N-glycosylation determines the abundance of the transient receptor potential channel TRPP2.
Glycosylation plays a critical role in the biogenesis and function of membrane proteins. Transient receptor potential channel TRPP2 is a nonselective cation channel that is mutated in autosomal dominant polycystic kidney disease. TRPP2 has been shown to be heavily N-glycosylated, but the glycosylation sites and the biological role of N-linked glycosylation have not been investigated. Here we show, using a combination of mass spectrometry and biochemical approaches, that native TRPP2 is glycosylated at five asparagines in the first extracellular loop. Glycosylation is required for the efficient biogenesis of TRPP2 because mutations of the glycosylated asparagines result in strongly decreased protein expression of the ion channel. Wild-type and N-glycosylation-deficient TRPP2 is degraded in lysosomes, as shown by increased TRPP2 protein levels upon chemical inhibition of lysosomal degradation. In addition, using pharmacological and genetic approaches, we demonstrate that glucosidase II (GII) mediates glycan trimming of TRPP2. The non-catalytic β subunit of glucosidase II (GIIβ) is encoded by PRKCSH, one of the genes causing autosomal dominant polycystic liver disease (ADPLD). The impaired GIIβ-dependent glucose trimming of TRPP2 glycosylation in ADPLD may explain the decreased TRPP2 protein expression in Prkcsh(-/-) mice and the genetic interaction observed between TRPP2 and PRKCSH in ADPLD. These results highlight the biological importance of N-linked glycosylation and GII-mediated glycan trimming in the control of biogenesis and stability of TRPP2.
From the Renal Division, Department of Medicine, University Medical Center Freiburg, Hugstetter Straβe 55, 79106 Freiburg, Germany, the Spemann Graduate School of Biology and Medicine (SGBM) and Faculty of Biology, Albert-Ludwigs-University Freiburg, 79106 Freiburg, Germany, and alexis.hofherr@uniklinik-freiburg.de.
Alexis
A
Hofherr
From the Renal Division, Department of Medicine, University Medical Center Freiburg, Hugstetter Straβe 55, 79106 Freiburg, Germany.
Claudius
C
Wagner
the Departments of Medicine and Genetics, Yale University School of Medicine, New Haven, Connecticut 06520.
Sorin
S
Fedeles
the Departments of Medicine and Genetics, Yale University School of Medicine, New Haven, Connecticut 06520.
Stefan
S
Somlo
From the Renal Division, Department of Medicine, University Medical Center Freiburg, Hugstetter Straβe 55, 79106 Freiburg, Germany, michael.koettgen@uniklinik-freiburg.de.
Michael
M
Köttgen
D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D009154:Mutation; D016891:Polycystic Kidney, Autosomal Dominant / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase
D047908:Intracellular Signaling Peptides and Proteins; C512949:Prkcsh protein, mouse; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D001216:Asparagine; D017346:Protein Serine-Threonine Kinases; D005959:Glucosidases
Genetic Diseases; Glycosylation; Kidney; Lysosomes; TRP Channels