Endocrine Abstracts

Background: Mutations in the UMOD gene, which encodes the Tamm-Horsfall Glycoprotein (THP), cause FJHN, an autosomal dominant disorder characterised by gout and renal failure. THP is a 640 amino acid glycosylphosphatidylinositol-anchored protein, containing three Epidermal Growth Factor (EGF)-like domains, a cysteine-rich region and a Zona Pelllucida (ZP) domain. THP is translated into the Endoplasmic Reticulum (ER) lumen, glycosylated in the Golgi apparatus, trafficked to the plasma membrane and excised from the cell. We have investigated the effects of THP mutants associated with FJHN on these cellular processes.

Methods: WT and 6 THP mutants located in the first and third EGF-like domains, the cysteine-rich region and the ZP domain, were transiently transfected into HeLa cells and studied using immunofluorescence and Western Blot analysis over a 48 h period.

Results: After 48 h, >90% of WT THP was present as the fully glycosylated mature ~97 kDa form at the plasma membrane and in conditioned medium. In contrast, THP mutants of the first (Cys32Trp) and third (Cys126Arg, Asn128Ser) EGF-like domains, and the cysteine-rich region (Asp196Asn, Cys223Arg), were not detected at the plasma membrane or in conditioned medium, but were retained in the ER as the unglycosylated immature ~84 kDa form. However, the THP mutant of the ZP domain (Gly488Arg) was retained to a lesser extent in the ER and was found to be trafficked to the plasma membrane and detected in conditioned medium.

Conclusion: Thus, our results demonstrate that THP mutants due to FJHN are retained in the ER, and are associated with reduced maturation and trafficking to the plasma membrane and excision from the cell. This aggregation of THP in the ER will likely result in cell stress, thereby indicating a mechanism by which THP mutants may cause the phenotype of FJHN.