ABSTRACT
The glycoprotein uromodulin (UMOD) is the most abundant protein in human urine and forms filamentous homopolymers that encapsulate and aggregate uropathogens, promoting pathogen clearance by urine excretion. Despite its critical role in the innate immune response against urinary tract infections, the structural basis and mechanism of UMOD polymerization remained unknown. Here, we present the cryo-EM structure of the UMOD filament core at 3.5 Å resolution, comprised of the bipartite zona pellucida (ZP) module in a helical arrangement with a rise of ~65 Å and a twist of ~180°. The immunoglobulin-like ZPN and ZPC subdomains of each monomer are separated by a long linker that interacts with the preceding ZPC and following ZPN subdomains by ß-sheet complementation. The unique filament architecture suggests an assembly mechanism in which subunit incorporation could be synchronized with proteolytic cleavage of the C-terminal pro-peptide that anchors assembly-incompetent UMOD precursors to the membrane.
Subject(s)
Uromodulin , Cryoelectron Microscopy , Humans , Models, Molecular , Polymerization , Protein Conformation, beta-Strand , Protein Domains , Uromodulin/chemistry , Uromodulin/metabolism , Uromodulin/ultrastructureABSTRACT
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, salt-dependent 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.
Subject(s)
Polymerization , Uromodulin/chemistry , Amino Acid Sequence , Animals , Blotting, Western , Crystallography, X-Ray , Disulfides/metabolism , Dogs , Extracellular Matrix Proteins/genetics , Fluorescent Antibody Technique , GPI-Linked Proteins/genetics , HEK293 Cells , Humans , Madin Darby Canine Kidney Cells , Maltose-Binding Proteins/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Mutation, Missense/genetics , Protein Multimerization , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Structural Homology, Protein , Uromodulin/ultrastructureABSTRACT
Uromodulin/Tamm-Horsfall protein is not immunostimulatory in the tubular lumen, but through unknown mechanisms it can activate dendritic cells and promote inflammation in the renal interstitium. Here, we noted that uromodulin isolated from human urine aggregates to large, irregular clumps with a crystal-like ultrastructure. These uromodulin nanoparticles activated isolated human monocytes to express costimulatory molecules and to secrete the mature proinflammatory cytokines, including IL-1ß. Full release of IL-1ß in response to uromodulin depended on priming of pro-IL-1ß expression by Toll-like receptors, TNF-α, or IL-1α. In addition, uromodulin-induced secretion of mature IL-1ß depended on the NLRP3 inflammasome, its linker molecule ASC, and pro-IL-1ß cleavage by caspase-1. Activation of NLRP3 required phagocytosis of uromodulin particles into lysosomes, cathepsin leakage, oxidative stress, and potassium efflux from the cell. Taken together, these data suggest that uromodulin is a NLRP3 agonist handled by antigen-presenting cells as an immunostimulatory nanoparticle. Thus, in the presence of tubular damage that exposes the renal interstitium, uromodulin becomes an endogenous danger signal. The inability of renal parenchymal cells to secrete IL-1ß may explain why uromodulin remains immunologically inert inside the luminal compartment of the urinary tract.
