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*Department of Pediatrics, Philipps University, Marburg, Germany; Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark; Institute of Human Genetics, Philipps University, Marburg, Germany; Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Correspondence to Dr. Martin Konrad, Department of Pediatrics, Philipps University Marburg, Deutschhausstrasse 12, D-35037 Marburg, Germany. Phone: 49-6421-2862789; Fax: 49-6421-2865724; E-mail: konradm{at}mailer.uni-marburg.de
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutations in PCLN1 were found to be associated with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC, OMIM 248250), a hereditary renal disease with urinary magnesium and calcium loss and progression to end-stage renal failure (4,7,8). As concluded from the phenotype of FHHNC patients, an important role of paracellin-1 in the regulation of paracellular transport of divalent cations in the TALH was suggested. Little is known about human magnesium transport mechanisms; therefore, the identification of paracellin-1 represented a major step to elucidate the mechanisms of renal magnesium reabsorption. The bovine PCLN1 gene was recently identified, and PCLN1 deletions were described in Japanese Black cattle affected by an autosomal-recessive renal disorder (9,10). Renal histology in these animals showed a tubular atrophy associated with interstitial nephritis, pointing to an additional role of paracellin-1 for renal epithelial integrity.
To provide the basis for further studies of paracellin-1 in suitable animal models we performed a cloning strategy of the homologous genes in mouse and rat. We here present the cDNA sequences of mouse and rat Pcln1, the murine genomic structure, and the results of RNA and protein expression studies.
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Materials and Methods |
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By 5'-RACE-PCR (rapid amplification of cDNA ends) the lacking 5'-region was amplified from mouse kidney Marathon Ready cDNA (Clontech, Palo Alto, CA) with the Advantage II Polymerase Mix (Clontech) by using the mouse gene-specific primer mmGSP1 (for all primer sequences see Table 1). A Southern blot procedure was used to identify the correct PCR-fragment (ECL-3'-oligolabeling and detection systems; Amersham Pharmacia Biotech, Uppsala, Sweden) (results not shown). A mouse gene-specific primer, mmGSP2, was designed from the 5'-RACE product, and an additional 3'-RACE-PCR performed to obtain the complete full-length downstream sequence. The 784-bp and 485-bp PCR fragments were agarose gel purified, subcloned into a pCR2.1-TOPO vector (Invitrogen, Groningen, The Netherlands), and sequenced from both strands by using the ABI PRISM 310 Genetic Analyzer (Applera, Norwalk, CT).
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The cDNA sequence of full-length murine Pcln1 was deposited with GenBank accession no. AF323748 and the full-length rat cDNA sequence with accession no. AF333099.
Analysis of cDNA and Amino Acid Sequences
The final cDNA and amino acid sequences obtained were compared with human and bovine PCLN1 by using the FASTA and BLAST programs that are supplied by NCBI and Infobiogen web sites (http://www.ncbi.nlm.nih.gov; http://www.infobiogen.fr). Protein pattern and statistical protein structure analysis were performed by using the PROSITE protein pattern search tools (www.expasy.ch/prosite).
Determination of Exon-Intron Boundaries of Mouse Pcln1
To determine the exon-intron boundaries of murine introns two, three, and four, exon-specific primers were designed and the intervening intron sequences amplified from mouse genomic DNA with the Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany). The fragments were run on an agarose gel, and the approximate sizes were determined by using a half-logarithmic function. The exon-intron boundaries of these introns were sequenced directly with exon-specific primers.
The exon-intron boundaries of intron one were determined by using the Mouse Genome Walker Kit (Clontech). The nested PCR reaction yielded a single fragment that was subcloned into the pCR2.1-TOPO vector and sequenced from both strands. Alignment with human PCLN1 was performed with the CLUSTALW-program (http://clustalw.genome.ad.jp). The human gene structure was obtained by search in human genomic databases with the human cDNA sequence and by location of the human PCLN1 gene in GenBank contigs AC009520 and AC073963.
Chromosomal Assignment of Mouse Pcln1
The genetic map position of mouse Pcln1 was determined by segregation analysis by using gene-specific amplification of clone DNA from the Whitehead Institute Center for Genomic Research mouse radiation hybrid mapping panel (11). The primer pair mm-Int4-F/R spanning intron 4 of mouse Pcln1 was used for amplification (fragment size, 
1350 bp). Data vectors, which were based on two independent PCR analyses of the entire panel, with data arranged in the order specified for the Whitehead Institute/MIT Center for Genome Research, Mouse EST RH Mapping Project, Public Data Release 3 (April, 2000) were submitted to two-point maximum-likelihood analysis (http://www.genome.wi.mit.edu/cgi-bin/mouse_rh/rhmap-auto/rhmapper.cgi). Mouse-human-synteny was analyzed by using NCBI and MGI mouse-human homology databases (http://www..ncbi.nlm.nih.gov:80/Homology/mouse16.html; http://www.informatics.jax.org/searches/linkmap.cgi).
Northern Blot Analysis of Mouse Pcln1 Transcription
Poly(A)+ RNA from eight adult mouse tissues was studied by multiple-tissue Northern analysis (Clontech). Hybridization was carried out overnight at 55°C by using 10 ng of a PCR-amplified fragment comprising the full-length mouse Pcln1 coding sequence, radiolabeled with Redivue 32P (Rediprime II random prime labeling system; Amersham Pharmacia Biotech, Buckinghamshire, UK). Rapid-hyb buffer (5 ml) (Amersham Pharmacia Biotech) was used for hybridization. The membrane was then washed four times with decreasing SSC concentrations (2X/0.1% sodium dodecyl sulfate [SDS]-0.1X/0.1% SDS) and increasing temperatures (from room temperature up to 65°C). The membrane was autoradiographed at -70°C for 72 h by using two intensifying screens and a BioMax MS-film (Eastman Kodak, Rochester, NY). A positive control (dot blot with linearized and denatured plasmid DNA containing the murine Pcln1 full-length coding sequence) and a negative control (dot blot with linearized and denatured plasmid DNA containing the murine Cldn1 full-length sequence encoding claudin-1) were hybridized and washed in the same hybridization bottle. Thereafter, the Northern blot was probed with a Rediprime II random prime labeled human ß-actin cDNA probe (PCR fragment supplied by the manufacturer) to compare loading in each lane.
Microdissection of Rat Nephron Segments
Kidneys from healthy male Wistar rats (180 to 200 g; M & B, Eiby, Denmark) were perfused with saline via the abdominal aorta before perfusion with collagenase A in Dulbecco’s modified Eagle’s medium (Life Technologies, Tåstrup, Denmark) with 0.1% bovine serum albumin, 1 ml/L penicillin-streptomycin solution (Sigma Chemical, Taufkirchen, Germany), and 660 U/L insulin. Kidney slices were further incubated for 25 min at 37°C with gentle shaking. The slices were moved to Dulbecco’s modified Eagle’s medium that contained 10% fetal calf serum, 1 ml/L penicillin-streptomycin solution, and 660 U/L insulin. Tubules were dissected on ice by using a Leica MZ 12.5 microscope (Leica, Bensheim, Germany).
RNA Extraction
From the dissected rat nephron segments, mRNA was extracted from single segments of approximately 1 mm in length. Segments were not pooled, and mRNA was extracted by using poly-T coated magnetic beads (Dynabeads mRNA DIRECT Micro kit; Dynal, Oslo, Norway).
Reverse Transcriptase-PCR
Reverse transcriptase-PCR (RT-PCR) was performed on mRNA (directly on the magnetic beads) by using SuperscriptII (Life Technologies). RT negative control reactions were performed on half of the extracted mRNA. A fluorescence multiplex PCR (30 cycles) was carried out by using sequence-specific primers for rat Pcln1 (rn-PC-RT-F/R) and ß-actin (rn-ßA-RT-F/R). ß-actin was included to confirm the presence of cDNA, and a PCR negative control reaction was performed. The forward primers were fluorescence labeled. PCR products were analyzed by capillary electrophoresis by using the ABI PRISM 3100 Genetic Analyzer (Applera).
Solubilization of Rat Kidney Proteins
Rat renal tissue was disrupted in ice-cold 50 mM Tris-HCl, pH 7.4, that contained 1% Triton X-100, 0.5% Na-deoxycholacid, 0.1% SDS, 5 mM pefablock, 25 µM pepstatin A, 10 µM leupeptin, 1mM Na-orthovanadat, 10% glycerol, and 2 mM ethylenediaminetetraacetic acid by the use of an ultra turax device and a Potter-Elvehjem homogenizer. Final homogenization was achieved by ultrasound. The resulting homogenate was centrifuged at 10,000 g for 30 min to separate undisrupted tissue. The supernatant containing solubilized proteins was stored at -80°C until use. Protein concentration was determined with bicinchoninic acid according to the protocol of the supplier (Pierce, Rockford, IL) with BSA as protein standard.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Immunoblot Assay
Protein samples (100 µg/lane) were run on 4 to 20% polyacrylamide gradient gels (GradiGels, Gradipore, North Ryde, Australia) and then transferred onto a nitrocellulose membrane by electroblotting with a discontinuous buffer system. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline (TBS; 25 mM Tris, 150 mM NaCl, pH 7.5) at 4°C overnight and then incubated in 5% normal goat serum for 30 min. After washing with 0.1% Tween 20/TBS (TTBS), a polyclonal antibody directed against paracellin-1 peptide (1:1000 in 5% goat serum/TTBS) was applied for 4 h at room temperature. This antibody was raised in rabbits against the C-terminal peptide 282-VSMAKSYSAPRTETAKMYAVD-302 of human paracellin-1 coupled to keyhole limpet hemocyanin. Peptide sequence homology to rat paracellin-1 was 95% (20/21). No sequence homology to mouse and rat claudin-1 (GenBank Accession BC002003 and AF195500, respectively) and mouse claudin-2 (GenBank Accession AF072128) was observed. For detection of bound antibodies, a goat anti-rabbit IgG peroxidase conjugate (1:7500 in 5% goat serum/TTBS) was added to the membranes and incubated at room temperature for 45 min. After four wash cycles with TTBS, visualization was achieved by enhanced chemiluminescence technique (Amersham Pharmacia Biotech) with exposure times of 30 s to an autoradiographic film (Hyperfilm ECL; Amersham Pharmacia Biotech). In a control experiment, the primary antibody was omitted. In this experiment, no protein bands were visible after the staining procedure (not shown).
Immunohistochemistry
Rat kidneys were fixed in 4% paraformaline and 7-µm-thick sections were cut on a microtome (Rotationsmikrotom 3455 Leitz; Leica). For staining of paracellin-1 protein with the polyclonal antibody described above the alkaline phosphatase anti-alkaline phosphatase technique was used as described earlier (12). The specificity of immunolabeling was tested by omitting the primary antibody.
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Results |
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Like human PCLN1, mouse Pcln1 comprises five coding exons. Analysis of the murine exon-intron boundaries revealed identical coding exon sizes for exons 2 to 5. The adjacent intronic sequences show high similarity between the murine and human genes (Figure 1).
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Statistical Analyses
In analogy to the human protein, hydrophilicity plots predict four transmembrane segments and intracellular N- and C-termini for both mouse and rat paracellin-1. Statistical protein analysis revealed three putative protein kinase C phosphorylation sites in mouse and rat paracellin-1 (AA position 40, 217, and 225) and a C-terminal microbody targeting signal (TRV, AA postion 233 to 235). Sequence comparison of rodent paracellin-1 in protein databases yielded similarity with a great number of claudins of different species and also to hypothetical proteins of Caenorhabditis elegans (ZK563.4; GenBank accession no. AAA81150) and Drosophila melanogaster (CG6398; GenBank accession no. AAF48766) (UCSC Computational Biology Target99 alignment (13)).
Chromosomal Assignment of Mouse Pcln1 and Mouse/Human Synteny Map
Radiation hybrid panel analysis for mouse Pcln1 indicated a direct linkage to marker D16Mit133 on mouse chromosome 16q. D16Mit133 maps 20.2 cM distal to the centromere of mouse chromosome 16. As shown in Figure 3, this mouse chromosomal region is syntenic to human chromosome 3q27-q28, which harbors the human PCLN1 gene.
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Discussion |
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As it was shown for human and bovine PCLN1, mouse and rat Pcln1 are also highly homologous, which indicates evolutionary sequence conservation. Homology is not only seen on the amino acid level but also on the DNA level including exon-intron boundaries. However, all four species differ considerably in their 5'-UTR. Both human and bovine PCLN1 have more than one in-frame start codon in a suitable Kozak consensus sequence, which determines the translational start point (14). For human paracellin-1, Simon et al. (4) already discussed the possibility of the second methionine in-frame (Met71) to be the original translational start site because it is analogous to the start sites of other claudins. Comparing human and bovine PCLN1, both share only one start codon in-frame encoding human Met71 and bovine Met1 (GenBank accession no. AB035210), before a downstream sequence of high homology (91% on the amino acid level).
Genetic analysis of PCLN1 in humans revealed a polymorphism at amino acid position 55 that would result in a preterminal translation stop and consecutively to a truncated paracellin-1 protein. This polymorphism is frequently found in healthy individuals (8); therefore, it is suggested that the underlying nucleotide sequence is not coding.
The sequence data of mouse and rat Pcln1 provide further arguments for paracellin-1 being shorter than reported in the GenBank submission AF152101, as both lack a methionine that corresponds to human Met1 but share a methionine that corresponds to human Met71 and bovine Met1 (GenBank accession no. AB035210). The similarity of the downstream PCLN1 sequence is extremely high among all four species. In addition, the multiple alignment of paracellin-1 and other members of the claudin family demonstrates that all claudins share the short cytoplasmic N-terminus in protein prediction models with rodent paracellin-1 (1,2).
Tissue-specific transcription of murine Pcln1 was studied by multiple-tissue Northern blot analysis, which revealed expression exclusively in the kidney. Interestingly, two transcripts of 1.2 kb and 1.0 kb were detected. As the murine Pcln1 cDNA sequence is 939 bp in length without polyadenylation, the 1.2 kb transcript is considered to represent 3'-polyadenylated full-length Pcln1-mRNA. The shorter fragment might be due to alternative splicing with a deletion of sequence information and/or differences in 5' or 3' UTR length. It is less likely that the shorter transcript represents a highly homologous, yet unidentified, gene that is exclusively expressed in the kidney.
A kidney-specific expression of PCLN1 was also observed by Northern blot analysis in humans (4). These findings correlate with the renal-specific phenotype of the majority of FHHNC patients. Nevertheless, a subgroup of FHHNC patients also presents with ocular symptoms such as horizontal nystagmus, severe myopia, or congenital coloboma (15,16), and it still remains to be studied whether defects in paracellin-1 are also responsible for these ocular abnormalities.
For bovine PCLN1, expression was observed in the kidney but also to a minor extent in the lung (9). However, cattle with complete PCLN1 deletions do not show pathophysiologic changes in their lungs (9), and the role of bovine PCLN1 for respiratory function is unknown. No pulmonary PCLN1 transcript was found in human and murine Northern blot analysis.
Immunoblotting of whole rat kidney preparations with a polyclonal antibody directed against a C-terminal peptide of the paracellin-1 protein demonstrated two specific protein bands with approximate molecular weights of 50 kD and 35 kD, respectively. In contrast, Western blot analysis of human paracellin-1 revealed a single band with an apparent molecular mass of 36 kD (4). However, this result was observed after overexpression of human paracellin-1 (hsMet1-Val305) in bacteria. The 50-kD band found in rat kidney corresponds to the doubled predicted molecular weight of 26 kD for rat paracellin-1 (rnMet1-Val235) and might represent paracellin-1 dimers.
Expression of paracellin-1 along the nephron studied by RT-PCR and immunohistochemical analysis was observed in cortical and medullary TALH, DT, and CD. Previous immunohistochemical studies of paracellin-1 in human kidney demonstrated expression predominantly in the TALH (4). In all species examined so far, TALH expression of paracellin-1 is confirmed. This expression pattern is in accordance with the phenotype of FHHNC-affected patients who present with a dysfunction of the paracellular reabsorption of divalent cations in this nephron segment.
However, there are differences between species concerning paracellin-1 expression in the CD. In contrast to RT-PCR and immunohistochemistry on rat kidney, no CD signal was seen in RT-PCR on rabbit nephron segments, and paracellin-1 expression has not been described so far in the CD of human kidney (4). One might speculate that there is a difference in the extension of the expression pattern with respect to more distal tubule segments among different species. As there is no major paracellular transport of magnesium and calcium in the distal convoluted tubule and CD, the maintenance of renal ion transport mechanisms does probably not depend on paracellin-1 expression in these segments. On the other hand, the phenotype of FHHNC-affected patients points to a possible role of paracellin-1 for tubular integrity as these patients frequently develop end-stage renal disease that is histologically characterized by renal calcium deposits, interstitial inflammation, glomerular sclerosis, and tubular atrophy. For FHHNC patients, loss of renal function has often been related to the progression of medullary nephrocalcinosis. On the other hand, various hereditary tubulopathies associated with severe nephrocalcinosis (e.g. hyperprostaglandin E syndrome/antenatal Bartter syndrome (17) and distal renal tubular acidosis (18)) do not proceed to renal insufficiency.
Loss of renal function with tubular atrophy and interstitial nephritis are also seen in Japanese black cattle affected by a complete loss of the PCLN1 gene. Taken together, these findings support the theory that paracellin-1 might represent an important structural protein beside its role for paracellular reabsorption of magnesium and calcium in the TALH.
In summary, the identification of the two rodent analogous genes of human PCLN1 provides a useful tool for further studies on paracellin-1 and tight junction physiology in suitable animal models. Magnesium handling in the kidney is precisely regulated and paracellin-1 is the first protein identified to be essentially involved in renal magnesium transport mechanisms; therefore, these studies will be helpful for understanding magnesium homeostasis. Furthermore, analysis of paracellin-1 in embryonal and fetal rodent tissues might give insight into expression patterns during early developmental stages and enlighten its possible role for renal development and tubular integrity.
The availability of the mouse gene structure will be valuable for the generation of a mouse model for FHHNC by gene targeting in embryonic stem cells. Site-directed mutagenesis of PCLN1 in a target vector might allow the investigation of phenotypic effects of single amino acid exchanges, truncated gene products, and complete loss of paracellin-1.
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Acknowledgments |
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