Figure 1. MDCK cells as a model to study physiological uromodulin shedding.
(A) Schematic representation of human uromodulin domain structure containing a leader peptide (predicted to be cleaved at residue 23), three EGF-like domains, a central domain with 8 conserved cysteines (D8C), a bipartite Zona Pellucida (ZP) domain (ZP-N/ZP-C) and a glycosylphosphatidylinositol (GPI)-anchoring site (predicted at position 614). Internal (IHP) and External (EHP) Hydrophobic Patches (Jovine et al., 2004; Schaeffer et al., 2009), Consensus Cleavage Site (CCS) and seven N-glycosylation sites (Ψ) are also indicated. (B) Immunofluorescence analysis of non-permeabilised MDCK cells expressing uromodulin. Polymers formed by the protein are clearly detected on the cell surface. Scale bar, 50 µm. (C) Electron microscopy analysis of uromodulin polymers purified from the medium of MDCK cells. The arrows indicate the typical protrusions of uromodulin filaments spaced about 130 Å. Scale bar, 100 nm. (D) Representative Western blot analysis of N-deglycosylated uromodulin secreted by transfected MDCK cells or purified from urine. A single isoform is clearly seen in the urinary sample. An isoform with similar molecular weight is released by MDCK cells (white arrowhead), which also secrete a longer and more abundant one (black arrowhead). (E) Representative tandem mass-spectrometry (MS/MS) spectrum confirming the identity of the C-terminal peptide 572DTMNEKCKPTCSGTRF587 of the short uromodulin isoform released by MDCK cells and table of fragmented ions. The C-terminal residue F587 is identical to the one that we mapped in human urinary protein (Santambrogio et al., 2008).