Human ADA2 deficiency is characterized by the absence of an intracellular hypoglycosylated form of adenosine deaminase 2

Abstract

Pathogenic variants inADA2underlie human deficiency of adenosine deaminase 2 (DADA2) and lead to impaired ADA2 protein secretion and reduced deaminase activity. Yet, the mechanisms driving the disease on a cellular level are poorly understood. Here, we report thatin vitrodifferentiation of macrophages from DADA2 CD14+ monocytes partially restores protein expression of mutant ADA2. Healthy control macrophages express a high-molecular-weight (HMW) secreted form of ADA2 and a hypoglycosylated low-molecular-weight (LMW) intracellular protein. LMW-ADA2 is absent in DADA2 macrophages, suggesting differential glycosylation of mutant ADA2. In HEK293T cells transfected with pathogenic ADA2 variants, HMW-ADA2 accumulates intracellularly. Interactome analysis of the pathogenic variant p.R169Q revealed increased binding to proteins of the protein folding machinery compared with wild-type ADA2. Pathogenic ADA2 variants form intracellular protein aggregates and show increased degradation upon inhibition of the secretory pathway. In conclusion, we show aberrant N-glycosylation of ADA2 expressed in DADA2 macrophages for the first time and establish the absence of LMW-ADA2 as a characteristic of these ADA2-deficient cells.Graphical abstractFigure 1:SummaryThis study identifies a previously undescribed hypoglycosylated intracellular form of ADA2 in human monocyte-derived macrophages. Absence of this low-molecular-weight form is a key feature of human ADA2 deficiency (DADA2), suggesting a role for intracellular ADA2 in the pathogenesis of the disease.