title: N-linked glycosylation is critical for the plasma membrane localization of nephrin.
abstract: The expression pattern, subcellular localization, and the role of glycosylation of the human nephrin was examined in transfected cells. Stable cell lines, constitutively expressing a full-length human nephrin cDNA construct, were generated from transfected immortalized mouse podocytes (IMP) and a human embryonic kidney cell line (HEK-293). Immunofluorescence confocal microscopy of transfected cells showed plasma membrane localization of the recombinant nephrin. Immunoblotting showed that the recombinant nephrin expressed in transfected cell lines migrated as a double band with a molecular weight of 185 kD. When cells were treated with the N-glycosylation inhibitor, tunicamycin, the molecular weight of nephrin was decreased to a single immunoband of 150 kD, indicating that the shift in the electrophoretic migration of nephrin is due to N-linked carbohydrate moieties. It was further shown that this glycosylation process is highly sensitive to inhibition by tunicamycin, which is a naturally occurring antibiotic, leading to retention of nonglycosylated nephrin molecules in the endoplasmic reticulum. It was concluded that N-glycosylation of nephrin is crucial for its proper folding and thereby plasma membrane localization; therefore, inhibition of this process might be an important factor in the onset of pathogenesis of some acquired glomerular diseases.
authors.affiliation: Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
authors.forename: Kunimasa
authors.initials: K
authors.lastname: Yan
authors.forename: Jamshid
authors.initials: J
authors.lastname: Khoshnoodi
authors.forename: Vesa
authors.initials: V
authors.lastname: Ruotsalainen
authors.forename: Karl
authors.initials: K
authors.lastname: Tryggvason
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D011506:Proteins / Q000378:metabolism*; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D008565:Membrane Proteins; D011506:Proteins; C113267:nephrin; D014415:Tunicamycin