EXPRESSION, PURIFICATION AND CHARACTERIZATION OF TWO RECOMBINANT FORMS OF HUMAN RENALASE, A NEW PROTEIN INVOLVED IN BLOOD PRESSURE AND CARDIOVASCULAR FUNCTION REGULATION

Cardiovascular complications represent one of the leading causes of morbidity and mortality in Chronic Kidney Disease (CKD). Even mild to moderate renal injuries, besides total functionality loss, are proven to correlate with increased cardiovascular risk. The hypothesis that the kidney may fill additional unknown assignments for its endocrine function, which may be very relevant for the development of cardiovascular diseases, prompted some authors (Xu et al., 2005) to sift the human genome to find novel proteins secreted by the kidney, finally leading to the discovery of renalase. The cDNA library of the Mammalian Gene Collection Project was screened for proteins with secretory features and low similarity with proteins of known function. A single gene, encoding a new human protein was found, named renalase, after its main secretion site. Based on the detection of renalase in plasma, on its putative monoamine oxidase activity on catecholamines in vitro, on the identification of a FAD-binding motif and a low degree of similarity with monoamine oxidases (MAOs), on the observation of in vivo effects on blood pressure and hemodynamics, and on the absence on plasma renalase in patients with severe renal failure, it has been proposed that renalase is a FAD-dependent enzyme secreted by the kidney, which metabolizes plasma catecholamines, resulting in the decrease of blood pressure and heart rate. For this reason, renalase is also considered as a new member of the MAO family of enzymes, and it has been also called MAO-C. The hypothesis of the fundamental role of renalase in the regulation of circulating catecholamine levels and its involvement in the control of blood pressure and cardiac function and, consequently, in the pathogenesis of cardiovascular diseases was supported by population genetic analysis and by in vivo studies (Zhao et al., 2007; Ghosh et al., 2006a; Ghosh et al., 2006b; Desir et al., 2008; Li et al., 2008; Desir et al., 2007). However, the data about the catalytic activity of renalase on catecholamines reported by Xu et al. (2005) were severely criticized (Boomsma and Tipton, 2007). Above that, the lack of a thorough biochemical characterization of renalase prompted us to start the study of this protein. Here we report the development and optimization of two different protocols for the expression of recombinant human renalase1 in E. coli, the predominant human isoform, and its purification in a holoprotein form (Pandini, Ciriello et al., 2010), and provide a detailed biochemical and structural characterization of this protein. The cDNA encoding renalase1 was purchased from imaGenes GmbH (Berlin, Germany), carried by an expression plasmid which drive the production of a fusion protein with an N-terminal His-tag in frame with the coding sequence of renalase1. Starting from this plasmid, we generated a second construct for the production of an N-tagged SUMO-renalase fusion protein, which is easily cleaved by a specific SUMO protease (Senp2 protease), resulting in a protein which carries only an additional serine residues at its N-terminus, followed by the native sequence of human renalase1 (Ser-renalase). Both recombinant forms were purified to homogeneity in amounts sufficient for their biochemical and structural characterization, and were found to be highly stable and monomeric. Considering the proposed MAO-like nature of renalase, its biochemical characterization has initially focused on the possibility that renalase could actually be ascribed to the MAO family of enzymes. We demonstrated for the first time that renalase contains non-covalently bound FAD, while the flavin cofactor in MAOs is bound in a covalent manner. Moreover, other biochemical differences emerged between renalase and monoamine oxidases, in terms of activity of the protein and reactivity of the flavin cofactor, suggesting that it is improper to classify it as a MAO-like enzyme. On the contrary, further experiments showed that renalase might rather be an NADPH-dependent flavoenzyme, with an enzymatic activity possibly similar to that of bacterial NAD(P)H-dependent flavin monooxygenases, such as p-hydroxybenzoate hydroxylase.