The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

Brunati, Martina; Perucca, Simone; Han, Ling; Cattaneo, Angela; Consolato, Francesco; Andolfo, Annapaola; Schaeffer, Céline; Olinger, Eric; Peng, Jianhao; Santambrogio, Sara; Perrier, Romain; Li, Shuo; Bokhove, Marcel; Bachi, Ángela; Hummler, Edith; Devuyst, Olivier; Wu, Qingyu; Jovine, Luca; Rampoldi, Luca

doi:10.7554/elife.08887

Cited by 100 publications

(103 citation statements)

References 61 publications

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“…Uromodulin has extremely high cysteine content and extensive disulfide bond formation, resulting in extremely slow transit through the ER. After being cleaved by a protease hepsin (46), uromodulin is released to the urine. UMOD mutations are clustered (94%) in exons 3 and 4 encoding for the N-terminal half of the protein, and ~50% of known UMOD mutations affect cysteine (13).…”

Section: Discussionmentioning

confidence: 99%

Elevated urinary CRELD2 is associated with endoplasmic reticulum stress–mediated kidney disease

Park¹,

Manson²,

Molina³

et al. 2017

JCI Insight

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Section: Discussionmentioning

confidence: 99%

Elevated urinary CRELD2 is associated with endoplasmic reticulum stress–mediated kidney disease

Park¹,

Manson²,

Molina³

et al. 2017

JCI Insight

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“…Furthermore, they have not met the inherent challenges of urine: wide ionic concentration and pH range, intra‐ and interindividual variability, artifacts caused by naturally occurring urinary pigments and proteins as well as protease effects all complicating their isolation and downstream analytical processes. Particularly, Tamm–Horsfall protein (THP; uromodulin), the most abundant and glycan‐rich glycoprotein in urine can naturally entrap a large portion of uEVs to form filamentous networks . Hence, THP represents a significant challenge as it distorts the spectrum of uEV classes recovered.…”

Section: Introductionmentioning

confidence: 99%

Management of Tamm–Horsfall Protein for Reliable Urinary Analytics

Barreiro

Musante

et al. 2019

Proteomics Clinical Apps

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PurposeUrinary extracellular vesicles (uEVs) are a novel source of biomarkers. However, urinary Tamm–Horsfall Protein (THP; uromodulin) interferes with all vesicle isolation attempts, precipitates with normal urinary proteins, thus, representing an unwanted “contaminant” in urinary assays. Thus, the aim is to develop a simple method to manage THP efficiently.Experimental designThe uEVs are isolated by hydrostatic filtration dialysis (HFD) and treated with a defined solution of urea to optimize release of uEVs from sample. Presence of uEVs is confirmed by transmission electron microscopy, Western blotting, and proteomic profiling in MS.ResultsUsing HFD with urea treatment for uEV isolation reduces sample complexity to a great extent. The novel simplified uEV isolation protocol allows comprehensive vesicle proteomics analysis and should be part of any urine analytics to release all sample constituents from THP trap.Conclusions and clinical relevanceThe method brings a quick and easy protocol for THP management during uEV isolation, providing major benefits for comprehensive sample analytics.

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“…Mass spectroscopy (MS) analysis of ZP-C disulfide linkages suggests that there are two types of ZP modules with different structures (13). Type II contains 10 conserved Cys (C 1-7,a,b,8 ) and both homopolymerizes (UMOD, GP2, and TECTA) and heteropolymerizes (ZP1, ZP2, and ZP4), whereas type I (ZP3) includes eight conserved Cys (C [1][2][3][4][5][6][7][8] ) and only heteropolymerizes with type II (7,13,14). However, MS studies of egg coat protein disulfides are contradictory (15), and type II disulfide linkages C 5 -C 6 , C 7 -C a , and C b -C 8 are compatible neither with the fold of ZP3 (3) nor with structures of the ZP-C domain of BG, whose ZP module contains 10 Cys (16,17).…”

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confidence: 99%

A structured interdomain linker directs self-polymerization of human uromodulin

Bokhove

Nishimura

Brunati³

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant human urinary protein, plays a key role in chronic kidney diseases and is a promising therapeutic target for hypertension. Via its bipartite zona pellucida module (ZP-N/ZP-C), UMOD forms extracellular filaments that regulate kidney electrolyte balance and innate immunity, as well as protect against renal stones. Moreover, saltdependent aggregation of UMOD filaments in the urine generates a soluble molecular net that captures uropathogenic bacteria and facilitates their clearance. Despite the functional importance of its homopolymers, no structural information is available on UMOD and how it self-assembles into filaments. Here, we report the crystal structures of polymerization regions of human UMOD and mouse ZP2, an essential sperm receptor protein that is structurally related to UMOD but forms heteropolymers. The structure of UMOD reveals that an extensive hydrophobic interface mediates ZP-N domain homodimerization. This arrangement is required for filament formation and is directed by an ordered ZP-N/ZP-C linker that is not observed in ZP2 but is conserved in the sequence of deafness/Crohn's disease-associated homopolymeric glycoproteins α-tectorin (TECTA) and glycoprotein 2 (GP2). Our data provide an example of how interdomain linker plasticity can modulate the function of structurally similar multidomain proteins. Moreover, the architecture of UMOD rationalizes numerous pathogenic mutations in both UMOD and TECTA genes.uromodulin | ZP2 | polymerization | zona pellucida domain | X-ray crystallography U romodulin (UMOD) is expressed in the thick ascending limb of Henle's loop as a GPI membrane-anchored precursor that consists of three EGF-like domains, a domain of unknown function (D8C), and a zona pellucida (ZP) module (1, 2) (Fig. 1A, Top). The latter, containing Ig-like domains ZP-N and ZP-C (3-5), is found in other medically important human glycoproteins linked to infertility (egg coat components ZP1-ZP4), nonsyndromic deafness [inner ear α-and β-tectorin (TECTA/B)], Crohn's disease [glycoprotein 2 (GP2)], and cancer [TGF-β coreceptors betaglycan (BG) and endoglin (ENG)] (6, 7). Upon processing by Ser protease hepsin (8) at a consensus cleavage site (CCS) C-terminal to the ZP module (9), UMOD sheds a C-terminal propeptide (CTP) that contains a polymerization-blocking external hydrophobic patch (EHP), exposing an internal hydrophobic patch (IHP). This event triggers homopolymerization into filaments that are excreted into the urine (4, 10), where UMOD performs a plethora of biological functions, including protection against urinary tract infections, prevention of kidney stones, and activation of innate immunity (1,2,11,12).Although UMOD activity is strictly linked to its supramolecular state (2), the mechanism of ZP module-dependent assembly remains unclear. Mass spectroscopy (MS) analysis of ZP-C disulfide linkages suggests that there are two types of ZP modules with different structures (13). Type II contains 10 conserved Cys (C 1-7,a,b,8 ) and bo...

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The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

Cited by 100 publications

References 61 publications

Elevated urinary CRELD2 is associated with endoplasmic reticulum stress–mediated kidney disease

Elevated urinary CRELD2 is associated with endoplasmic reticulum stress–mediated kidney disease

Management of Tamm–Horsfall Protein for Reliable Urinary Analytics

A structured interdomain linker directs self-polymerization of human uromodulin

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