A structured interdomain linker directs self-polymerization of human uromodulin

Abstract

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...

Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy

“…Although there is no structural data for ZP1 a structure of the ZP2 ZP-C domain is available. 80 It is possible that metal-binding surface residues on these proteins could participate in Zn 2+ binding along with those from ZP3. Crystal structures of the full length proteins will enable further investigation into potential zinc binding sites.…”

Urinary glycated uromodulin in diabetic kidney disease

“…For example, mutations of uromodulin have been shown to cause congenital hyperuricemia and cystic kidney disease [41]. These mutations result in misfolding of uromodulin, with misfolded proteins retained in the endoplasmic reticulum and not secreted in the urine.…”

“…Uromodulin is found in the urine as a high molecular weight polymer assembled into filaments, matrices, or gel-like structures [41]. Polymeric uromodulin associates with exosomes and can be pelleted by high speed centrifugation (200000 × g) [32].…”

“…Uromodulin proteins are present in the urine as high molecular weight polymers [41] and can be readily isolated by centrifugation. We examined whether urinary uromodulin and glcUMOD proteins were present in the supernatant and/or pellet fractions by centrifuging urine samples from two healthy control participants and two patients with DKD at 18000 × g. Uromodulin in the supernatant and pellet fractions were then immunoprecipitated with an anti-uromodulin antibody, and the immunoprecipitants were subjected to Western blotting with anti-AGE or anti-uromodulin antibodies.…”

Serum uromodulin is associated with impaired glucose metabolism

“…[3] In urine, uromodulin forms extracellular filaments and aggregations via self-polymerization which capture pathogenic bacteria. [4,5] This protection mechanism against urinary tract infection has been first reported in 1980. [6] More recently, it has also been hypothesized that uromodulin might act as kind of a guardian against kidney disease and hypertension.…”