Mono-ADP-ribosylation sites of human CD73 inhibit its adenosine-generating enzymatic activity

Abstract

CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD⁺ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD⁺ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD⁺ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD⁺ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity.

Keywords: Adenosine; CD296; Mono-ADP-ribosylation; NT5e; Post-translational modification.

Fig. 1

Ribosylation of CD73 by ARTC1. a Scheme of extracellular nucleotide metabolism. b–c Human recombinant CD73 was incubated with ARTC1 in the presence of 320 µM etheno-labelled NAD⁺ (eNAD⁺) for 16 h at 30 °C. Transfer of etheno label from eNAD⁺ on CD73 and ARTC1 was detected by multi-colour immunoblotting using etheno-adenosine (eADO)-, CD73-, and ARTC1-specific primary antibodies in combination with fluorochrome-labelled secondary antibodies. Representative blots are shown in b. In the top panel, an overlay of CD73, ARTC1, and eADO signals is displayed. Dashed lines indicate where the blot membrane was cut to allow separate antibody incubation (upper part, α-eADO with α-CD73; lower part, α-eADO with α-ARTC1). eADO signals co-localized with CD73 were quantified and normalized to CD73 signals as shown in c. Means ± SD (n = 4 independent experiments). d Human recombinant CD73 was ribosylated as described in (a) and analysed for enzymatic activity by HPLC. Generation of ADO was quantified after 5 min of incubation with 20 µM AMP. Means ± SD (n = 4 independent experiments). One-way ANOVA and Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01. HI heat-inactivated ARTC1

Fig. 2

Sites of ARTC1-mediated ribosylation in CD73. a Human recombinant CD73 was incubated with ARTC1 in the presence of 320 µM etheno-labelled NAD⁺ for 16 h at 30 °C. Ribosylated arginines in CD73 were identified by mass spectrometry (n = 3 independent ribosylation reactions). Only sites observed in all three analysed ribosylated CD73 samples are shown. ADP-ribosylation sites, negative logarithmic p-values, peptide spectrum matches (PSM), and the calculated normalized abundances are given. b Environment of the identified arginines in open and closed CD73 conformations