Renalase may be cleaved in blood. Are blood chymotrypsin-like enzymes involved?

Renalase (RNLS) is an intriguing protein, which plays different roles inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase (EC 1.6.3.5), while extracellular RNLS lacks its N-terminal peptide, FAD cofactor, and exhibits various protective effects in a non-catalytic manner. Numerous ELISA-based studies revealed altered RNLS levels in blood of patients with various chronic renal diseases (CRD) and cardiovascular diseases (CVD), including hypertension. However, our mass spectrometry data (Medvedev et al., Int. J. Cardiol., 2020, 307: 179) indicate the lack of an internal fragment located in the central part of RNLS. This means that plasma/serum RNLS is not an intact protein secreted into extracellular space. Our hypothesis is that reaching systemic circulation RNLS undergoes proteolytic cleavage by plasma proteases. In order to verify this hypothesis we incubated purified human recombinant RNLS with plasma samples from healthy volunteers and evaluated the remaining full-length RNLS by Western blot with polyclonal antibodies. These experiments revealed a significant decrease of the full-length RNLS. In an attempt to find putative enzymes responsible for RNLS cleavage in human blood, we have analyzed possible involvement of proteases in the cleavage of the RNLS amino acid sequence. The bioinformatics analysis performed by means of PeptideCutter and Procleave programs revealed several candidates that would be able to cleave RNLS in humans as well as blood proteases unable to cleave RNLS (e.g. thrombin). Direct experiments with purified enzymes have shown that in addition to trypsin, the classical cutting protease used in proteomics, only chymotrypsin effectively cleaved RNLS, while thrombin was ineffective. Since chymotrypsin is a protease normally acting in the small intestine we hypothesize that related chymotrypsin like enzymes secreted into blood can perform initial cleavage of blood RNLS with subsequent formation of RNLS-derived peptides possessing various biological effects. An understanding of the fact that blood RNLS can be effectively cleaved by some plasma proteases would help to asses actual levels of this potential biomarker in norm and disease.