Disrupted N-linked glycosylation as a disease mechanism in deficiency of ADA2

Abstract

Deficiency of adenosine deaminase 2 is characterized by vasculitis, early-onset strokes, immunodeficiency, and bone marrow failure. We describe a novel pathogenic mutation affecting a consensus N-linked glycosylation sequence and illustrate the essential role of glycosylation in the biology of ADA2.

Figure 1.

Deficiency of ADA2 associated with a novel mutation that disrupts N-linked glycosylation. A) Photographs of erythematous papules and livedo reticularis. B) Non-contrast CT images of the brain demonstrating intracranial hemorrhage episodes occurring 9 days apart. C) Quantitation of plasma ADA2 activity using a spectrophotometric assay. D) Pedigree illustration of CECR1 variants in the patient and parents. NM, no mutation. *Paternal genotype is inferred. E) Chromatograms show patient’s complementary DNA (top) and genomic DNA (middle) sequences for the c.385A>C variant. Deletion break-point sequence is shown in the bottom panel. F) ELISA of ADA2 in the medium and G) western blot of intracellular ADA2 expression in 293T cells. Wedge indicates a gradient of plasmid concentration. H) Western blot of wild-type and mutant ADA2. I) ELISA of secreted wild-type ADA2 with or without tunicamycin (TM; 1 μM). J) western blot of intracellular ADA2 expression with or without TM (1 μM). K) Confocal microscopy of ADA2 and giantin staining. Arrow indicates the Golgi apparatus. L) Confocal microscopy of ADA2 and ERp72 staining. Images are representative of 3 independent experiments. *p < 0.05.

Figure 2.

N-linked glycosylation at three sites is required for ADA2 secretion. A) Western blot of ADA2 mutants with disrupted N-glycosylation sites. B) Confocal microscopy of ADA2 and ERp72 staining. Arrow indicates perinuclear foci consistent with Golgi apparatus. C) ELISA of ADA2 mutants. D) Enzymatic activity of wild-type and mutant ADA2 quantified using a spectrophotometric assay. ADA activity is coupled to consumption of the substrate NADH measured at OD340. E) Enzymatic activity of ADA2 mutants in the medium of transfected cells normalized to the amount of secreted protein. Data are representative of 3 independent experiments. *p < 0.05.