N-linked glycosylation is critical for the plasma membrane localization of nephrin

J Am Soc Nephrol. 2002 May;13(5):1385-9. doi: 10.1097/01.asn.0000013297.11876.5b.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • Cell Membrane / metabolism*
  • Cloning, Molecular
  • Glycosylation
  • Humans
  • Immunoblotting
  • Kidney / cytology
  • Membrane Proteins
  • Mice
  • Microscopy, Fluorescence
  • Proteins / metabolism*
  • Transfection
  • Tunicamycin / pharmacology

Substances

  • Membrane Proteins
  • Proteins
  • nephrin
  • Tunicamycin