism measurements were performed at 25 °C on an Aviv Model 215 CD spectrometer (Lakewood, NJ) using 0.1 mm cells, at 0.5 nm intervals from 260 to 180 nm. Concentration values for each protein solution were determined by amino-acid analysis. Buffer, 20 mM Tris, pH 8.5. Deconvolution and analysis of the secondary structure presented in Table 1 was performed using the variable selection method.50 Data are expressed as Delta-epsilon per mole per cm.
