Aberrant N-glycosylation in pathogenic variants of adenosine deaminase 2 underlying human ADA2 deficiency

Human deficiency of adenosine deaminase 2 (DADA2) is an autoinflammatory disease caused by pathogenic variants in ADA2 that lead to impaired deaminase activity. Recently, a lysosomal function of ADA2 has been proposed but an intracellular form of the protein has not yet been characterized. Here, we analyze protein expression of mutant ADA2 in human monocyte-derived macrophages from 10 DADA2 patients. We identify an intracellular low-molecular-weight (LMW) form of ADA2 that undergoes glycan trimming by -mannosidases and is absent in DADA2 macrophages. Subcellular fractionation and immunofluorescence microscopy demonstrate that LMW-ADA2 is localized in the lysosomes. By overexpression of 34 ADA2 variants in HEK293T and U-937 cells, we show that absence of LMW-ADA2 strongly correlates with reduced deaminase activity and predicts variant pathogenicity. In conclusion, we describe a previously unreported intracellular hypoglycosylated form of ADA2 and establish the absence of this LMW-ADA2 as a cellular characteristic of DADA2. Thereby, we introduce a protein correlate of the recently described lysosomal form of ADA2. SummaryEhlers et al. demonstrate that mutant ADA2 fails to undergo glycan processing beyond the ER leading to the absence of intracellular low-molecular-weight (LMW) ADA2 in DADA2 patients macrophages. LMW-ADA2 localizes to the lysosomes and can be generated from extracellular wild-type ADA2.