Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease, caused by mutations of at least nine different genes. Several extrarenal manifestations characterize this disorder, including cerebellar defects, situs inversus and retinitis pigmentosa. While the clinical manifestations vary significantly in NPHP, mutations of NPHP5 and NPHP6 are always associated with progressive blindness. This clinical finding suggests that the gene products, nephrocystin-5 and nephrocystin-6, participate in overlapping signaling pathways to maintain photoreceptor homeostasis. To analyze the genetic interaction between these two proteins in more detail, we studied zebrafish embryos after depletion of NPHP5 and NPHP6. Knockdown of zebrafish zNPHP5 and zNPHP6 produced similar phenotypes, and synergistic effects were observed after the combined knockdown of zNPHP5 and zNPHP6. The N-terminal domain of nephrocystin-6-bound nephrocystin-5, and mapping studies delineated the interacting site from amino acid 696 to 896 of NPHP6. In Xenopus laevis, knockdown of NPHP5 caused substantial neural tube closure defects. This phenotype was copied by expression of the nephrocystin-5-binding fragment of nephrocystin-6, and rescued by co-expression of nephrocystin-5, supporting a physical interaction between both gene products in vivo. Since the N- and C-terminal fragments of nephrocystin-6 engage in the formation of homo- and heteromeric protein complexes, conformational changes seem to regulate the interaction of nephrocystin-6 with its binding partners.
Nephronophthisis (NPHP) is a heterogenetic autosomal recessive disorder, characterized by renal cysts at the corticomedullary border and characteristic extrarenal manifestation, including retinitis pigmentosa, liver fibrosis, cerebellar vermis aplasia and situs inversus (1,2). Although a rare disease, NPHP is the most frequent genetic cause of kidney failure during the first three decades of life. Mutations of more than nine genes have been identified that can cause NPHP. The NPHP gene products (nephrocystins) are quite dissimilar, but share a unique subcellular localization, the cilium–centrosome complex. Multiple protein–protein interactions, facilitated by Src homology 3 (SH3), coiled-coil, ankyrin-repeat or tetratricopeptide domains, characterize the different members of the NPHP gene family (3–7), suggesting that nephrocystins participate in the formation of complex protein networks. NPHP has been categorized as a ciliopathy; however, the molecular functions of the NPHP gene products in the cilium have remained largely elusive. Several nephrocystins have been identified in the connecting cilium of the photoreceptor, a zone that joins the outer segment of the photoreceptor to the photoreceptor's cell body (8,9). As protein synthesis is confined to the cell body, all peptides have to be transported through the connecting cilium to reach the outer segment. The presence of nephrocystins in this segment suggests that they form part of a multiprotein complex that regulates the transport of cargo molecules to the outer segment of the photoreceptor. A disruption of this transport has deleterious consequences for the structural and functional integrity of the photoreceptor, resulting in the loss of photoreceptors and degeneration of the retina. Retinal degeneration is a common clinical manifestation in NPHP, and eye abnormalities are present in all patients with either NPHP5 or NPHP6 mutations.
Nephrocystin-5 localizes to the connecting cilia of photoreceptors and the cilia of renal epithelial cells. Containing two calmodulin-binding IQ domains that flank a central coiled-coil region, nephrocystin-5 binds calmodulin and is found in a complex with the retinitis pigmentosa GTPase regulator (RPGR) (8). Nephrocystin-6, a protein with multiple coiled-coil domains, has been reported to associate with RPGR and ATF4, a transcription factor implicated in cAMP-dependent renal cyst formation (9,10). Hypomorphic mutations of NPHP6, for example the common c.2991+1655A-G splice mutation, seem to represent the most frequent cause of childhood blindness (11,12).
In the present study, we demonstrate that nephrocystin-5 can recognize and bind nephrocystin-6, explaining the overlapping clinical phenotype and the strong genetic interaction of both gene products during zebrafish and Xenopus embryogenesis. Furthermore, we found that several domains of nephrocystin-6 engage in homo- and heterodimeric interactions, suggesting that conformational changes of nephrocystin-6 regulate the interaction with other proteins and the assembly of multiprotein complexes.
Depletion of zebrafish NPHP5 (zNPHP5) causes pronephric cysts in combination with cerebral abnormalities
We identified the zebrafish homologue of human NPHP5 (NM001008622; ZFIN gene: zgc 101792) on chromosome 6, and targeted the second exon by an antisense morpholino-oligonucleotide (MO5) to induce aberrant splicing between the second and third exon (Fig. 1A). This strategy, which deletes exon 2 and results in a premature stop codon at the beginning of exon 3, was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis. Knockdown of zNPHP5 caused significant developmental changes, including an abnormal body curvature (Fig. 1B), cerebral abnormalities, characterized by a defective mid-to-hindbrain boundary with the development of hydrocephalus (Fig. 1B and C), and the presence of pronephric cysts (Fig. 1D). Rescue experiments using in vitro transcribed human NPHP5 mRNA ameliorated the observed phenotypes and developmental abnormalities (Fig. 1E), confirming the specificity of the MO-mediated knockdown of zebrafish NPHP5.
The combined knockdown of NPHP5 and NPHP6 during zebrafish development is synergistic
Depletion of zebrafish NPHP6 (zNPHP6) by an antisense morpholino-oligonucleotide (MO6) recapitulates the renal, retinal and cerebellar phenotypes of Joubert syndrome (9). We noted that an abnormal mid-to-hindbrain region was associated with hydrocephalus in NPHP6-deficient zebrafish embryos (Fig. 2A); this phenotype developed in a dose-dependent fashion (Fig. 2B). Pronephric cysts were already present at low MO6 concentrations (0.1 mm), and increased to 37.5% at 0.6 mm (Fig. 3). The individual knockdown of either zNPHP5 by MO5 or zNPHP6 by MO6 had little effect on pronephric cyst formation at low MO concentrations (0.05–0.1 mm) (<15%). However, a combination of 0.05 mm MO5 and MO6 caused pronephric cysts in more than 39% of embryos, suggesting a genetic interaction between NPHP5 and NPHP6 (Fig. 4).
Nephrocystin-5 recognizes and directly binds a defined domain of nephrocystin-6
Nephrocystin-6 was found to interact with several centrosomal/ciliary proteins, including the RPGR, but not with nephrocystin-5 (10). Since the combined knockdown of NPHP5 and NPHP6 supports a genetic interaction between both gene products, we analyzed the biochemical interaction of nephrocystin-6 with nephrocystin-5. Since expression of full-length nephrocystin-6 did not yield sufficient protein levels, we divided this large coiled-coil protein into several fragments, and tested the interaction of each fragment with nephrocystin-1, -2, -3, -4 and -5. As demonstrated in Figure 5, the fragment of nephrocystin-6, spanning amino acids 665–1288, interacted with nephrocystin-5, but not with any of the other nephrocystins. Further truncational analysis limited the interacting domain of nephrocystin-6 to a region between amino acid 696 and 896 (Fig. 6A). Pull-down experiments, using this fragment of nephrocystin-6 fused to GST (GST–NPHP6696–896) suggested that this interaction was direct (Fig. 6B). Taken together, these findings demonstrate that nephrocystin-5 can specifically recognize a domain of nephrocystin-6, encompassing the third coiled-coil domain (CC III) of nephrocystin, as well as part of the SMC (structural maintenance of chromosomes) homology domain.
The nephrocystin-5-binding fragment of nephrocystin-6 prevents neural tube closure in Xenopus laevis
We discovered that knockdown of Xenopus nephrocystin-5 prevented neural tube closure during early tadpole development (Fig. 7A and B), and speculated that the nephrocystin-5-binding domain of nephrocystin-6 (amino acids 665–1288) should exert a dominant-negative effect, and interfere with the function of nephrocystin-5. Indeed, injection of NPHP6 (665–1288) mRNA into Xenopus laevis embryos resulted in severe neural tube closure defects (Fig. 7A and C). This defect was rescued by simultaneous co-expression of nephrocystin-5, reducing the neural tube closure defect from 82% to 43% (Fig. 7D). These findings further support our hypothesis that the genetic link between NPHP5 and NPHP6 is mediated by a physical interaction between both proteins.
N- and C-terminal fragments of nephrocystin-6 form homo- and heterodimers
Coiled-coil domains do not only mediate interaction with other proteins, but can also induce intramolecular interactions between different regions of the same protein. We determined therefore, whether nephrocystin-6 fragments can form homo- or heterodimers. As shown in Figure 8, the N-terminal fragment of nephrocystin-6 (amino acids 1–695) interacted with itself and the C-terminal fragment of nephrocystin-6 (amino acids 1966–2479), which also formed homodimers. This finding suggests that the C-terminal end of nephrocystin-6 can fold back on the N-terminal fragment; alternatively, nephrocystin-6 can form multimers through homodimerization of the N- and C-terminal domains.
NPHP6 mutations are associated with isolated cystic kidney disease (13) or retinal dystrophy (Leber congenital amaurosis) (11), but can also manifest as a multisystemic disease with brain stem malformation (Joubert syndrome), extensive embryonal defects and perinatal lethality (Meckel-Guber syndrome) (9,14–16). The gene product of NPHP6, nephrocystin-6, forms a complex with several ciliary and centrosomal proteins, including RPGR and RPGR-interacting protein 1 (RPGRIP1; 10). The rd16 mouse develops early-onset retinal degeneration due to the deletion of base pairs 5073–5969 (amino acids 1599–1897) of NPHP6/CEP290. This mutant binds RPGR more avidly than wild-type nephrocystin-6, suggesting that an abnormal interaction between both proteins contributes to the development of retinal disease.
Since all patients with NPHP5 or NPHP6 mutations develop retinal dysfunction, we compared the developmental defects caused by either NPHP5 or NPHP6 depletion in zebrafish embryos. Knockdown of either nephrocystin-5 or nephrocystin-6 in zebrafish embryos produced almost identical abnormalities, including hydrocephalus, developmental eye defects and pronephric cysts. Furthermore, the combined knockdown of both proteins using low morpholino-oligonucleotide (MO) concentrations synergistically augmented these phenotypes. To understand the molecular basis for this genetic interaction, we analyzed the biochemical properties of nephrocystin-5 and nephrocystin-6 in more detail. Nephrocystin-6 does not interact with nephrocystin-5 in retinal extracts (10); however, nephrocystin-5 specifically recognized a domain of nephrocystin-6 located between amino acids 696 and 896. The specificity of this interaction is underlined by the observation that neither of the other four nephrocystins nor RPGR recognized any of the nephrocystin-6 domains.
If nephrocystin-5 and -6 form a functional protein complex, expression of the nephrocystin-5-binding fragment of nephrocystin-6 should exert a dominant-negative effect, and interfere with the function of NPHP5. We noted that NPHP5 and NPHP6 mRNA are detectable during early Xenopus development (data not shown). Expression of the nephrocystin-5-binding domain of nephrocystin-6 inhibited neural tube closure during early embryogenesis; a similar phenotype was observed after MO-mediated knockdown of Xenopus NPHP5, suggesting that both approaches target the same signaling pathway. This interpretation is supported by the finding that co-expression of nephrocystin-5 reversed the neural tube closure defects caused by the nephrocystin-6 fragment.
Nephrocystin-6 consists of multiple long-stretched coiled-coil domains. Recent findings suggest that the C. elegans kinesin-2 motor protein OSM-3 is regulated by intramolecular folding that involves the coiled-coil stalk and a central hinge region of OSM-3. This intramolecular folding results in autoinhibition of OSM-3, which is presumably relieved by IFT cargo-binding (17). Thus, access of nephrocystin-5 to the nephrocystin-5-binding domain in nephrocystin-6 may be strongly influenced by parallel or antiparallel intramolecular coupling of nephrocystin-6 domains. Our finding that the N- and C-terminal domains of nephrocystin-6 engage in homo- and heterodimeric interactions supports this possibility (Supplementary Material, Fig. S1). Thus, access of interacting proteins such as nephrocystin-5 and RPGRIP1 may be regulated by conformational changes of nephrocystin-6. Based on our findings, we propose that the rd16-associated loss of amino acids 1599–1897 causes a conformational change of nephrocystin-6 that increases the recruitment of RPGR (10). If the function of nephrocystin-6 is significantly influenced by its overall conformation, mutations of nephrocystin-6 that result in conformational changes of nephrocystin-6, may cause extensive clinical disease, while mutations that maintain the overall conformation of nephrocystin-6, may only affect tissue-specific functions. Our results together with the absence of nephrocystin-5 in nephrocystin-6-containing retinal precipitates suggest that access of nephrocystin-5 to nephrocystin-6 is precluded at least in some tissues. Although future work needs to address how conformational changes of nephrocystin-6 are initiated, it is interesting to note that the NPHP8 gene product, RPGRIP1L, also contains multiple coiled-coil domains. Our study suggests that extensive coiled-coil domains may regulate the assembly of functional nephrocystin complexes through intra- and intermolecular coupling of their coiled-coil domains.
MATERIALS AND METHODS
Reagents and plasmids
FLAG-tagged full-length versions of nephrocystin-1, -2, -3, -4 and -5 were used as previously described (4,7,18). FLAG- and V5-tagged truncations of nephrocystin-6 were created by PCR and standard cloning techniques, and fused to YFP (eYFP-C1, Clontech, Mountain View, CA), and GST- (pGEX4-T1, GE Healthcare, Freiburg, Germany). Antibodies used in this study, included mouse M2 antibody to FLAG (Sigma, Hamburg, Germany), mouse antibody to V5 (Serotec), mouse antibody to GFP (MBL, Woburn, MA), and mouse antibody to GST (Amersham, Buckinghamshire, UK). Membrane-targeted RFP-pCS2+ was provided by J. Wallingford (Austin, TX, USA). Blast searches with human nephrocystin-5 identified MGC81507 (Acc BC068847) as the Xenopus laevis homologue of nephrocystin-5. The corresponding clone was obtained from ImaGenes GmbH, Berlin, and isolated by PCR. Truncations of NPHP6 and full-length xNPHP5 were subcloned into a modified pxT7Hi, linearized with SalI, and transcribed with T7 RNA polymerase (Ambion, Austin, TK). Sox3 was amplified from stage 21 X. laevis cDNA using the following primers: sox3-fp (5′-CGCGGGACGCGTATG TATAGCATGTTGGACACCGACA-3′) and sox3-rp (5′-GAATGCGGCCGCTTATATGTGA GTGAGCGGTACCG TGCCA-3′). The resulting product was cloned into pGEM-T using the Easy Vector System (Promega, Madison, Wisconsin).
Zebrafish lines and manipulations
Zebrafish manipulations were performed as recently described (19). Total RNA was isolated from wild-type and MO-injected zebrafish embryos using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The znphp5 fragment was amplified by nested PCR, using the following primers: outer pair: zNPHP5-RT out-fp (5′-AGAGCTCAAGGATTTGGTGGAAGAC-3′, forward), zNPHP5-RT-rp1 (5′-CCTGAAGATATCGTTCCACAAGGTG-3′, reverse); inner pair: zNPHP5-RT-fp 5′-GATTTGGTGGAAGACACCAGAGAAA-3′, forward), zNPHP5-RT-rp2 (5′-TCGTTCCACAA GGTGACAGTAACAA-3′, reverse). PCR products were ligated into pCRII vector (Invitrogen, Carlsbad, CA), and sequenced to characterize the altered mRNA splicing products. Fertilized eggs were microinjected with 4.6 nl of injection solution at the one- to two-cell stage with antisense MO (Gene Tools LLC, Philomath, OR) diluted in 200 mm KCl, 0.1% Phenol Red and 10 mm HEPES, pH 7.5). The following MO were used: zNPHP5-Ex2donor, (MO5: 5′-TCAAATCTGAATACCTGAGGAGGTC-3′), and the zNPHP6-Ex42donor MO (MO6: 5′-TGAAAAGTTGCACCTACAGAGGGTC-3′) (9). For rescue experiments, capped human NPHP5 mRNA was generated using the mMessage mMachine Kit in combination with SP6 RNA polymerase (Ambion, Austin, TX). Zebrafish embryos were analyzed with a Leica MZ16 stereomicroscope; images were taken with a SPOT Insight fire wire system (Diagnostic Instruments, Inc., Sterling Heights, MI), and processed with the SPOT imaging software (version 4.1) and Adobe Photoshop. For histology analysis, embryos were fixed in 4% PFA in phosphate-buffered saline, and embedded in Technovit 7100 resin (Heraeus, Wehrheim, Germany). Sections of 5 µm were stained with Methylene Blue/Azure II.
Xenopus embryonic manipulation and microinjection
Eggs were obtained from X. laevis females, fertilized and staged as recently described (20). In vitro synthesis of capped mRNA was performed using the Ambion mMESSAGE mMACHINE kit. RNA or MO were injected into both dorsal blastomeres of four-cell stage embryos. An ATG-blocking MO was obtained for NPHP5 (Gene Tools LLC, Philomath, OR, USA) (NPHP5-Mo: 5′-GGTCAACAGTGTCCGCCATTGTTC-3′; control MO: 5′-CCTCTTACCTCAG TTACAATTTATA-3′).
Whole mount in situ hybridization
Whole mount in situ hybridization was performed as described (21). Embryos were hybridized with antisense RNA probes for sox3. The probe was transcribed with SP6 RNA polymerase, and labelled with digoxigenin (DIG; Roche, Mannheim, Germany). Staining was carried out using anti-DIG-AP Fab fragments (Roche) and 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP). After fixation in Bouin's fix, embryos were bleached (1% H2O2, 5% formamide, 0.5X SSC) to remove pigmentation.
Co-immunoprecipitation and GST pulldown
Co-immunoprecipitation experiments were carried out as described (22). For GST pulldown experiments, the fusion proteins were expressed in Escherichia coli BL21 (λDE3) strain, lysed in 50 mm Tris–HCl pH 7.5, 100 mm NaCl, 10 mm EDTA, 5 mm DTT and 0.5 mm PMSF, and purified using GSH-Sepharose (Amersham, Buckinghamshire, UK). Human embryonic kidney cells (HEK) 293T were transfected with FLAG-NPHP5 (pcDNA6) or empty vector, and lysed after 24 h in 50 mm Tris–HCl pH 7.5, 150 mm NaCl and 0.5% Triton X-100. Cellular protein (200 µg) was incubated with 10 pmol of purified protein for 30 min at 4°C, followed by incubation with 30 µl of GSH-beads for another 30 min, and subsequently washed three times with 1 ml of lysis buffer before analyzing the samples using SDS–PAGE and western blotting.
This work was supported by grants of the European Union (EUCILIA) to G.W.
We thank the members of the Renal Division for helpful discussions, and Christina Engel, Temel Kilic, Nelli Schetle and Simone Diederichsen for excellent technical assistance.
Conflict of Interest statement. None declared.
- situs inversus
- amino acids
- cystic kidney
- retinitis pigmentosa
- signs and symptoms
- xenopus laevis
- medullary cystic disease
- autosomal recessive inheritance
- signal pathway
- signal transduction pathways
- gene interaction
- binding (molecular function)