Question:

Autosomal dominant polycystic kidney disease is the most common monogenetic lethal disorder in humans. Mutations in the PKD2 gene account for approximately 30 % of cases. PKD2 encodes the transient receptor channel polycystin-2 (TRPP2), a non-selective Ca²⁺-permeable cation channel. The physiological function of proteins is affected by a battery of specific enzyme-catalyzed modifications on their amino acid side chains. N-linked glycosylation, i.e. the attachment of a glycan to a nitrogen of arginine or asparagine side-chains, is the most common type of glycosidic bond. It has been described to modulate protein stability, function and interaction. So far, several studies have shed light on specific post-translational modifications of TRPP2. However, a comprehensive analysis of posttranslational glycosylation of TRPP2 is lacking.

Methods:

Here we characterize the N-linked glycosylation of TRPP2 using liquid chromatography tandem mass spectrometry. Endogenous TRPP2 was isolated from canine and mouse cell lines as well as mouse kidneys by immuno-precipitation.

Results:

The collected peptide data translated to a 62.6 % coverage of TRPP2. We identified five N-linked glycosylation sites in TRPP2. Mutation of these sites abolished N-linked glycosylation completely and significantly changed the biochemical properties of TRPP2. Protein stability of TRPP2 mutants was reduced by 80 %.

Conclusions:

These results may be a starting point for a more detailed analysis of the functional implications of post-translational TRPP2 modifications. Our data highlight impaired TRPP2 protein stability as a mechanism for polycystic disorders in humans.