FIGURE 2.

Three of the four common DOA Alleles have altered function.

(A) Schematic of the DOA common alleles relative to the DOA*0101 gene. DOA contains the following domains as indicated: a signal sequence (SS), a MHCII alpha-like (alpha-like), an immunoglobulin-like (Ig), a transmembrane (TM), and a cytoplasmic tail (Ct). The other three common alleles have single missense mutations that result in single amino acid changes which are shown. Numbering indicates amino acid number starting after signal sequence cleavage and the amino acid encoded by the DOA*0101 gene is shown in the rectangle domains followed by an arrow showing amino acid changes for DOA*0102, DOA*0103, and DOA*0104 alleles. For DOA*0104 a frameshift mutation results in the addition of 25 random amino acids prior to a stop codon (see Fig. 4).

(B) HeLa.CIITA cells were transiently transfected with a vector encoding DOB*0101-AcGFP (the most common DOB allele) together with a control empty vector (EV-mRuby), or vectors encoding DOA*0101, DOA*0102, DOA*0103, or DOA*0104. Seventy-two hours later, the transiently transfected cells were stained as in Figure 1 prior to analysis by flow cytometry. Histogram overlays on left show representative data for MHCII and DM and on right for MHCII-CLIP and DO after gating as indicated for MHCII⁺ and DM⁺, respectively, in cells expressing both the DOA and DOB expression constructs (mRuby⁺ AcGFP⁺). Shaded histograms are levels obtained for unstained cells as a negative control. Colors used in histogram overlays for each DOA common allele and empty vector control (EV-mRuby) are shown on right. gMFI values for MHCII, DM, MHCII-CLIP, and DO are shown on each histogram for each corresponding combination, color-coded as on right. Data are representative of one of four to five independent experiments.

(C) Quantification of independent experiments shown in (B). Background gMFI for MHCII-CLIP and DO was eliminated by subtracting the gMFI value of the EV-mRuby/DOB*0101-AcGFP control from each sample. gMFI values measured for MHCII, DM, MHCII-CLIP, and DO were normalized to the value obtained after transfection of DOA*0101 (with DOB*0101) to allow for analysis across independent experiments (black bars).

(D) The relative function of each DOA allele was determined by calculating the ratio of MHCII-CLIP to DO from the values obtained in (C) to correct for different DO protein levels relative to the value obtained for DOA*0101 (with DOB*0101). Blue bars indicate alleles that have increased function relative to DOA*0101 (with DOB*0101; black bar). Data were combined from 4-5 individual experiments. Symbols and error bars represent the mean ± SD. Significance was calculated using an unpaired, two-tailed Student’s t test. *, p=0.0147; ****, p<0.0001.