title: Characterization of a short isoform of the kidney protein podocin in human kidney.
abstract: BACKGROUND
  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. 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.
  
  
  METHODS
  We 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.
  
  
  RESULTS
  Mass 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.
  
  
  CONCLUSIONS
  A 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.
authors: Department 2 of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Kerpener Strasse 62, Cologne, Germany.
authors: Linus A
authors: LA
authors: Völker
authors: Eva-Maria
authors: EM
authors: Schurek
authors: Markus M
authors: MM
authors: Rinschen
authors: Judit
authors: J
authors: Tax
authors: Barbara A
authors: BA
authors: Schutte
authors: Tobias
authors: T
authors: Lamkemeyer
authors: Denise
authors: D
authors: Ungrue
authors: Bernhard
authors: B
authors: Schermer
authors: Thomas
authors: T
authors: Benzing
authors: Martin
authors: M
authors: Höhne
mesh_terms: D000595:Amino Acid Sequence; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D007668:Kidney / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D000091402:Prohibitins; D020033:Protein Isoforms / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C407223:NPHS2 protein; C000717328:PHB protein, human; D000091402:Prohibitins; D020033:Protein Isoforms
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