Characterization of a short isoform of the kidney protein

37 Background/Aims: Steroid resistant nephrotic syndrome is a severe hereditary disease often caused 38 by mutations in the NPHS2 gene. This gene encodes the lipid binding protein podocin which localizes 39 to the slit diaphragm of podocytes and is essential for the maintenance of an intact glomerular 40 filtration barrier. Podocin is a hairpin-like membrane-associated protein that multimerizes to recruit 41 lipids of the plasma membrane. Recent evidence suggested that podocin may exist in a canonical, 42 well-studied large isoform and an ill-defined short isoform. Conclusive proof of the presence of this 43 new podocin protein in the human system is still lacking. 44 Methods: We used database analyses to identify organisms for which an alternative splice variant has 45 been annotated. Mass spectrometry was employed to prove the presence of the shorter isoform of 46 podocin in human kidney lysates. Immunofluorescence, sucrose density gradient fractionation and 47 PNGase-F assays were used to characterize this short isoform of human podocin. 48 Results: Mass spectrometry revealed the existence of the short isoform of human podocin on protein 49 level. We cloned the coding sequence from a human kidney cDNA library and showed that the 50 expressed short variant was retained in the endoplasmic reticulum while still associating with 51 detergent-resistant membrane fractions in sucrose gradient density centrifugation. The protein is 52 partially N-glycosylated which implies the presence of a transmembranous form of the short isoform. 53 Conclusions: A second isoform of human podocin is expressed in the kidney. This isoform lacks part 54 of the PHB domain. It can be detected on protein level. Distinct subcellular localization suggests a 55 physiological role for this isoform which may be different from the well-studied canonical variant. 56 Possibly, the short isoform influences lipid and protein composition of the slit diaphragm complex by 57 sequestration of lipid and protein interactors into other cell compartments. 58