Key result
L351Q ADA2 variant abrogates catalytic activity and impairs secretion, driving constitutive Type I interferon expression.
Why the study?
A unique c.1052T>A (p.Leu351Gln) variant in the catalytic domain of ADA2 was recently identified in patients with Deficiency of ADA2 (DADA2), but its functional impacts remained to be characterized.
Answers are grounded in this paper and cite their sources.
The L351Q variant in ADA2 impairs protein secretion and elevates type I interferon-responsive gene expression, providing mechanistic insight into the severe phenotype of DADA2 patients.
Background Adenosine deaminase 2 (ADA2) is a homodimeric, extracellular enzyme and putative growth factor that is produced by cells of the myeloid lineage and, catalytically, deaminates extracellular adenosine to inosine. Loss-of-(catalytic)-function variants in the ADA2 gene are associated with Deficiency of ADA2 (DADA2), an autosomal recessive disease associated with an unusually broad range of inflammatory manifestations including vasculitis, hematological defects and cytopenia. Previous work by our group led to the identification of ADA2 variants of novel association with DADA2, among which was a unique c.1052T>A (p.Leu351Gln; herein referred to as L351Q) variant located in the catalytic domain of the protein. Methods Mammalian (Flp-IN CHO) cells were engineered to stably express wild-type ADA2 and ADA2 protein variants, including the pathogenic L351Q variant identified in DADA2 patients. An enzyme assay and immunoblotting were used to assess ADA2 catalytic activity and secretion, respectively, and the outcome of experimentally induced inhibition of protein processing (Golgi transport and N-linked glycosylation) was assessed. Reverse transcription quantitative real-time PCR (RT-qPCR) was applied to determine the relative expression of Type I Interferon stimulated genes (ISGs), IFIT3 and IRF7. Results In addition to abrogating catalytic activity, the L351Q variant impaired secretion of L351Q ADA2 resulting in an intracellular accumulation of L351Q ADA2 protein that was not observed in cells expressing wild-type ADA2 or other ADA2 protein variants. Retention of L351Q ADA2 was not attributable to impaired glycosylation on neighboring asparagine residues and did not impact cell growth or integrity. Constitutive expression of Type I ISGs IFIT3 and IRF7 was observed in cells expressing L351Q ADA2. Conclusions The impaired secretion of L351Q ADA2 may be an important factor leading to the severe phenotype observed in patients with this variant further emphasizing the importance of assessing impacts beyond catalytic activity when evaluating genotype-phenotype relationships in DADA2.
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Bowers et al. (2022) studied Deficiency of ADA2 (DADA2). L351Q ADA2 variant vs. Wild-type ADA2 and other ADA2 protein variants was evaluated on ADA2 catalytic activity, secretion, and relative expression of Type I Interferon stimulated genes (IFIT3 and IRF7). The L351Q ADA2 variant abrogated catalytic activity, impaired secretion resulting in intracellular accumulation, and led to constitutive expression of Type I interferon-stimulated genes IFIT3 and IRF7.
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