Renalase Secreted by Human Kidney HEK293T Cells Lacks its N-Terminal Peptide: Implications for Putative Mechanisms of Renalase Action

Abstract

Background/Aims: Renalase is a recently discovered flavoprotein involved in regulation of blood pressure. Altered renalase levels have been found in blood of patients with end stage renal disease. The antihypertensive effect of circulating renalase is attributed to putative FAD-dependent monoamine oxidase activity demonstrated by some authors. Being synthesized as an intracellular flavoprotein renalase requires the presence of its N-terminal peptide for FAD accommodation. However, conventional routes of export of secretory proteins outside the cell usually include cleavage of their N-terminal peptide. The aim of this study was to investigate whether renalase is secreted by НЕK293T cells as a full length protein (via proposed nonconventional pathway) or its export is accompanied by the loss of its N-terminal peptide. Methods: We have expressed human recombinant renalase-1 in human kidney НЕK293T cells and analyzed this protein inside the cells and in the extracellular medium for the presence of the N-terminal peptide by using high resolution targeted MS/MS. Results: Intracellular renalase contained clearly detectable N-terminal peptide, which was absent in extracellular renalase. Conclusions: Lack of the N-terminal peptide, the structural precondition for FAD binding, suggests that extracellular (circulating) renalase acts in a FAD-independent manner and mechanisms of its action are not associated with FAD.

A Simplified Method for Obtaining cDNA of Low-Copy and Silent Eukaryotic Genes Using Human Renalase as an the Example

“…Данный подход клонирования генов мы использовали при клонировании гена реналазы-1 и -2 [21,22], а также при клонировании различных форм реналазы в плазмидные вектора pET-28 и pcDNA4 (Fedchenko et al, in preparation).…”

Extracellular renalase protects cells and organs by outside‐in signalling

“…They also found that human recombinant renalase-1 secreted by HEК293T cells also lacks the N-terminal signal peptide. These results suggest renalase's signal peptide is cleaved upon secretion and that extracellular (blood and urine) renalase acts in a FADindependent manner [24]. However, the possibility of additional proteolytic processing of extracellular renalase should be considered [25].…”

A new method for quantitative determination of renalase based on mass spectrometric determination of a proteotypic peptide labelled with stable isotopes

“…Renalase is a secretory protein which attracts much attention in the context of blood pressure regulation . It is secreted by the kidney and kidney‐derived cell cultures, and renalase deficiency frequently develops in patients with end‐stage renal disease (ESRD), patients with other chronic renal diseases and related experimental animal models, thus determining development of hypertension complications. There are many reports of altered levels of renalase in biological body fluids (plasma and urine) in these and other groups of patients (or animal models) .…”

“…Recently we successfully used mass spectrometry for qualitative detection of renalase in human urine 14,15 and cultivated HEK293T cells. 7 In the case of renalase detection in urine, ammonium sulfate precipitation provided a convenient approach for concentrating full-length renalase up to detectable limits and eliminating possible renalase-derived peptides. 14,15 In the study reported here, we developed a new method for quantitative determination of renalase based on mass spectrometric determination of a proteotypic peptide containing С-terminal…”