Human urinary renalase lacks the N-terminal signal peptide crucial for accommodation of its FAD cofactor

Fedchenko, V. I.; Buneeva, O. A.; Kopylov, Arthur T.; Veselovsky, A. V.; Zgoda, Victor G.; Медведев, А. Е.

doi:10.1016/j.ijbiomac.2015.04.023

Cited by 17 publications

(39 citation statements)

References 25 publications

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“…It overlaps with the minimal Rossman fold (residues 2-32) responsible for accommodation of the adenine nucleotide moiety of FAD [5,7,12]. The presence of non-covalently bound FAD was originally demonstrated by the Aliverti's group in purified recombinant renalase [7,12] and further confirmed by other laboratories [6,13,15].…”

Section: Introductionmentioning

confidence: 83%

“…After washing and destaining gel was dried under vacuum and rehydrated in 10 µL of trypsin 100 ng/µL in the supplied 1 mM hydrochloric acid for 5 min at 4 o C following addition of 20 µL of 75 mM TEAB. The reaction of tryptic digestion was performed overnight at 37 o C under stirring at 1200 rpm for 5 min every 45 min [15]. Peptides were extracted from gel pieces via three changes by 0.5% TFA.…”

Section: In-gel Digestion With Trypsinmentioning

confidence: 99%

“…Recently, using the full-length recombinant renalase as control of tryptic patterns, we found that human urinary renalase isolated by means of immunoaffinity chromatography lacked the N-terminal peptide, while in the recombinant protein this peptide was clearly detected [15]. As shown by molecular simulations performed using the renalase crystal structure obtained by the Aliverti's group [12], the absence of the N-terminal signal peptide excludes the possibility of FAD binding by the truncated protein [15].…”

Section: Introductionmentioning

confidence: 99%

“…As shown by molecular simulations performed using the renalase crystal structure obtained by the Aliverti's group [12], the absence of the N-terminal signal peptide excludes the possibility of FAD binding by the truncated protein [15].…”

Section: Introductionmentioning

confidence: 99%

“…Although possible formation of the truncated renalase during urine storage may be ruled out [15], cleavage of the N-terminal peptide may occur: (a) during secretion of intracellular renalase from the cell; (b) in blood circulation; (c) during urinary excretion of proteins. Studying adrenaline induced renalase secretion by human renal proximal tubular epithelial cells (HK2) Wang et al [22] detected this protein in the extracellular medium.…”

Section: Introductionmentioning

confidence: 99%

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

Fedchenko

Kopylov

Kozlova

et al. 2016

Kidney Blood Press Res

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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.

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