Renalase (RNLS) is a recently discovered protein involved in blood pressure regulation. It exists both as an intracellular catalytically active flavoprotein (EC 1.6.3.5 dihydro-NAD(P):oxygen oxidoreductase) and an extracellular protein that demonstrates various cell protecting effects. Using a twenty-membered peptide corresponding to the residues 220-239 of the renalase sequence (RP-220) and the HK-2 cell line Wang et al. identified a renalase-binding protein, which was considered as a receptor for extracellular renalase crucial for MAPK signaling (Wang et al., 2015) [1]. In this study we have investigated profiles of renalase binding proteins in HEK293 cells by using affinity based proteomic profiling with full-length recombinant human RNLS-1 and human RNLS-2 as affinity ligands followed by analysis of bound proteins by liquid chromatography-mass spectrometry. Both renalases (RNLS-1 and RNLS-2) contain the RP-220 sequence (residues 220-239) but differ in their C-terminal region (residues 293-342 and 293-325, respectively). Profiling of HEK293 proteins resulted in identification of two different sets of proteins specifically bound to RNLS-1 and RNLS-2, respectively. We thus demonstrate that the C-terminal region is crucial for specific binding of renalase to its targets and/or receptors.
Keywords: Affinity-based proteomic profiling; LC–MS; RP-220 peptide; Renalase; Renalase-binding protein.