ID: 182: Characterization of IL22RA2, a risk locus for multiple sclerosis

Abstract

IL22RA2 (IL-22 binding protein, IL-22BP) is a soluble high-affinity antagonist of the cytokine IL-22, and has emerged recently with genome-wide significance as a risk locus for multiple sclerosis (MS). We performed a single nucleotide polymorphism (SNP) screen to identify putative causative variants, and identified a non-synonymous (NS) SNP significantly associated with MS in a multi-center screen including over 16,000 MS patients and controls. In order to evaluate if this SNP affected IL22RA2 protein processing and secretion, a site-directed mutagenesis was carried out, and both constitutive CMV-promoter-based and Tet-Express inducible vectors expressing C-terminally FLAG-tagged wild-type and mutant proteins were generated. Vectors were transfected in HEK293 cells and the intracellular and secreted protein fractions were analyzed by western blot. This showed that this NS SNP modulates secretion levels of the protein, with lower levels observed for the mutant than for the wild-type protein. As part of this work we performed a characterization of the N-glycosylation status of the recombinant protein and demonstrate that it contains uniquely high-mannose chains (43 kD) which are converted into complex sugar chains in the secreted form augmending its Mr to 50 kD. We have also investigated transcription, secretion and N-glycosylation of IL22RA2 produced by monocyte-derived dendritic cells and have characterized in detail the intracellular and secreted forms of the natural protein. We will present both a detailed overview of the genetic study and of the characterization of recombinant and natural IL22RA2.