Figure 4.

ADA2 protein expression, enzyme activity and macrophage responses in cell line models of DADA2. (A, B) ADA2-KO THP-1 cells were transduced with a lentivirus encoding wild-type ADA2 (WT-ADA2), or with ADA2 p.G47R or p.R169Q mutants and protein expression assessed; results of blot testing shown in 4A and cumulative protein quantification in 4B. This resulted in detectable ADA2 protein expression for EFS-WT ADA2 and EFS-G47R ADA2, EFS-R169Q ADA2 vectors. (C) There was restoration of ADA2 enzyme activity in the supernatants of cells transduced with EFS-WT ADA2, compared to no recovery of ADA2 activity when cells were treated with EFS-GFP; or when the EFS-G47R ADA2, EFS-R169Q ADA2 vectors were used. (D) EFS-ADA2-GFP transduction of these cells to express wild type ADA2 also reduced the levels of TNFα released in culture supernatants compared to no change observed in EFS-GFP or EFS-G47R ADA2, EFS-R169Q ADA2 treated cells. Blots were performed in cell lysates. TNFα, tumour necrosis factor-α; IL, interleukin; EFS, the elongation factor 1α short; GFP, green fluorescent protein; HUVEC, human umbilical vein endothelial cells; UT, untransduced: ADA2, adenosine deaminase 2; KO, knock out; WT, wild type.