ABSTRACT. Nephrin and NEPH1, the gene products of NPHS1 andNEPH1, are podocyte membrane proteins of the Ig superfamily.Similar to the nephrin knockout, mice lacking NEPH1 show severeproteinuria leading to perinatal death. To identify the ligandof NEPH1, the extracellular domain of NEPH1 was fused to humanIgG. This NEPH1-Ig fusion protein labeled the glomerular capillarywall of mouse kidneys in a staining pattern identical to NEPH1and nephrin, prompting speculation that that NEPH1 might formhomodimers and/or heterodimers with nephrin. In coimmunoprecipitationand pull-down assays, the NEPH1-Ig fusion protein precipitatedwild-type NEPH1 from overexpressing HEK 293T cells. Truncationalanalysis revealed that the adhesive properties were not confinedto a single Ig domain of NEPH1. Fusion proteins containing twoIg domains of NEPH1 were sufficient to immobilize NEPH1, butthey failed to interact with control protein containing thephylogenetically related PKD repeats of polycystin-1. NEPH1also precipitated nephrin, a protein with eight Ig domains anda fibronectin-like domain. Truncational analysis of nephrinrevealed a very similar mode of interaction, i.e., two nephrinIg domains fused to human IgG precipitated either nephrin orNEPH1, but not the control protein. Both NEPH1 and nephrin interactionswere strictly dependent upon posttranslational glycosylation,and bacterially expressed protein failed to bind NEPH1. Thesefindings demonstrate that the Ig domains of NEPH1 and nephrinform promiscuous homodimeric and heterodimeric interactionsthat may facilitate cis- and trans- homodimerizations and heterodimerizationsof these molecules at the glomerular slit diaphragm.
Renal filtration of small solutes and water without loss oflarger molecules is intimately linked to the glomerular basementmembrane and the slit diaphragm between interdigitating podocytes.Alterations of these structures, either acquired or hereditary,commonly lead to proteinuria.
Hereditary nephrotic syndromes are a heterogeneous group, displayingsevere proteinuria and renal failure. Best-characterized isthe congenital nephrotic syndrome of the Finnish type, causedby mutations in NPHS1, the gene encoding nephrin. Affected individualsexhibit massive proteinuria in utero and nephrosis at birth(1). Nephrin is an integral membrane protein located at adjacentsites of secondary foot processes of podocytes, a specializedepithelial cell that ensures size and charge selective ultrafiltration(reviewed in reference 2). The precise function of nephrin isunknown; however, it appears to form a zipper-like filter structurewithin the approximately 40-nm-wide slits between two foot processes(3).
In mice, the deletion of NEPH1 causes severe proteinuria andperinatal death (4). Like nephrin, NEPH1 is a transmembraneprotein of the Ig superfamily expressed by podocytes and localizesto the slit diaphragm by electron microscopy (5). The extracellulardomain of NEPH1 contains five Ig domains and the integrin recognitionmotif RGD. The third Ig domain is similar to the PKD repeatfound in polycystin-1, an evolutionary distinct Ig-like foldthat appears to mediate strong homophilic interactions (6).Deletion of either nephrin or NEPH1 causes an almost identicalphenotype in mice (4,7); therefore, it has been speculatedthat nephrin and NEPH1 participate in identical or overlappingpathways.
In Drosophila, the nephrin- and NEPH1-related proteins hibris(Hbs), kirre/dumbfounded (Duf), irregular chiasm-roughest (IrreC-rst),and sticks and stones (Sns), have been implicated in myoblastfusion and myotube guidance (8). Human nephrin is most closelyrelated to Sns (28% identities) and Hbs (28% identities), whereasNEPH1 is more closely related to Duf (33% identities) and IrreC-rst(31% identities). Interestingly, the deletion of Sns, or combineddeletions of Duf and IrreC-rst, abrogate myoblast fusion (9),whereas ectopic overexpression, but not deletion of Hbs, affectsmuscle development (8). Furthermore, S2 cell aggregation assaysrevealed a heterotypic interaction of Hbs and Sns with Duf,indicating that the interaction between these proteins may becrucial for muscle development in Drosophila.
These findings prompted us to speculate that NEPH1 is a structuralcomponent of the zipper-like structure formed between opposingpodocytes (3). To identify ligands of NEPH1, we generated aNEPH1 Ig fusion protein (NEPH1.ec.Fc) and used this fusion proteinto stain renal tissue. The NEPH1.ec.Fc fusion protein specificallyrecognized an antigen along the glomerular capillary wall, closelyresembling the staining pattern obtained for NEPH1 and nephrin.This result led to the hypothesis that NEPH1 and nephrin mayform homodimeric and heterodimeric interactions at the slitdiaphragm of glomerular podocytes.
Plasmids and Antibodies
Full-length nephrin and NEPH1 cDNAs were recently described(10,11). The Fc fusion proteins contain the CH2 and CH3 domainof human IgG. The control protein sFc.7 contains the leadersequence of CD5, followed by the CH2 and CH3 domain of humanIgG, and the membrane anchor of CD7. In the sFc fusion proteins,the CD7 anchor was replaced by NEPH1 or nephrin sequences asindicated. All truncations were generated by PCR, and verifiedby automated sequencing. To generate Nephrin.ec.Fc and Nephrin.Ig1–3,the 5'-oligonucleotide 5'-CGCGGGAAGCTTGCCACCATGGCCCTGGGGACGACGCTCAGGGCTTCTCTCCTGCTCwas used with the 3'-oligonucleotides 5'-CGCGGGCAGATCTGAAGGTGGCTCTGTGGGCAGand 5'-CGCGGGCAGATCTTTCTGGGCGGGATAT TTTAC, respectively. The5'-oligonucleotide 5'-CGCGGGCGAGATCTGGGATCTGCATCCCAG and the3'-oligonucleotide 5'-CGCG GGGCGGCCGCGCTAGATCGTCACGTTAGTTGGwere used to generate sFc.Nephrin.Ig3+4, the 5'-oligonucleotide5'-CGCGGGCGAGATCTGGCCAACGCATCCGCA and the 3'-oligonucleotide5'-CGCGGGGCGGCCGCAGACTATGTCCACAG AA were used to generate sFc.Nephrin.Ig5+6,and the 5'-oligonucleotide 5'-CGCGGGCGAGATCTCCAGGACCCCACTGAGand the 3'-oligonucleotide 5'-CGCGGGGCGGCCGCCAGAAGGCTGGTGGAGAwere used to generate sFc.Nephrin.7-9. NEPH1.ec.Fc and NEPH1.Ig1+2.Fcwere generated using the 5'-oligonucleotide 5'-CGCGGGAAGCTTGCCACCATGACTCTGGAGAGCCCTand the 3'-oligonucleotides 5'-CGCGGGCGGATCCACAGGTAACACCTCTCGCTCand 5'-CGCGGGCGGATCCACATCAAGCTCAATGGATGT, respectively. The5'-oligonucleotide 5'-CGCGGGGATCCCAATGGCAAGGAGACA and the 3'-oligonucleotide5'-CGCGGGGCGGCCGCGCTAGGCCCGGCAGGTATAGGTGCCAGC were used to generatesFc.NEPH1.Ig3+4. The domain structures of Nephrin and NEPH1were predicted using the programs SMART (http://smart. embl-heidelberg.de)and motif scanner (http://scansite.mit.edu/). The NEPH1 antiserumhas recently been described (11); the M2 anti-FLAG was obtainedfrom Sigma-Aldrich (Taufkirchen, Germany).
Purification of NEPH1-Fusion Protein
A stable cell line, expressing the NEPH1.ec.Fc fusion protein,was established using the pLXSN retroviral vector system asdescribed earlier (12). In brief, the retroviral vector wasproduced by cotransfection of HEK 293T cells with three plasmids(2.5 µg of pMD-G, 7.5 µg of pMD-gp, and 10 µgof the retroviral transfer vector containing NEPH1.ec.Fc) usingthe calcium phosphate method. The supernatant was harvestedafter 3 d, centrifuged, filtered, and applied to COS M6 cellsin the presence of 8 µg/ml polybrene for 4 h. Transducedcells were selected 3 to 4 d after viral transduction in neomycin(500 µg/ml). To purify the secreted NEPH1.ec.Fc fusionprotein, supernatants were harvested after 10 to 14 d, centrifuged,filtered, and applied to an anti-human IgG column (ICN Biomedicals,Eschwege, Germany). Bound protein was eluted using 0.1 M glycine(pH 3.0), neutralized in TRIS-buffer, and dialyzed against PBSover night. Concentrations were determined photometrically at280 nm and by an ELISA using goat-anti human IgG (ICN Biomedicals)coupled to 96-well ELISA plates (Greiner GmbH, Frickenhausen,Germany).
Coimmunoprecipitation
Coimmunoprecipitations were performed as described (10). Briefly,HEK 293T cells were transiently transfected using the calciumphosphate method. After incubation for 24 h, cells were washedtwice and lysed in a 1% Triton X-100 lysis buffer (20 mM Tris-HCl,pH 7.5; 1% Triton X-100; 50 mM NaCl; 50 mM NaF; 15 mM Na4P2O7;0.1 mM EDTA; 2 mM Na3VO4; and protease inhibitors). After centrifugationat 15,000 x g (15 min, 4°C) and ultracentrifugation at 100,000x g (30 min, 4°C), cell lysates containing equal amountsof total protein were precleared with protein G-sepharose andthen incubated for 1 h at 4°C with the appropriate antibody,followed by incubation with 30 µl of protein G-sepharosebeads for approximately 3 h. The beads were washed extensivelywith lysis buffer, and bound proteins were resolved by 10% SDS-PAGE.For endogenous interaction, eight mouse kidneys were homogenizedusing a glass potter, cleared by centrifugation, and solubilizedin lysis buffer supplemented with 20 mM CHAPS and 3 mM ATP.Before immunoprecipitation, cellular lysates were further preclearedby ultracentrifugation and absorption to protein G beads. Allkidneys were freshly prepared and perfused in situ with ice-coldphosphate-buffered saline before lysis. Control samples wereeither incubated with a nonspecific rabbit antiserum beforeimmunoprecipitation or with protein G beads alone.
Pull-Down Experiments
For pull-down experiments HEK 293T cells were transiently transfected,harvested, and lysed as described above. Lysates were preclearedand incubated with the recombinant protein (10 µg) for2 h at 4°C followed by incubation with 30 µl of proteinG-sepharose beads for approximately 3 h. As in coimmunoprecipitations,the beads were washed with lysis buffer, and bound proteinswere resolved by 10% SDS-PAGE.
Glycosylation Assays
HEK 293T transiently overexpressing NEPH1.ec.Fc were harvested,washed, and lysed as described above. The fusion protein wasimmobilized from lysates using G-sepharose beads. For each reaction,30 µl of immobilized protein were added to 10 µlof 5x assay buffer (250 mM NaHPO4, pH 7.5) and 2.5 µlof denaturation buffer (2% SDS, 1 M -mercaptoethanol) followedby incubation at 95°C for 5 min. After cooling down, 2.5µl of Triton-X100 were added to each tube together with2 µl N-glycosidase F (PNGase F, Calbiochem, Bad Soden,Germany) for the test samples, and 2 µl H2O for the controls.After incubation at 37°C for five hours, deglycosylationwas verified by monitoring the size-shift on a 10% SDS-PAGE.
Immunohistochemisty
Frozen mouse kidneys were embedded in OCT, cut into 5-µmsections, and fixed with ice-cold acetone for 2 min before staining.The blocking solution was 10 mM TRIS-HCl (pH 7.4), 100 mM MgCl2,0.5% Tween 20, 1% bovine serum albumin, and 5% fetal calf serum,the washing buffer was PBS containing 0.04% Triton-X100 and0.35% DMSO. All incubations were performed in a moist chamberat room temperature. For indirect immunoperoxidase stainings,we used the Vectastain ABC kit (Vector Laboratories, Burlingame,CA) following the manufacturers’ protocol. Endogenousperoxidase activity was quenched with 0.3% H2O2 for 30 min.
Fusion protein staining was performed by incubating cryosectionswith NEPH1.ec.Fc at 20 µg/ml for 1 h, followed by incubationwith FITC-goat anti human (ICN). Controls were incubated withidentical concentrations of human IgG. For paraffin sections,tissues were fixed with methanol/DMSO (4:1 vol/vol) at 4°Cover night, dehydrated, paraffin-wax embedded, cut into 5-µmsections, mounted on Super Frost Plus slides (Menzel, Braunschweig,Germany), dewaxed, and rehydrated. For all NEPH1 stainings,the antiserum was diluted 1:10. To improve antibody penetrationand antigen recognition, paraffin sections were boiled in 10mM citric acid for 5 min. An Axiophot 2 microscope (Zeiss, Jena,Germany) equipped with a CCD camera was used for image documentation.
The Ligand of NEPH1 Co-Localizes to the Glomerular Capillary Wall
To characterize the ligand of NEPH1, we generated Ig fusionproteins that contained the extracellular domain or single Igdomains of NEPH1 and nephrin fused to the CH2 and CH3 domainof human IgG (Figure 1). To ensure correct glycosylation ofthe NEPH1 fusion protein containing the entire extracellulardomain of NEPH1 (NEPH1-ec.Fc), the fusion protein was affinity-purifiedfrom retrovirally transformed COS M6 cells or transiently transfectedHEK 293T cells (Figure 2). Frozen sections of mouse kidneyswere incubated with NEPH1.ec.Fc or an identical concentrationof human IgG. As shown in Figure 3, the NEPH1 fusion protein,but not human IgG, specifically outlined the glomerular capillarywall, using immunohistochemistry or indirect immunofluorescence.The NEPH1 fusion protein labeled the mouse glomerulus in a stainingpattern identical to NEPH1; we therefore speculated that theNEPH1 fusion protein binds to NEPH1 at the glomerular slit diaphragm.
Figure 1. Generation of nephrin and NEPH1 fusion proteins. The Ig and fibroncectin (FN)-like domains were predicted using the SMART prediction program (http://smart.EMBL-Heidelberg.de/). All constructs containing the amino-terminal domains of either NEPH1 or nephrin include the authentic signal peptide (SP). The carboxy-terminal Ig domains were fused to the carboxy-terminus of the CH2 and CH3 domain of human IgG. To target these constructs to the secretory pathway, the fusion proteins were preceded by the leader sequence of CD5 (black box) (10,11). The constructs Nephrin.ec.Fc and NEPH1.ec.Fc contain the entire extracellular (ec) domain of either nephrin or NEPH1.
Figure 2. Expression of nephrin and NEPH1 fusion proteins. (A) Expression of NEPH1 and control fusion proteins in HEK 293T cells. Lysates were prepared after 24 h of incubation and separated on SDS-PAGE. Western blot analyses were performed with a HRP-coupled antiserum directed against human IgG. The control protein sFc.7, a fusion protein containing the CH2 and CH3 domain of human IgG, is extremely well expressed, typically resulting in a smear. (B) Expression of nephrin and control fusion proteins in HEK 293T cells. Lysates were prepared after 24 h of incubation and separated on SDS-PAGE. Western blot analyses were performed with an HRP-coupled antiserum directed against human IgG.
Figure 3. Glomerular localization of NEPH1 and the ligand of NEPH1. (A and B) Glomerular distribution of NEPH1 in the mouse kidney at 4 wk of age by immunohistochemistry. The anti-NEPH1 antiserum outlines the glomerular basement membrane in a pattern similar to the reported distribution of nephrin and podocin. The control rabbit antiserum reveals no significant staining of mouse renal tissue. (C and D) Glomerular distribution of NEPH1 in the mouse kidney at 4 wk of age by immunofluorescence. The anti-NEPH1 antiserum outlines the glomerular basement membrane, while the control rabbit antiserum reveals very little background staining. (E and F) Glomerular distribution of the putative ligand of NEPH1. Frozen sections of the mouse kidney were incubated with affinity-purified NEPH1.ec.Fc (E) or human IgG (F) at a concentration of 20 µg/ml. Both sections were subsequently labeled with a FITC-conjugated secondary antibody directed against human IgG. The NEPH1.ec.Fc, but not the control serum, revealed a highly specific labeling of the glomerular basement membrane in a staining pattern identical to the staining pattern obtained for the anti-NEPH1 antiserum (C).
NEPH1 Forms Homodimers
Using wild-type NEPH1 in combination with either NEPH1.ec.Fcor Flag-tagged NEPH1, we tested whether constructs containingthe extracellular domain of NEPH1 can precipitate full-lengthNEPH1 from transiently transfected HEK 293T cells. We used twocontrol proteins, sFc.7, a fusion protein containing the CH2and CH3 domains of human IgG (10,11), and PKD.Fc, a fusionprotein containing amino acid 1 to 480 of the N-terminal domainof polycystin-1 (13), including the first PKD repeat, and theCH2 and CH3 domain of human IgG. PKD repeats are Ig-like domainspresent in the extracellular domain of polycystin-1 (13) andmediate homodimeric interactions (6). While the NEPH1-Ig fusionprotein (NEPH1.ec.Fc) precipitated full-length NEPH1, both controlproteins failed to immobilize NEPH1 using identical detergentand washing conditions (Figure 4).
Figure 4. Homodimeric and heterodimeric interactions of NEPH1. (A) The NEPH1 fusion proteins precipitate NEPH1. The NEPH1 fusion proteins, containing either the entire extracellular domain of NEPH1 (NEPH1.ec.Fc) or truncations of the extracellular domain of NEPH1, were co-expressed with the Flag-tagged version of NEPH1 (NEPH1.F) in transiently transfected HEK 293T cells. Cellular lysates were incubated with protein G to precipitate the NEPH1 fusion proteins. NEPH1.F, bound to NEPH1.ec.Fc, was detected by Western blot analysis, using the M2 anti-Flag monoclonal antibody. All three NEPH1 fusion proteins, NEPH1.ec.Fc, containing the entire extracellular domain of NEPH1, NEPH1.Ig1+2.Fc, containing the N-terminal three Ig domains of NEPH1, and sFc.NEPH1.Ig3+4, containing the third and fourth Ig domain, precipitated Flag-tagged NEPH1. In contrast, both control proteins, sFc.7 and PKD. Fc, failed to immobilize NEPH1.F. (B) The NEPH1 fusion proteins precipitate nephrin. The NEPH1 fusion proteins were co-expressed with Flag-tagged nephrin (Nephrin.F) in HEK 293T cells. Again, all NEPH1 fusion proteins precipitated Nephrin.F, whereas the two control proteins failed to interact with nephrin. (C) Nephrin fusion proteins precipitate NEPH1. Nephrin fusion proteins were generated to test the NEPH1-nephrin interaction after immobilization of nephrin. Co-expression of Nephrin.ec.Fc, containing the entire extracellular domain of nephrin, or fusion proteins, containing individual nephrin Ig domains as indicated, immunoprecipitated the Flag-tagged NEPH1. Both control proteins failed to precipitate NEPH1, although the NEPH1 expression was higher in the controls than in the presence of the nephrin fusion proteins. (D) Interaction between NEPH1 and nephrin in vivo. Lysates prepared from mouse kidneys were incubated with anti-nephrin antiserum and subsequently precipitated with protein G. Immobilized NEPH1 was detected by Western blot analyses, using a NEPH1-specific antiserum, indicating that at least a small fraction of nephrin and NEPH1 appears to interact in vivo (upper panel). Addition of ATP to the lysis buffer appeared to increase the amount of immobilized NEPH1 (lower panel).
The Adhesive Properties of NEPH1 Are Not Confined to a Single Ig Domain of NEPH1
In several cell surface receptors of the Ig superfamily, theligand binding area can be mapped to a linear motif, often locatedat the N-terminus of the molecule. To test whether the N-terminaltwo Ig domains mediate the NEPH1-NEPH1 interaction, we generatedfusion proteins containing the two N- or two C-terminal Ig domains.These Ig domains were fused to the N- or the C-terminus of humanCH2 and CH3. As shown in Figure 4A, both fusion proteins weresufficient to precipitate NEPH1, but a similarly constructedcontrol protein failed to interact with NEPH1.F. These resultssuggest that the homodimerization of NEPH1 molecules is notmediated by a linear peptide motif, but most likely involvesmultiple contact sites along the entire extracellular domainof NEPH1.
NEPH1 Forms Heterodimers with Nephrin
The targeted deletion of either NEPH1 or nephrin in mice ischaracterized by heavy proteinuria and death in the early perinatalperiod. These almost identical phenotypes suggest that bothmolecules have similar or overlapping functions. We thereforetested whether NEPH1 and nephrin form physical interactions.As demonstrated in Figure 4B, the NEPH1-Ig fusion proteins precipitatednephrin containing a C-terminal Flag-tag. Furthermore, a nephrin-Igfusion protein containing either the full-length or truncationsof the extracellular domain of nephrin precipitated Flag-taggedNEPH1 (Figure 4C). These findings indicate that NEPH1 and nephrincan engage in heterodimeric interactions. However, the interactionis surprisingly promiscuous, involving different Ig domainsof both molecules. Nevertheless, immunoprecipitation of nephrinfrom mouse kidneys with anti-nephrin antiserum, but not withnormal rabbit antiserum, precipitated a band at the predictedsize of NEPH1, suggesting that the interaction between nephrinand NEPH1 is present in vivo (Figure 4D). This interaction wasonly detectable after solubilization of kidney lysates with20 mM CHAPS, indicating that a substantial fraction of bothproteins is associated with lipid rafts. Addition of 3 mM ATPto the lysis buffer appeared to increase the amount of NEPH1,precipitated with anti-nephrin antiserum versus protein G alone(Figure 4D, lower panel), indicating that association with bothlipid rafts (14) and the cytoskeleton (15) reduces the solubilityof NEPH1 and/or nephrin and limits the experimental approachesto verify this interaction in vivo.
Nephrin Forms Homodimers
It has been proposed that the electron-dense central regionof the slit diaphragm is formed by interdigitating nephrin moleculesthat span the distance between two neighboring podocyte footprocesses. Using coimmunoprecipitations of transiently transfectedproteins, we tested whether the nephrin fusion protein immobilizeswild-type nephrin. As demonstrated in Figure 5, the nephrin-Igfusions precipitated membrane-anchored, Flag-tagged nephrin.Again, this interaction was not confined to a specific Ig domainof nephrin. These findings confirm that the Ig domains of NEPH1and nephrin form promiscuous homodimeric and heterodimeric interactions.
Figure 5. Nephrin forms homodimers. HEK 293T cells were transiently transfected with Flag-tagged nephrin and the nephrin fusion proteins as indicated. The Nephrin.ec.Fc fusion contains the entire extracellular domain of nephrin fused to the CH2 and CH3 domain of human IgG, whereas the other fusion proteins contain the Ig domains 1 to 3, 3 and 4, 5 and 6, or 7 to 9 fused to either the N-terminus of CH2 (Fc fusions) or the C-terminus of CH3 (sFc fusions). Protein G was used to immobilize the fusion proteins, and Western blot analyses were performed to detect precipitated nephrin, utilizing the Flag-specific M2 monoclonal antibody. (A and B) Flag-tagged nephrin (Nephrin.F) interacts with nephrin fusion proteins that include either the entire extracellular domain of nephrin or fragments of the extracellular domain of nephrin. In contrast, the two control proteins, sFc.7 and PKD.Fc, did not interact with nephrin.
The Nephrin-NEPH1 Interaction Is Glycosylation-Dependent
PNGase F treatment of immunoprecipitated NEPH1 significantlydecreased the molecular weight of NEPH1, revealing that NEPH1is heavily glycosylated (Figure 6A). Correct folding of theextracellular domain of surface proteins and interaction withligands or other surface proteins commonly requires adequateglycosylation. We therefore suspected that NEPH1 expressed ineukaryotic cells, but not bacterially expressed NEPH1, can precipitateFlag-tagged nephrin in pull-down experiments. Figure 6B demonstratesthat purified NEPH1-ec.Fc immobilized with protein G retainsFlag-tagged NEPH1, whereas MBP-NEPH1, a maltose-binding proteinfusion with NEPH1 generated in Escherichia coli, failed to immobilizeeither nephrin or NEPH1. Furthermore, purified NEPH1-ec.Fc expressedin HEK 293T cells, failed to immobilize NEPH1 or nephrin aftertreatment with PNGase F (Figure 6, C and D). These findingsdemonstrate that the NEPH1-NEPH1 as well as the nephrin-NEPH1interactions require correct glycosylation of NEPH1.
Figure 6. The NEPH1-nephrin interactions require post-translational glycosylation. (A) NEPH1 is N-glycosylated. HEK 293T cells were transiently transfected with Flag-tagged NEPH1 and precipitated with the Flag-specific M2 monoclonal antibody. Treatment with PNGase F resulted in a significant decrease of the molecular weight of NEPH1, confirming that this molecule is heavily glycosylated. (B) The extracellular domain of NEPH1, expressed in HEK 293T cells (NEPH1.ec.Fc), but not recombinant NEPH1, expressed as a maltose-binding protein fusion (MBP) in Escherichia coli (MBP.NEPH1.ec) interacts with nephrin or NEPH1. Cellular lysates of HEK 293T cells, expressing either Flag-tagged nephrin or NEPH1, were incubated with the control protein sFc.7, NEPH1.ec.Fc or MBP.NEPH1.ec, immobilized to either protein G or amylose resin. NEPH1.F only interacted with NEPH1.ec.Fc, but not with either the control protein, or the recombinant MBP fusion protein. (C and D) PNGase F treatment abolished the interaction of NEPH1 and nephrin with the NEPH1 fusion protein. The NEPH1.ec.Fc was immobilized to protein G and treated with PNGase F or buffer, lacking the enzyme. PNGase F-treated NEPH1.ec.Fc failed to precipitate either Flag-tagged nephrin (Nephrin.F) or NEPH1 (NEPH1.F), expressed in HEK 293T cells.
Several proteins, altered in hereditary nephrotic syndromesor experimental proteinuria, localize to the glomerular slitdiaphragm. This structure, formed between interdigitating podocytes,together with the glomerular basement membrane and the endothelialcell layer is a filtration barrier for proteins of higher molecularweights. On the basis of electron micrographs of tannic acid-stainedand glutaraldehyde-fixed rat glomerular, Rodewald and Karnovsky(16) suggested that the slit diaphragm is formed by a zipper-likeorganization of integral membrane proteins. The domain architectureof nephrin prompted Ruotsalainen et al. (3) to speculate thatnephrin contributes to the formation of this zipper-like structurethrough homophilic interactions, typcial for several cell surfacemolecules of the Ig superfamily (17,18). A head-to-head assemblyof nephrin was suggested, postulating that the N-terminal sixIg domains of opposing nephrin molecules form interdigitatingassemblies. The average length of the nephrin Ig domains wasestimated at 35Å; therefore, the eight Ig domains togetherwith the fibronectin type III domain and spacer regions of nephrincould bridge most of the estimated 35- to 45-nm width of theslit diaphragm (3). Our results presented here provide the firstevidence that nephrin forms homodimers. Yet, the self-associationof nephrin Ig domains was far more promiscuous than anticipated.All tested combinations of Ig domains precipitated full-lengthnephrin and appear to contribute to the homodimerization ofnephrin molecules. Interestingly, there are several cell adhesionmolecules (CAM) of the Ig superfamily that display similar properties(reviewed in references 17 and 18). For example, members ofthe L1 family of CAM involved in axonal growth and path findingform homodimers and heterodimers involving several Ig domain.Furthermore, several CAM that mediate neurite outgrowth, associatewith lipid rafts, couple to Fyn, and activate canonical signalingcascades, thereby closely resembling some of the currently knownproperties of nephrin (10,19). Type II and desmosomal membersof the cadherin protein superfamily share a similar domain architecturewith NEPH1, containing five tandem extracellular cadherin domains(reviewed in reference 20). Again, several intermolecular interfacesand contact sites appear to form the zipper-like superstructuretypical of these cell adhesion molecules (21,22).
Although NEPH1 coimmunoprecipitates nephrin, the NEPH1 fusionprotein immobilized on plastic surfaces, failed to bind NEPH1/nephrin-transfectedHEK 293T, or differentiated mouse podocytes retrovirally transducedto overexpress nephrin (data not shown). Therefore, additionalfactors may be required for NEPH1/nephrin to mediate the cell-celladhesion reported for the related Drosophila proteins Hbs andDuf (8). It is important to note that several cell adhesionmolecules of the Ig superfamily associate with the cytoskeleton,and their adhesive strength critically depends on this interaction(18,23). Thus, recruitment to lipid rafts and anchorage ofNEPH1 or nephrin with the cytoskeleton may be mandatory forefficient homodimerization and heterodimerization. In addition,antiparallel trans interaction of dimers on opposing cells mayrequire prior cis homodimerization or heterodimerization ofNEPH1 and nephrin. Clearly, cis homodimerization is a mandatorystep for subsequent trans interactions in C-cadherin-mediatedcell contacts (24). It is interesting to note that the Ca2+-independentIg-like cell-cell adhesion molecule nectin appears to utilizedifferent Ig domains for different functions. While the N-terminalIg domain is essential for trans-dimer formation, cis homodimerization,an essential step for subsequent trans homodimerization, ismediated by the second Ig domain of nectin (25).
Although immunoprecipitation of endogenous nephrin from mousekidneys immobilized NEPH1, this finding has to be interpretedwith caution. On the basis of the comparison with other Ig CAM,we anticipate that most of the NEPH1 and nephrin molecules engagedin homogenic and heterogenic interactions are associated withlipid rafts and/or the cytoskeleton and are thus detergent-resistant(15). It is therefore possible that the small amounts of immobilizedNEPH1 result from a minor fraction of protein that has not beentargeted to lipid rafts.
Although our findings confirm and extend some of the earlierspeculations by Tryggvason (2,26,27), they also raise newquestions. First, the interactions between NEPH1/nephrin moleculesare surprisingly promiscuous and not confined to single Ig domains,suggesting that additional factors such as lipid raft recruitmentand cytoskeletal anchorage may regulate the affinity and specificityof the NEPH1/nephrin interactions in vivo. Second, althoughhomodimers and heterodimers of NEPH1 and nephrin are clearlydetectable by immunoprecipitation, future work needs to demonstrate,whether these molecules can engage in trans interactions thatare sufficient to mediate cell-cell adhesion.
Acknowledgments
We thank L.B. Holzman for the generous gift of the anti-nephrinantiserum, O. Beltcheva and K. Tryggvason for helpful discussions,P. Stunz and B. Wehrle for expert technical assistance, andthe members of the Walz laboratory for valuable suggestions.Some of our results were presented at the ASN meeting 2002 inPhiladelphia, Pennsylvania. We acknowledge that similar findingswere reported by L.B. Holzman’s laboratory at this meeting.This work was supported by DFG grant WA 597.
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Received for publication November 21, 2002.
Accepted for publication January 4, 2003.
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Top
Abstract
Introduction
-mercaptoethanol) followed by incubation at 95°C for 5 min. After cooling down, 2.5 µl of Triton-X100 were added to each tube together with 2 µl N-glycosidase F (PNGase F, Calbiochem, Bad Soden, Germany) for the test samples, and 2 µl H2O for the controls. After incubation at 37°C for five hours, deglycosylation was verified by monitoring the size-shift on a 10% SDS-PAGE. 
