Department 2 of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Kerpener Strasse 62, Cologne, Germany.
BMC Nephrol. 2013 May 6;14:102. doi: 10.1186/1471-2369-14-102.
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.
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.
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.
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.
激素耐药性肾病综合征是一种严重的遗传性疾病，通常由 NPHS2 基因突变引起。该基因编码脂质结合蛋白足细胞蛋白 podocin，其定位于足细胞的裂孔隔膜，对于维持完整的肾小球滤过屏障至关重要。Podocin 是一种发夹状的膜相关蛋白，可多聚化以募集质膜的脂质。最近的证据表明，Podocin 可能存在一种规范的、研究充分的大亚型和一种定义不明确的短亚型。在人体系统中存在这种新的 Podocin 蛋白的明确证据仍然缺乏。
我们使用数据库分析来鉴定已注释有替代剪接变异体的生物体。采用质谱法证明人肾裂解物中存在较短的 Podocin 同工型。免疫荧光、蔗糖密度梯度分级分离和 PNGase-F 测定用于表征人 Podocin 的这种短同工型。
质谱法在蛋白质水平上揭示了人 Podocin 短同工型的存在。我们从人肾 cDNA 文库中克隆了编码序列，并表明表达的短变体在内质网中保留，同时仍与蔗糖梯度密度离心中的去污剂抗性膜部分结合。该蛋白部分 N-糖基化，这意味着短同工型存在跨膜形式。
人 Podocin 的第二种同工型在肾脏中表达。该同工型缺失部分 PHB 结构域。它可以在蛋白质水平上检测到。不同的亚细胞定位表明该同工型具有生理作用，可能与研究充分的规范变体不同。可能，短同工型通过将脂质和蛋白相互作用物隔离到内质网中，影响裂孔隔膜复合物的脂质和蛋白组成。
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