title: Human urinary renalase lacks the N-terminal signal peptide crucial for accommodation of its FAD cofactor.
abstract: Renalase is a recently discovered secretory protein involved in the regulation of blood pressure. Cells synthesize all known isoforms of human renalase (1 and 2) as flavoproteins. Accommodation of FAD in the renalase protein requires the presence of its N-terminal peptide. However, in secretory proteins, such peptides are usually cleaved during their export from the cell. In the present study, we have isolated human renalase from urinary samples of healthy volunteers and human recombinant renalases 1 and 2 expressed in Escherichia coli cells. In these proteins, we investigated the presence of the renalase N-terminal peptide and the FAD cofactor and performed computer-aided molecular analysis of the renalase crystal structure to evaluate possible consequences of removal of the N-terminal peptide. In contrast to human recombinant renalase isoforms 1 and 2 containing non-covalently bound FAD and clearly detectable N-terminal peptide, renalase purified from human urine lacks both the N-terminal signal peptide and FAD. The computer-aided analysis indicates that the removal of this peptide results in inability of the truncated renalase to bind the FAD cofactor. Thus, our results indicate that human renalase secreted in urine lacks its N-terminal peptide, and therefore catalytic activities of urinary renalase reported in the literature cannot be attributed to FAD-dependent mechanisms. We suggest that FAD-dependent catalytic functions are intrinsic properties of intracellular renalases, whereas extracellular renalases act in FAD- and possibly catalytic-independent manner.
authors.affiliation: Department of Proteomic Research and Mass Spectrometry, Institute of Biomedical Chemistry, 10 Pogodinskaya Street, Moscow 119121, Russia.
authors.forename: Valerii I
authors.initials: VI
authors.lastname: Fedchenko
authors.affiliation: Department of Proteomic Research and Mass Spectrometry, Institute of Biomedical Chemistry, 10 Pogodinskaya Street, Moscow 119121, Russia.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Buneeva
authors.affiliation: Department of Proteomic Research and Mass Spectrometry, Institute of Biomedical Chemistry, 10 Pogodinskaya Street, Moscow 119121, Russia.
authors.forename: Arthur T
authors.initials: AT
authors.lastname: Kopylov
authors.affiliation: Department of Proteomic Research and Mass Spectrometry, Institute of Biomedical Chemistry, 10 Pogodinskaya Street, Moscow 119121, Russia.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Veselovsky
authors.affiliation: Department of Proteomic Research and Mass Spectrometry, Institute of Biomedical Chemistry, 10 Pogodinskaya Street, Moscow 119121, Russia.
authors.forename: Victor G
authors.initials: VG
authors.lastname: Zgoda
authors.affiliation: Department of Proteomic Research and Mass Spectrometry, Institute of Biomedical Chemistry, 10 Pogodinskaya Street, Moscow 119121, Russia. Electronic address: professor57@yandex.ru.
authors.forename: Alexei E
authors.initials: AE
authors.lastname: Medvedev
mesh_terms: D000595:Amino Acid Sequence; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008995:Monoamine Oxidase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D008995:Monoamine Oxidase; C521557:renalase
keywords: FAD cofactor; N-terminal signaling peptide; Renalase