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*
Division of Matrix Biology, Department of Medical Biochemistry and
Biophysics, Karolinska Institute, Stockholm, Sweden
Developmental Biology Research Program, Institute of Biotechnology,
University of Helsinki, Finland.
Correspondence to Dr. Karl Tryggvason, Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-17177 Stockholm, Sweden. Phone: +46 8 728 7720; Fax: +46 8 316 165; E-mail: karl.tryggvason{at}mbb.ki.se
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--isoform of the intracellular tight junction protein zonula
occludens-1, located at sites where the slit diaphragm meets the lateral cell
membrane of the foot processes of podocytes
(7,8). We have recently identified a novel protein, termed nephrin (9), and shown its specific localization to the slit diaphragm (10). The nephrin gene is mutated in congenital nephrotic syndrome (NPHS1) (9,11), a disease characterized by massive proteinuria and edema (12). Based on Northern and in situ hybridization, nephrin has been shown to be expressed by the glomerular podocytes (9). Using immunoelectron microscopy, we recently localized nephrin to the slit diaphragm where nephrin is surmised to form a zipper-like isoporous filter structure (10) similar to that proposed by Rodewald and Karnovsky based on transmission electron microscopic observations (13). Because nephrin resides in the slit diaphragm, and mutations in the gene cause severe nephrotic syndrome, this protein must have a crucial role in the filtration mechanism of the glomeruli and may participate more generally in the pathogenesis of proteinuria and renal failure.
To further characterize nephrin and facilitate studies on its involvement in renal physiology and disease, we have now determined the primary structure of both mouse and rat nephrin based on cDNA sequences, determined the structure of the mouse gene, and studied its expression pattern both by Northern and in situ hybridization analyses during mouse embryogenesis.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]dCTP
(3000 Ci/mmol) (Amersham Pharmacia Biotech), using random priming (Life
Technologies). Positive fragments, 2.4-kb EcoRI (positive for exons 2
to 3) and 7.2-kb BamHI (positive for exons 26 to 29), were purified
with the Gel Extraction kit (Qiagen, Chatsworth, CA) and subcloned into the
pBluescript SK(+) vector. The plasmids were isolated using the QIAprep spin
miniprepkit (Qiagen) and sequenced on an ABI 310 Genetic Analyzer (Perkin
Elmer). For identification of exons, the sequences were compared with those of
the human nephrin cDNA sequence using the FASTA program. This comparison
yielded exons 1 to 4 from an EcoRI fragment and exons 24 to 30 from a
BamHI fragment of the genomic BAC clone.
Cloning of Mouse Nephrin cDNA
Mouse nephrin exon-specific primers were designed from genomic sequence
data obtained from the subcloned BAC clone fragments. Two primer pairs were
used for PCR to amplify the cDNA from mouse kidney QUICK-Clone cDNA (Clontech,
Palo Alto, CA) with Advantage cDNA polymerase mix (Clontech): m(forw)1 from
the 5' untranslated region (UTR) 5'-GAC AGC AAC AAA CAA GCT GCT
GG-3' together with m(rev)1 primer from exon 29 5'-TCA CAC CAG ATG
TCC CCT CAG C-3'; and the same m(forw)1 primer together with m(rev)2
primer from the 3'UTR region 5'-GGA AAC AGG TGT CGT GAA GAG
TC-3'. The PCR products were agarose-gel purified and subcloned into the
pCR-Script SK(+) vector (Stratagene, La Jolla, CA). Two clones from each
amplification were sequenced from both strands using either ABI 310 or 377
Genetic Analyzers. The sequences were compared to each other using the FASTA
program, and nonmatching sequence segments were reamplified using Vent DNA
polymerase (New England Biolabs, Beverly, MA) from mouse kidney QUICK-Clone
cDNA in two rounds. The PCR fragments were agarose-gel purified and sequenced
directly from both strands. The final mouse cDNA sequence was determined by
comparing the sequences to that of the gene. Furthermore, the 5' region
of the cDNA was amplified (rapid amplification of cDNA ends [5' RACE])
in two separate reaction sets in two rounds from a mouse kidney Marathon-Ready
cDNA (Clontech) with the Advantage cDNA polymerase mix. In the first round,
AP1 primer was used together with exon 2-specific m(rev)3 primer 5'-CAG
AAG CAG CCC ATC CTT AGC-3' and m(rev)4 primer 5'-CAG GTA ACT GTG
CTT CCT GCC TC-3'. The second, nested amplification round was performed
with AP2 primer together with exon 2-specific m(rev)3b primer 5'-CAG ATA
GAG CCC AGA AGC CTC G-3' and m(rev)3 primer that is upstream to the
m(rev)4 primer, respectively. The nested PCR round products were agarose
gel-purified and subcloned into pCR-Script SK(+) vector. Three subclones were
sequenced from both strands.
Cloning of Rat Nephrin cDNA
To obtain the rat nephrin cDNA, a rat kidney 
gt10 cDNA library
(Clontech) was screened using a human nephrin cDNA fragment comprising exons 3
to 29 as a probe, according to the manufacturer's instructions. Three positive
primary plaques were obtained and, after secondary screening, three
positive-phage DNA were isolated using a lambda mini kit (Qiagen), digested
with EcoRI and subcloned into pBluescript SK(+). Three clones, 3.32,
5.92, and 6.21, were sequenced from both strands and the sequences were
analyzed with the FASTA program. A missing segment between clones 3.32 and
5.92 was amplified using Vent DNA polymerase from the rat kidney gt10
cDNA library using two primer pairs: r(forw)1 5'-CAT CCT GGC CAA CTC GTC
CG-3' together with r(rev)1 5'-GGA GTA GGC TGA TCC ACC
TG-3'; and r(forw)2 5'-CAG GCG ACG CCT TGA ACT TG-3'
together with r(rev)2 5'-GCA AAT CGG ACG ACA AGA CG-3'. The
fragments were agarose-gel purified and subcloned into the pCR-Script SK(+)
vector. Two clones from each reaction were sequenced from both strands. To
obtain the missing 5' end, two sets of amplification reactions were
performed from the rat kidney Marathon-Ready cDNA (Clontech) using Vent DNA
polymerase (5' RACE). First, a round using primer pair AP1 together with
r(rev)3 5'-AGC ACG ATC TCC TTC CAG GC-3' was carried out, followed
by a second nested round using primer pair AP2, together with r(rev)4
5'-GAA GCC TGG CAT CTT CGG G-3'. The resulting PCR fragments were
agarose gel-purified and sequenced directly from both strands.
Analysis of cDNA and Amino Acid Sequences
The final cDNA and amino acid sequences were compared with the human ones
using the FASTA and BLAST programs. Alignment of amino acid sequences was
performed with the PileUp program (Genetics Computer Group, Madison, WI), and
signal peptide prediction with the SPScan program (Wisconsin Package version
10.0, Genetics Computer Group). Protein patterns were searched using the
PROSITE protein pattern search tool
(16).
Determination of Exon-Intron Boundaries of the Mouse Nephrin
Gene
To determine the exon-intron boundaries, exon-specific primers were
designed and intervening intron sequences were PCR amplified from BAC clones
using HotStarTaq DNA polymerase (Qiagen). The fragments were size-determined
and agarose-gel purified. The exonintron boundaries were sequenced from both
strands either from the amplified fragment or from the subcloned BAC-fragments
described above. The smallest introns were sequenced entirely. For intron 23,
the BAC Southern nylon filter was hybridized at 65°C with a mouse nephrin
cDNA fragment comprising exons 23 to 24. A positive 8.7-kb EcoRI
fragment was agarose gel-purified, subcloned into pBluescript SK(+), and
sequenced with exons 23 and 24-specific primers to find the exon-intron
junction. A region with high GC content in the boundary of exon 23 and intron
23 was sequenced until a suitable site for PCR primer annealing was found. The
rest of intron 23 was PCR-amplified with intron 23-specific primers.
Northern Analysis of Mouse Nephrin Expression
By using multiple-tissue Northern analysis, poly(A)+ RNA from
eight adult mouse tissues and 7-, 11-, 15-, and 17-d-old mouse embryos were
studied (Clontech). Hybridizations were carried out at 65°C using a
PCR-amplified fragment comprising exons 23 to 29 as probe, as described above.
The Northern blots were also probed with similarly labeled human ß actin
cDNA to compare the loading in each lane.
In Situ Hybridization
For in situ hybridization, a probe from the 3' end of the
gene was amplified from a mouse kidney QUICK-Clone cDNA using exon 29 specific
m(forw)2 primer 5'-GAC CCC TAT GAC CTT CGC TG-3', together with a
3'UTR-specific m(rev)5 primer 5'-CAG ATG TCA GCT GGA GTC
TTC-3'. The amplification was performed using Advantage cDNA polymerase
mix. The resulting fragment was subcloned into the pCR-Script SK(+) vector and
sequenced from both strands. The 219-bp sense BamHI and antisense
NotI 35S-labeled cRNA probes were transcribed, and in
situ hybridization was performed essentially as described by Wilkinson
and Green (17) with some
modifications. Briefly, E11 to newborn whole mouse embryos and isolated
kidneys were fixed in 4% paraformaldehyde and processed for paraffin
histology. Sections at 7 µm were cut, treated in prehybridization solutions
as described (17), and finally
hybridized overnight at +65°C. After high stringency washes at +65°C,
the sections were dehydrated, air-dried, dipped into NTB-2 emulsion (Eastman
Kodak), and exposed at +4°C in the dark for 10 to 14 d. The sections were
developed in D19 (Eastman Kodak), fixed in sodium fixative (Eastman Kodak),
counterstained in hematoxylin (Shandon, Pittsburgh, PA), and mounted. Sections
were analyzed and photographed with Olympus Provis AX microscope equipped with
a charge-coupled device camera (Photometrics Ltd.). The dark-field images were
inverted, stained red, and combined with the bright-field images in the Adobe
PhotoShop 4.0 program.
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Southern hybridization of the APLP1-positive BAC clones with different fragments of the human nephrin cDNA demonstrated that the entire mouse nephrin gene was present in two of the three clones obtained. Two restriction fragments of 2.4 and 7.2 kb hybridizing with the human cDNA were sequenced and shown to contain sequences corresponding to exons 1 to 4 and 24 to 29 in the human gene, respectively. Primers based on these exon sequences were then used for amplification of the mouse nephrin cDNA from pooled kidney mRNA, and the full-length cDNA could be compiled and used to determine the exon structure.
The mouse nephrin gene spans about 27 kb (Figure 1). To determine the exon-intron boundaries of the gene, exon-specific primers were used to amplify the intervening intron sequences from BAC clones except for intron 23, which was cloned separately (see Materials and Methods). Unlike human NPHS1, which has 29 exons, the mouse gene has 30 exons, the last exon encoding only for the major part of the 3' UTR. The exon sizes range from 25 to 216 bp (Figure 2) and are similar between human and mouse, except for exons 3, 8, 13, and 28, which differ by 1 to 6 bp (11). The codon for translation initiator methionine is in the first exon, and the translational stop codon TGA is in exon 29.
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In human, an unusual donor site starting with GC instead of the GT exists at exon 23, which encodes part of the fibronectin type III module (11). This turned out to be the case at mouse exon 23 as well. All other acceptor (AG) and donor (GT) sites are conventional.
Determination of Mouse and Rat Nephrin cDNA Sequences
The coding sequence of the full-length mouse cDNA was shown to be 3768
nucleotides. The codon for the first possible initiator methionine was more
5' than that of the human sequence
(Figure 3). This was verified
using a 5' RACE reaction, as well as by analyzing the sequence of the
5' end of the gene (Figure
2). The analyzed genomic exon sequences of the mouse gene were all
found in the sequenced mouse cDNA clones.
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The sequence of the rat nephrin cDNA was determined from three cDNA clones, 3.32 containing bases 270-1819, 5.92 containing bases 2526-3840, and 6.21 containing bases 2730-3971 (not shown). The missing parts were amplified either from the same cDNA library or with 5' RACE. The full-length rat cDNA contained 3756 bp. Two codons for methionine are present in the 5' end, the first located at the same site as that in the mouse cDNA and the other 12 bp downstream of the codon for the initiator methionine in the human cDNA. Because the nucleotides around the upstream methionine codon better fit the Kozak's consensus sequence (20) for translation initiation than the downstream one, the upstream codon is probably used for the initiator methionine. This is also supported by the high identity (82%) in nucleotide sequence between the mouse and rat from the first rat methionine codon to the second.
Comparison of cDNA-Deduced Mouse, Rat, and Human Nephrins
While the mouse and rat amino acid sequences show 93% identity, both the
mouse and rat amino acid sequences show 83% identity with the overlapping
areas of the human nephrin sequence. The most notable difference is at the
amino termini, where the mouse and rat proteins are 14 residues longer than in
human (Figure 3). Mouse nephrin
has 1256 amino acid residues with a predicted molecular weight of 136 kD
without posttranslational modifications, whereas rat nephrin has 1252 residues
with a predicted molecular weight of 136 kD. Human nephrin has previously been
shown to contain 1241 residues, with a molecular weight of 135 kD
(9).
The signal peptide cleavage site in human nephrin is located between residues 22 and 23, whereas it is predicted to be between residues 36 and 37 in mouse and rat. All 10 potential N-glycosylation sites are conserved between the three species (Figure 3). Of the seven serine-glycine (SG) doublets that are potential heparan sulfate attachment sites, only two are conserved between the three species.
Similar to Ig motifs in other proteins (21), all eight Ig motifs in nephrin of the three species contain two conserved cysteines that form disulfide bonds within the motif. In addition, the mouse and rat nephrin molecules contain three cysteine residues that are conserved between the species and, depending on the species, one or two unconserved residues. The conserved residues are one in the first Ig motif, one in the spacer domain, and one in the transmembrane domain. The unconserved residues include one cysteine in Ig motif 4 in the mouse, one each in Ig motifs 2 and 4 in the rat, and one residue in the fibronectin III domain in human nephrin. Furthermore, both the mouse signal peptide and the rat intracellular part have one cysteine residue each (Figure 3).
The intracellular domain in human has nine tyrosine residues that are tentative phosphorylation sites for intracellular signaling, while the corresponding domains of mouse and rat contain 10 and eight residues, respectively. Six of those tyrosine residues are conserved between the three species. However, the sequences around the tyrosines are completely conserved between the three species only in the case of three of the residues. According to the numbering of mouse nephrin, these are tyrosines 1128, 1208, and 1232. Tyrosine phosphorylation of nephrin is still an open question, so the naming of any specific interactors is purely speculative. Yet, comparison to the optimal recognition motifs for SH2 domains from different proteins reveals that if tyrosine 1208 is phosphorylated, a binding site for an adapter protein Nck might be generated. This motif pYDEV, and the one containing tyrosine 1232, pYDQV, may also provide a binding site for tyrosine kinases of the Src family (22,23). Furthermore, two potential conserved protein kinase C phosphorylation sites with the consensus sequence Ser/Tyr-X-Arg/Lys (Figure 3) were detected by PROSITE protein pattern search tool (16).
Developmental Expression of Mouse Nephrin
The overall expression pattern of mouse nephrin was studied by Northern
analysis, using RNA from whole mouse embryos of different ages and adult mouse
tissues. No signals above background could be observed with
poly(A)+ RNA from four different aged mouse embryos (not shown).
With poly(A)+ RNA from eight adult mouse tissues, the analysis
revealed a signal only with RNA from kidney
(Figure 4). RNA from heart,
brain, spleen, lung, liver, skeletal muscle, and testis were all negative.
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Because Northern analyses may not reveal the presence of transcripts with highly restricted expression, we carried out in situ hybridization on whole mouse embryos and isolated metanephric and mesonephric kidneys. At E11, when the metanephric differentiation is initiated, no signal could be detected from the mesenchymal cells of the presumptive kidney (Figure 5C). However, nephrin expression was seen in the E11 mesonephric kidney, where cranial tubules with podocyte-like structures revealed strong expression, while the mesonephric mesenchyme and less differentiated caudal tubules remained negative (Figure 5, A and B). A highly specific expression was observed in the podocytes of the developing kidney beginning from the E13 mouse embryos (Figure 5, E and F). Early S-shaped bodies, as well as the metanephric mesenchyme, remained negative at this stage. Surprisingly, high expression of nephrin mRNA was observed in the hindbrain and spinal cord (Figure 6A). At E11 in the brain, the expression was limited to a subset of neurons in the hindbrain area and continued from there dorsally, confined to the mantle layer neurons of the spinal cord (Figure 6B). A similar expression pattern was observed at E13 (Figure 6C). From E13 to E17, nephrin mRNA was detected in neuroepithelium of cerebellum primordium at the roof of the fourth ventricle, but the epithelial cells of developing choroid plexus, which are involved in the formation of cerebrospinal fluid, remained negative. Expression was not observed in any other tissues of the embryo.
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The present analyses of the mouse gene demonstrated its similarity to the human one, except for the 3' UTR, which is encoded by two exons (exons 29 and 30) in the mouse gene as opposed to one (exon 29) in the human gene (11). The gene sequence was also important for verification of the mouse cDNA sequence, which differs particularly in the 5' end, that is, in the signal peptide from the human gene. The presently elucidated mouse gene structure will be valuable for enabling the generation of a mouse model for congenital nephrotic syndrome, using gene targeting in embryonic stem cells.
The mouse and rat cDNA sequences determined here showed that mouse and rat nephrins have 93% sequence identity, while human nephrin gene shows a considerably lower sequence identity of 83% with the two rodent species. These sequences of nephrin from three mammalian species are of considerable value in several respects. First, the conserved residues suggest significant structural motifs and functional importance for the molecule. Second, the availability of the sequences is important not only for the interpretation of the impact of the numerous sequence variants found thus far both in patients with nephrotic syndrome and in the disease carriers, but also for the numerous homozygous and heterozygous allele variants found in the general population with no family history of nephrotic syndrome (9,11).
Several conclusions can be made from the comparison of the three primary structures of nephrin. Thus, the three conserved, apparently free cysteines in the Ig motif 1, the spacer domain, and the transmembrane domain are probably crucial for the function of the protein. This supports our hypothetical model for the zipper-like nephrin structure of the slit membrane (10), where nephrin molecules from two opposite foot processes interact in an interdigitating manner through homophilic interactions reinforced by intermolecular disulfide bonds between the two free extracellular cysteines in Ig motif 1 and the spacer domain. Furthermore, the glycoprotein nephrin has 10 conserved N-glycosylation sites, suggesting that controlled glycosylation is important for the protein.
There are six conserved tyrosine residues in the intracellular domain of nephrin. It is not yet known whether any of those serves as a phosphoacceptor site. If this turns out to be the case, one could envision several roles for the tyrosine phosphorylation. First, the phosphorylation could have a role in the activation of signal transduction pathways upon binding of the extracellular ligand(s). Interestingly, residues immediately carboxy-terminal to three of the tyrosines are completely conserved between the three species. Thus, comparison to optimal binding sequences for SH2 domains suggests that signaling molecules, such as an adaptor protein Nck and Src-family protein kinases, may be recruited to nephrin if tyrosines 1208 and 1232 are phosphorylated. Second, it is also possible that tyrosine phosphorylation of nephrin plays either a positive or negative role in the assembly of the glomerular slit diaphragm. In this regard, it is noteworthy that tyrosine phosphorylation of the intracellular domain of neurofascin, which also is a transmembrane protein of the Ig superfamily, abolishes its binding to the membrane skeletal protein ankyrin and reduces coupling of neurofascin to the cytoskeleton (24). Yet another possibility is that tyrosine phosphorylation regulates extracellular ligand specificity of nephrin, as might be the case with platelet endothelial cell adhesion molecule-1, another member of the Ig superfamily (25).
Thus far, 21 missense mutations resulting in amino acid changes, mainly in
the Ig modules, have been reported in patients with congenital nephrotic
syndrome
(9,11).
All of the mutated amino acid residues, except for two, are conserved between
the three mammalian species analyzed in the present study. The two missense
mutations affecting unconserved amino acid residues are Ile 173 
Asn in
Ig 2 and Arg 1140 Cys in the intracellular domain. Although not all
missense mutations involve conserved amino acids, it is apparent that one can
suspect individuals with allelic changes in codons for conserved amino acids
to be carriers for congenital nephrotic syndrome. Thus, the three sequences
can indeed have direct practical value.
Recently, cDNA and amino acid sequences were reported for the mouse (26) and rat (27) nephrin homologues. Comparison of the amino acid sequences between the mouse sequence reported here and in that by Holzman et al. (26) reveals differences in the 5' end or in the signal peptide region. In the sequence reported by Holzman et al. (26), the first 10 amino acids are 100% identical to the human sequence. The 5' end mouse sequence reported here, however, is longer, and it differs significantly from the human one. Comparison of the 5' end of the mouse sequence with that of the mouse genomic sequence showed that the sequence reported in this study is correct. Thus, the reason for the difference between the two mouse sequences is unknown. Otherwise, only two amino acids were different in the two mouse amino acid sequences: Thr763 reported here was Ala in the Holzman study, and Leu1076 was reported to be Gln in their study (26). Both Thr763 and Leu1076 are conserved in human and rat, indicating that they are also conserved in mouse. With regard to the rat sequences, we found a longer signal peptide than that reported by Ahola et al. (27). Furthermore, Ahola et al. reported that there is Asn in position 1229, whereas we found it to be Asp between all three species studied here.
Nephrin mRNA was located by in situ hybridization both to the embryonic mesonephric and metanephric kidneys. The undifferentiated metanephric mesenchyme and first epithelial structures, the comma- and early S-shaped bodies, remained negative. The first mRNA were detected in late S-shaped bodies in the definitive podocytes. A similar expression pattern was noticed in the glomerular structures of the mesonephric kidney, which reveals that the mouse cranial mesonephric nephrones (28) could be secretory. Instead, the caudal mesonephric nephrones did not express nephrin mRNA.
The presence of nephrin mRNA in a specific region of the brain and spinal cord raises the question about a role of this protein in neuronal development and function. Thus far, severe extrarenal symptoms have not been reported in NPHS1 patients who have received kidney transplants (29). However, of approximately 50 transplanted Finnish patients, about 10% have congenital neurologic symptoms, such as ataxia, not normalizing after transplantation. These symptoms cannot be explained by thrombosis or other secondary insults due to NPHS1 (Christer Holmberg, personal communication). It is quite plausible that these neurologic symptoms are related to absence of normal nephrin in brain tissue, and that this function cannot be compensated for in all individuals. Participating in guidance of neurons (30,31), axonal fasciculation (32), and growth and stabilization of synapses (33), many of the members of the Ig superfamily have been shown to possess multiple roles in neuronal differentiation. Because congenital ataxias are often associated with impaired development of the cerebellum and its connections (34), the expression of nephrin in the nervous system could indeed indicate a role in neurogenesis.
The recent evidence on interaction of the cytosolic CD2-associated protein (CD2AP) with nephrin (35) is of great interest. First and foremost, the interaction provides information on potential linkage mechanisms between nephrin and the actin cytoskeleton of podocyte foot processes (36). Furthermore, removal of this gene was shown to cause a nephrotic syndrome similar to NPHS1. However, the CD2AP-deficient mice die at 6 to 7 wk of age (35), while nephrin-deficient mice die on their first day of life (H. Putaala, unpublished information). Therefore, it is possible that nephrin has multiple associating ligands that could have a role in the filtration mechanism.
Note Added in Proof: After submitting this work, mAb 5-1-6 was shown to be directed against the extracellular domain of rat nephrin (Topham PS, Kawachi H, Haydar SA, Chugh S, Addona TA, Charron KB, Holzman LB, Shimizu F, Salant DJ. J Clin Invest 104: 1559-1566, 1999).
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Journal of the American Society of Nephrology
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). Conserved
tyrosine residues in the intracellular domain are indicated with a dot
(•), and two possible protein kinase C phosphorylation sites with the
consensus sequence Ser/Tyr-X-Arg/Lys are marked with a star (*).
