Abstract
BackgroundSteroid resistant nephrotic syndrome is a severe hereditary disease often caused by mutations in the NPHS2 gene. This gene encodes the lipid binding protein podocin which localizes to the slit diaphragm of podocytes and is essential for the maintenance of an intact glomerular filtration barrier. Podocin is a hairpin-like membrane-associated protein that multimerizes to recruit lipids of the plasma membrane. Recent evidence suggested that podocin may exist in a canonical, well-studied large isoform and an ill-defined short isoform. Conclusive proof of the presence of this new podocin protein in the human system is still lacking.MethodsWe used database analyses to identify organisms for which an alternative splice variant has been annotated. Mass spectrometry was employed to prove the presence of the shorter isoform of podocin in human kidney lysates. Immunofluorescence, sucrose density gradient fractionation and PNGase-F assays were used to characterize this short isoform of human podocin.ResultsMass spectrometry revealed the existence of the short isoform of human podocin on protein level. We cloned the coding sequence from a human kidney cDNA library and showed that the expressed short variant was retained in the endoplasmic reticulum while still associating with detergent-resistant membrane fractions in sucrose gradient density centrifugation. The protein is partially N-glycosylated which implies the presence of a transmembranous form of the short isoform.ConclusionsA second isoform of human podocin is expressed in the kidney. This isoform lacks part of the PHB domain. It can be detected on protein level. Distinct subcellular localization suggests a physiological role for this isoform which may be different from the well-studied canonical variant. Possibly, the short isoform influences lipid and protein composition of the slit diaphragm complex by sequestration of lipid and protein interactors into the endoplasmic reticulum.
Highlights
Steroid resistant nephrotic syndrome is a severe hereditary disease often caused by mutations in the NPHS2 gene
Recently, it was suggested that the kidney disease protein podocin may exist in two isoforms that differ in size [6,7]
Since intron/exon boundaries are conserved between mouse and human, we speculated that a similar murine short isoform existed, yet no such EST could be found in our database searches
Summary
Steroid resistant nephrotic syndrome is a severe hereditary disease often caused by mutations in the NPHS2 gene This gene encodes the lipid binding protein podocin which localizes to the slit diaphragm of podocytes and is essential for the maintenance of an intact glomerular filtration barrier. The protein encoded by the NPHS2 gene, is mutated in hereditary steroid-resistant nephrotic syndrome (SRNS) and essential for an intact kidney filtration barrier [1,2,3]. Palmitoylated residue missing in variant evidence so far has been based on antibody-mediated detection and RT-PCR, making it uncertain whether the protein is expressed. This is interesting with regard to the importance of a functional PHB domain. We show that the short isoform is expressed in human kidneys and localizes to the endoplasmic reticulum raising the intriguing hypothesis that podocin may serve distinct functions in different parts of the podocyte
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