Abstract
Mouse T cells express the ecto-ADP-ribosyltransferase ARTC2.2, which can transfer the ADP-ribose group of extracellular nicotinamide adenine dinucleotide (NAD+) to arginine residues of various cell surface proteins thereby influencing their function. Several targets of ARTC2.2, such as P2X7, CD8a and CD25 have been identified, however a comprehensive mouse T cell surface ADP-ribosylome analysis is currently missing. Using the Af1521 macrodomain-based enrichment of ADP-ribosylated peptides and mass spectrometry, we identified 93 ADP-ribsoylated peptides corresponding to 67 distinct T cell proteins, including known targets such as CD8a and CD25 but also previously unknown targets such as CD73. We evaluated the impact of ADP-ribosylation on the capability of CD73 to generate adenosine from adenosine monophosphate. Our results show that extracellular NAD+ reduces the enzymatic activity of CD73 HEK cells co-transfected with CD73/ARTC2.2. Importantly, NAD+ significantly reduced CD73 activity on WT CD8 T cells compared to ARTC2ko CD8 T cells or WT CD8 T cells treated with an ARTC2.2-blocking nanobody. Our study provides a comprehensive list of T cell membrane proteins that serve as targets for ADP-ribosylation by ARTC2.2 and whose function may be therefore affected by ADP-ribosylation.
Highlights
Ecto-ADP-ribosyltransferases (ARTCs) are cell surface enzymes that utilize extracellular nicotinamide adenine dinuleotide (NAD+) to covalently attach the ADP-ribose group of NAD+ to arginine residues of various cell surface proteins under the release of nicotinamide [1, 2]
It has been shown that ADP-ribosylation of CD25 dampens interleukin 2 (IL-2) signalling by regulatory T cells, as the presence of NAD+ reduced STAT1 phosphorylation in response to IL-2 stimulation [12]
In this study we investigated the T cell ADP-ribosylome with a focus on ARTC2.2-mediated ADP-ribosylation of T cell surface proteins
Summary
Ecto-ADP-ribosyltransferases (ARTCs) are cell surface enzymes that utilize extracellular nicotinamide adenine dinuleotide (NAD+) to covalently attach the ADP-ribose group of NAD+ to arginine residues of various cell surface proteins under the release of nicotinamide [1, 2]. ARTC2.1 and ARTC2.2 are the ARTCs predominantly expressed by cells of the murine immune system [4]. ARTC2.1 is highly expressed on the cell surface. Mouse T Cell ADP-Ribosylome of innate immune cells such as macrophages and microglia [5] and to some extent on T cells [6]. ARTC2.2 is highly expressed on most T cell populations. It is worth noting that the ARTC2.1 encoding gene, Art2a, is inactivated by a premature stop codon in the C57BL/6 (B6) mouse strain, whereas other strains such as Balb/c carry an intact Art2a gene [7]. In B6 mice, ecto-ARTC activity in the immune system is limited to the T cell compartment
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