Background. Podocin is a membrane-integrated protein that is located at the glomerular slit diaphragm and directly interacts with nephrin. The gene encoding podocin, NPHS2, is mutated in patients ...with autosomal-recessive steroid-resistant nephrotic syndrome (SRN). In order to study a potential pathomechanism of massive proteinuria in patients with SRN, we have investigated the trafficking and subcellular localization of five common disease-causing missense mutants of human podocin. Methods. Site-directed mutagenesis was applied to generate cDNA constructs encoding five different missense mutations of human podocin (P20L, G92C, R138Q, V180M, and R291W). To identify the subcellular localization of each mutant in transfected human embryonic kidney (HEK)293 cells, we have generated and characterized a rabbit polyclonal antibody against the human podocin. Specificity of the antibody was determined by light and immunoelectron microscopy, as well as immunoblot analysis using human glomeruli. Confocal microscopy was applied to determine subcellular localization of the wild-type and the mutated podocin molecules, as well as wild-type nephrin in transfected cells. Immunoprecipitation and pull-down studies were carried out to investigate the molecular interaction of podocin mutants and wild-type nephrin. Results. Immunofluorescence and confocal microscopy showed that wild-type podocin located to the plasma membrane when expressed in HEK293 cells. Two missense mutations, P20L and G92C, located at the N-terminus part of the molecule, were also present at the plasma membrane, indicating that these mutations did not affect the subcellular localization of the mutated podocin molecules. In contrast, subcellular localization of three other missense mutants located in the proximal C-terminus part of the protein was drastically altered, in which R138Q was retained in the endoplasmic reticulum (ER), V180M formed inclusion bodies in the cytoplasm, and the R291W mutant was trapped both in the ER and in small intracellular vesicles. Interestingly, this abnormal subcellular localization of podocin missense mutants also resulted in alteration in protein trafficking of wild-type nephrin in cotransfected cells through the strong protein binding between both molecules. Conclusion. In patients with SRN, some missense mutations in the NPHS2 gene not only lead to misfolding and mislocalization of the mutated podocin, but they can also interfere with slit diaphragm structure and function by altering the proper trafficking of nephrin to the plasma membrane.
Full-length cDNA for starch branching enzyme (SBE) II of potato was isolated and sequenced. In potato, similarly to most other investigated plants, the SBE-II isoform differs from SBE-I by having an ...acidic amino-terminal extension and a shorter carboxyterminus. Two forms of SBE-II, migrating as 98 and 95 kDa proteins in 6% SDS-polyacrylamide gels, were associated to tuber starch. The latter form was 16 amino acids shorter in the amino terminus. Transcript of SBE-II was present in leaf tissue, whereas significant expression was not seen in tubers. On the other hand, a significant amount of SBE-I transcript was detected in tuber tissue but not in leaves.
Western blot analysis showed the presence of three forms of starch-branching enzyme (SBE), with apparent molecular masses of 103, 97 and 80 kDa, in extracts of leaves and stored tubers of Solanum ...tuberosum. The 80-kDa form was absent in extracts of fresh tuber. Active 80-kDa enzyme was partially purified from stored tubers and sequence analysis showed that it, similar to the two larger enzyme forms, was an SBE-I isoform. Limited proteolysis of isolated 103-kDa SBE-I under native conditions removed approximately 200 amino acid residues from the carboxy terminus. A stable intermediate with an apparent molecular mass of approximately 80 kDa was formed. Since the 80-kDa form displayed full enzymatic activity and its circular-dichroism spectrum did not differ significantly from that of the 103-kDa enzyme, the carboxy-terminal portion of the enzyme was suggested to have an extended, unordered structure and therefore to be easily accessible to proteolysis. A cDNA sequence encoding a mature SBE-I was amplified from tuber mRNA of S. tuberosum by means of PCR. The 3' end of this sequence differed significantly from that of previously published data. PCR amplification and DNA sequencing of the 3' ends of the steel gene showed that four steel alleles exist in the cultivar studied. Two of these four alleles. sbeIa and sbeIb, had slightly longer 3' ends compared with the other two, sbeIc and sbeId. The difference between the two groups of alleles was due to a partial deletion in sbeIc and sbeId of a segment duplicated in all alleles. All four alleles were expressed in leaf and tuber.
Monoclonal antibodies to human nephrin Ruotsalainen, Vesa; Reponen, Paula; Khoshnoodi, Jamshid ...
Hybridoma and hybridomics,
02/2004, Volume:
23, Issue:
1
Journal Article
Nephrin is a 180-200-kDa transmembrane protein of the immunoglobulin superfamily. In the kidney, nephrin localizes to the slit diaphragm (SD) between interdigitating podocyte foot processes and ...mutations in the nephrin gene cause congenital nephrotic syndrome. In addition to this rare genetic disorder, recent reports indicate that nephrin is more generally involved in the pathogenesis of glomerular disease. In this report, we describe production and characterization of mouse monoclonal antibodies to human nephrin, and discuss their applications. Recombinant human nephrin variants were produced in both prokaryotic and eukaryotic expression systems and purified proteins were used in an immunization protocol. A total of 16 antibodies were characterized for their reactivity with the nephrin by using ELISA, Western blots, immunoprecipitation and immunostaining of frozen and formaldehyde-fixed paraffin embedded tissue sections. The antibody epitopes were mapped using a variety of recombinant human nephrin variants. The detailed screening and characterization proved to be essential in order to find the most suitable antibody for each application. These antibodies will find wide use in studies of human nephrin and its involvement in kidney disease.
Semimembranosus tendon avulsion fractures are an uncommon occurrence and are often associated with anterior cruciate ligament (ACL) and medial meniscus tears. We present the imaging features of an ...unusual case of semimembranosus avulsion fracture of the posteromedial tibial plateau associated with posterior cruciate ligament (PCL) tear, medial meniscus tear, and capsular rupture in a 26-year-old man with a football injury.
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