Anales RANF

P.26 MONO-ADP-RIBOSYLATION OF CD73 – A NEW LEVEL OF PURI-NERGIC CONTROL J. Hesse 1 , M. K. Rosse 1 , B. Steckel 1 , B. Blank-Landeshammer 2 , A. Sickmann 2 , N. Sträter 3 , J. Schrader 1 1 . Heinrich Heine University Düsseldorf, Düsseldorf, Germany; 2 . Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany; 3 . Leipzig University, Leipzig, Germany. CD73-derived adenosine plays a major role in damage-induced tissue responses by modulating inflammation and tissue remodelling. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the release of ATP and NAD and upregulate the expression of the nucleotide-degrading purinergic ecto-enzyme cascade, including CD73. Extracellular NAD can either be degraded to adenosine by this cascade or serve as substrate for mono-ADP-ribosylation. In humans, this post-translational protein modification of arginine residues at the cell surface is mediated by ADP- ribosyltransferase 1 (ART1) and has been shown to regulate protein function of membrane proteins as well as soluble factors. To explore, whether CD73 activity is regulated by mono-ADP-ribosylation, human recombinant CD73 was studied in the presence of ART1 using etheno-labelled NAD as substrate. Multi-colour Western Blot analysis showed an ART1-mediated transfer of ADP-ribose onto CD73. Mass spectrometry of in vitro- ribosylated CD73 identified six ribosylation sites of which two are likely to inhibit the enzyme in model analysis. UPLC analysis of the adenosine- generating activity of in vitro -ribosylated CD73 revealed that activity is inhibited by about 60% in comparison to non-ribosylated CD73. Flow cytometric analysis of human peripheral blood immune cells identified a CD73+/ART1+ double-positive B cell population, in which ART1-mediated mono-ADP-ribosylation of CD73 might be of functional relevance. Our study is the first to identify human CD73 as target for ART1- mediated mono-ADP-ribosylation, which importantly can modulate its adenosine- generating activity. Therefore, CD73 activity on ART1-expressing cell types might be subject to an NAD-dependent regulation, implicating that high CD73 expression, e.g. after tissue damage, is not necessarily associated with enhanced adenosine generation and signalling.