N-linked glycosylation is critical for the plasma membrane localization of nephrin.
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.
Yan
Kunimasa
K
Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Khoshnoodi
Jamshid
J
Ruotsalainen
Vesa
V
Tryggvason
Karl
K
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
D008565:Membrane Proteins; D011506:Proteins; C113267:nephrin; D014415:Tunicamycin