Urinary uromodulin carries an intact ZP domain generated by a conserved C-terminal proteolytic cleavage Sara Santambrogio a,1 , Angela Cattaneo b,1 , Ilenia Bernascone a , Thomas Schwend c , Luca Jovine c , Angela Bachi b , Luca Rampoldi a, * a Dulbecco Telethon Institute, Molecular Genetics of Renal Disorders, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy b Functional Proteomics, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy c Karolinska Institutet, Department of Biosciences and Nutrition, Hälsovägen 7, S-141 57 Huddinge, Sweden article info Article history: Received 14 March 2008 Available online 28 March 2008 Keywords: Uromodulin Zona pellucida domain Mass Spectrometry abstract Uromodulin (or Tamm–Horsfall protein) is the most abundant protein in human urine under physiolog- ical conditions. Little is known about the molecular mechanism of uromodulin secretion. By extensive Mass Spectrometry analyses we mapped the C-termini of human and murine urinary proteins demon- strating that urinary uromodulin is generated by a conserved C-terminal proteolytic cleavage and retains its entire ZP domain. Ó 2008 Elsevier Inc. All rights reserved. Uromodulin (or Tamm–Horsfall protein) is the most abundant protein in human urine under physiological conditions [1]. It is exclusively expressed by epithelial cells of the thick ascending limb of Henle’s loop (TAL) and of the distal convoluted tubule (DCT) where it is shed from the apical plasma membrane into the tubule lumen. Uromodulin is found in urine as a high molecular weight polymer that can be dissociated into monomers of about 95 kDa. Despite being extensively studied during the past 50 years the biological function of uromodulin is still not fully understood. Studies on umod knock-out mice demonstrated that uromodulin has a protective role against urinary tract infections and urothelial damage induced by calcium oxalate crystals [2,3]. Moreover, uro- modulin has been recently suggested to play a dual role in renal immune response by acting both as an adjuvant and an auto-anti- gen itself [4]. Due to its ability to assemble into a meshwork of fi- bres forming a gel-like structure it has been proposed to have also a role in water/salt balance in the TAL and DCT [5]. Research on uromodulin has received a considerable boost since mutations in its gene UMOD have been identified in Medullary Cystic Kidney Disease type 2 (MCKD2) and Familial Juvenile Hyperuricemic Nephropathy (FJHN) patients [6]. MCKD2/FJHN are autosomal dominant renal disorders characterised by alteration of urine con- centrating ability, tubulo-interstitial fibrosis, hyperuricemia and gout, medullary cysts, and progressive kidney failure frequently leading to end-stage renal disease. Several studies link MCKD2/ FJHN pathogenesis to defective intracellular trafficking and secre- tion of mutant uromodulin [7–9]. Uromodulin protein sequence contains a N-terminal signal peptide, three epidermal growth factor (EGF)-like domains, a zona pellucida (ZP) domain and a glycosyl phosphatidylinositol (GPI) anchor attachment site. The ZP domain is a conserved module of about 260 amino acids present in many secreted or extracellu- larly exposed eukaryotic proteins including zona pellucida sperm receptors ZP1, ZP2, and ZP3 and tectorial membrane components alpha and beta tectorins [10]. The ZP domain has been shown to be a ‘‘polymerisation module” that is responsible for the assembly of ZP domain proteins into filaments or matrices [11]. Single transmembrane domains also play a crucial role for assembly of ZP domain proteins and are missing in the secreted mature proteins. Little is known about the molecular mechanism of uromodulin release from the plasma membrane. Previous findings suggested that a proteolytic cleavage at the juxtamembrane-ectodomain is responsible for uromodulin urinary excretion [12]. To gain new insights into the mechanism of uromodulin shedding from the plasma membrane we carried out MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time Of Flight) MS (Mass Spectro- metry) and nanoLC (Liquid Chromatography)–MS/MS analyses on human and murine urinary proteins. Our data demonstrate that urinary uromodulin is generated by a conserved C-terminal proteolytic cleavage such that it retains the entire ZP polymerisa- tion domain. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.03.099 * Corresponding author. Fax: +39 0226434767. E-mail address: rampoldi.luca@hsr.it (L. Rampoldi). 1 These authors contributed equally to the work. Biochemical and Biophysical Research Communications 370 (2008) 410–413 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc