Fig 2: A single nucleotide polymorphism (SNP) screen of the IL22RA2 locus in the Bilbao dataset. SNPs, depicted with dots in different colors depending on r2 values with respect to the index SNP rs202573, are plotted as a function of their log-converted p-value (left Y axis) and their position on chromosome 6 according to hg19 assembly of the human genome (X axis). The recombination rate across the locus is provided on the right Y axis. The red line represents the significance threshold (p = 0.05). SNPs genotyped in the primary screening [3] are shown in italics. rs202573, which was genotyped both in the previous and in the present study, is underlined.

Fig 3: Prediction of the effect of the p.Leu16Pro amino acid change on the IL-22BP signal peptide structure and cleavage site of mature IL-22BP. (a) The signal peptide cleavage site indicated with green (wt, Leu16) or red (Pro16) arrows, was predicted using SignalP-3.0, Phobius and PsiPred software. The cleavage site for the canonical sequence is predicted to occur between positions 21 and 22 by the three software applications used. The p.Leu16Pro variant causes a shift in the predicted cleavage site to between position 20 and 21 according to Phobius but not SignalP 3.0 and PsiPred each of which predicted identical cleavage sites to the canonical sequence. All software coincided in predicting a decrease in the length of the H-region in the mutant form. (b) Representation of the composition, hydrophobicity and charge of the amino acids that comprise the signal peptide of IL-22BP. The three domain structures of the IL-22BP signal peptide are represented based on the overall results obtained in (a), and consist of the N-region, a hydrophilic positively charged N-terminal region; the H-region, a hydrophobic core region; and the C-region, a polar uncharged C-terminal region that is recognized by signal peptidase.

Fig 4: Linkage disequilibrium structure and haplotypes of IL22RA2. On the left, LD plot of the 15 SNPs analyzed in the Bilbao cohort. Blocks were calculated using the confidence interval algorithm implemented in Haploview [25,26]. Only samples genotyped for all SNPs were considered for the haplotype calculation. The numbers inside the squares indicate r2 values, and darker shades of gray represent higher degrees of linkage disequilibrium. On the right, all the haplotypes that were present in the Bilbao cohorts considering all SNPs in a single LD block are represented. The only haplotype containing the risk (C) allele of the non-synonymous SNP rs28385692 is boxed. SNPs with significant associations and rs28385692 are indicated on top of the right panel.

Fig 5: The Pro16 variant in the SP of the three IL-22BP isoforms is associated with decreased secretion levels compared to the Leu16 variant. (a) HEK293 cells were transfected with the indicated expression plasmids, 24 h later cells were lysed and the conditioned medium collected. Intracellular and secreted IL-22BP isoform protein levels were measured by ELISA (mean ± SEM; n = 3; p-values by unpaired t-test). (b) HEK293 cells were transfected with the indicated expression plasmids, 24 h later cells were lysed and the conditioned medium was subjected to acetone precipitation (AP). Both cell lysates (CL) and AP were resolved by SDS-PAGE under non-reducing conditions and immunoblotted against FLAG (Ponceau staining served as loading control). For AP, the immunoblot membrane was subjected to longer exposure times. (c) HEK293 cells were transiently transfected with the indicated expression vectors (EV denotes empty vector), 24 h later cells were lysed and RNA purified. Intracellular IL-22BP protein was immunoaffinity-purified with FLAG agaroses and detected by WB following FLAG purification and in cell lysates (CL) and pass through fraction (PT). GRP94 detection and Ponceau staining served as loading controls. Transfection efficiency was measured by IL22RA2 RT-qPCR relative to the housekeeping gene ACTB. Mean ± SEM of three technical replicates. Note that as previously observed [20], immunoreactive bands corresponding to intracellular IL-22BP isoforms appear as a series of 43 to 56 kDa bands due to differential N-glycosylation, with secreted IL-22BPi2 gaining ~8 kDa (56 vs. 48 kDa) due to complex N-glycosylation.