Primary Gene Structure and Expression Studies of Rodent Paracellin-1

J Am Soc Nephrol 12: 2664–2672, 2001 Primary Gene Structure and Expression Studies of Rodent Paracellin-1 STEFANIE WEBER,* KARL P. SCHLINGMANN,* MELANIE PETERS,* LENE NIEMANN NEJSUM,† SØREN NIELSEN,† HARTMUT ENGEL,‡ KARL-HEINZ GRZESCHIK,‡ HANNSJO¨ RG W. SEYBERTH,* HERMANN-JOSEPH GRO¨ NE,§ ROLF NU¨ SING,* and MARTIN KONRAD* *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; and §Deutsches Krebsforschungszentrum, Heidelberg, Germany. Abstract. The novel member of the claudin multigene family, lengths and splice-site loci. By radiation hybrid mapping, the paracellin-1/claudin-16, encoded by the gene PCLN1, is a renal murine Pcln1 gene was assigned directly to marker D16Mit133 tight junction protein that is involved in the paracellular trans- on mouse chromosome 16 (syntenic to a locus on human port of magnesium and calcium in the thick ascending limb of chromosome 3q27, which harbors the human PCLN1 gene). Henle’s loop. Mutations in human PCLN1 are associated with Mouse multiple-tissue Northern blot showed Pcln1 expression familial hypomagnesemia with hypercalciuria and nephrocal- exclusively in the kidney. The expression profile along the cinosis, an autosomal recessive disease that is characterized by nephron was analyzed by reverse transcriptase–PCR on micro- severe renal magnesium and calcium loss. The complete cod- dissected nephron segments and immunohistochemistry of rat ing sequences of mouse and rat Pcln1 and the murine genomic kidney. Paracellin-1 expression was restricted to distal tubular structure are here presented. Full-length cDNAs are 939 and segments including the thick ascending limb of Henle’s loop, 1514 bp in length in mouse and rat, respectively, encoding a the distal tubule, and the collecting duct. The identification and putative open-reading frame of 235 amino acids in both species characterization of the rodent Pcln1 genes provide the basis for with 99% identity. Exon-intron analysis of the human and further studies of paracellin-1 function in suitable animal mouse genes revealed a 100% homology of coding exon models. Members of the claudin multigene family have been identified fied in the kidney, especially in the thick ascending limb of as essential components of the endothelial and epithelial tight Henle’s loop (TALH) (4). junction barrier. In 1998, claudin-1 and claudin-2 were first Mutations in PCLN1 were found to be associated with described by Furuse et al. (1) and were colocalized with the familial hypomagnesemia with hypercalciuria and nephrocal- ubiquitous tight junction protein occludin to cell-to-cell adhe- cinosis (FHHNC, OMIM 248250), a hereditary renal disease sions in chicken liver. Since then, a great number of proteins with urinary magnesium and calcium loss and progression to with related structure were identified in tight junction strands end-stage renal failure (4,7,8). As concluded from the pheno- of multiple tissues (2,3); some of them associated with hered- type of FHHNC patients, an important role of paracellin-1 in itary diseases in humans (4–6). All claudins represent mem- the regulation of paracellular transport of divalent cations in brane proteins with four transmembrane domains, two extra- the TALH was suggested. Little is known about human mag- cellular loops, and intracellular N- and C-termini. Recently, nesium transport mechanisms; therefore, the identification of paracellin-1/claudin-16, which is encoded by the gene PCLN1, paracellin-1 represented a major step to elucidate the mecha- was characterized as a new member of this protein family in nisms of renal magnesium reabsorption. The bovine PCLN1 humans. Although other members of the claudin family show gene was recently identified, and PCLN1 deletions were de- a wide tissue distribution, paracellin-1 was exclusively identi- scribed in Japanese Black cattle affected by an autosomal- recessive renal disorder (9,10). Renal histology in these ani- mals showed a tubular atrophy associated with interstitial nephritis, pointing to an additional role of paracellin-1 for renal Received May 25, 2001. Accepted July 10, 2001. Correspondence to Dr. Martin Konrad, Department of Pediatrics, Philipps epithelial integrity. University Marburg, Deutschhausstrasse 12, D-35037 Marburg, Germany. To provide the basis for further studies of paracellin-1 in Phone: 49-6421-2862789; Fax: 49-6421-2865724; E-mail: konradm@ suitable animal models we performed a cloning strategy of the mailer.uni-marburg.de homologous genes in mouse and rat. We here present the S.W. and K.P.S. contributed equally to this work. cDNA sequences of mouse and rat Pcln1, the murine genomic 1046-6673/1212-2664 Journal of the American Society of Nephrology structure, and the results of RNA and protein expression Copyright © 2001 by the American Society of Nephrology studies. J Am Soc Nephrol 12: 2664–2672, 2001 Paracellin-1 in Mouse and Rat 2665 Materials and Methods BLAST programs that are supplied by NCBI and Infobiogen web sites Cloning of Mouse and Rat Pcln1 cDNA (http://www.ncbi.nlm.nih.gov; http://www.infobiogen.fr). Protein pat- To obtain the cDNA sequences of mouse and rat Pcln1, a search tern and statistical protein structure analysis were performed by using with the coding sequence of human PCLN1 was performed in mouse the PROSITE protein pattern search tools (www.expasy.ch/prosite). and rat EST databases, which yielded five different mouse EST clones (EMBL/GenBank/DDBJ Accession no. BB499021, BB501602, BB Determination of Exon-Intron Boundaries of Mouse 0496375, BB498218, and AV380228) sequenced from the 3'-end Pcln1 corresponding to bases 790 to 989 of the human PCLN1 cDNA To determine the exon-intron boundaries of murine introns two, (EMBL/GenBank/DDBJ Accession no. AF152101) and two different three, and four, exon-specific primers were designed and the inter- rat EST clones comprising the 3'-end of rat Pcln1 (EMBL/GenBank/ vening intron sequences amplified from mouse genomic DNA with DDBJ Accession no. AI412107 and AW142781). the Expand Long Template PCR System (Roche Diagnostics GmbH, By 5'–RACE-PCR (rapid amplification of cDNA ends) the lacking Mannheim, Germany). The fragments were run on an agarose gel, and 5'-region was amplified from mouse kidney Marathon Ready cDNA the approximate sizes were determined by using a half-logarithmic (Clontech, Palo Alto, CA) with the Advantage II Polymerase Mix function. The exon-intron boundaries of these introns were sequenced (Clontech) by using the mouse gene–specific primer mmGSP1 (for all directly with exon-specific primers. primer sequences see Table 1). A Southern blot procedure was used to The exon-intron boundaries of intron one were determined by using identify the correct PCR-fragment (ECL-3'–oligolabeling and detec- the Mouse Genome Walker Kit (Clontech). The nested PCR reaction tion systems; Amersham Pharmacia Biotech, Uppsala, Sweden) (re- yielded a single fragment that was subcloned into the pCR2.1-TOPO sults not shown). A mouse gene–specific primer, mmGSP2, was vector and sequenced from both strands. Alignment with human designed from the 5'-RACE product, and an additional 3'-RACE-PCR PCLN1 was performed with the CLUSTALW-program (http://clust- performed to obtain the complete full-length downstream sequence. alw.genome.ad.jp). The human gene structure was obtained by search The 784-bp and 485-bp PCR fragments were agarose gel purified, in human genomic databases with the human cDNA sequence and by subcloned into a pCR2.1-TOPO vector (Invitrogen, Groningen, The location of the human PCLN1 gene in GenBank contigs AC009520 Netherlands), and sequenced from both strands by using the ABI and AC073963. PRISM 310 Genetic Analyzer (Applera, Norwalk, CT). The 5'– and 3'–RACE-PCR from rat kidney cDNA were performed Chromosomal Assignment of Mouse Pcln1 analogously with rat gene–specific primers rnGSP1 and rnGSP2, The genetic map position of mouse Pcln1 was determined by respectively. The 814-bp and 871-bp PCR fragments were subcloned segregation analysis by using gene-specific amplification of clone and sequenced as described above. DNA from the Whitehead Institute Center for Genomic Research The cDNA sequence of full-length murine Pcln1 was deposited mouse radiation hybrid mapping panel (11). The primer pair mm-Int4- with GenBank accession no. AF323748 and the full-length rat cDNA F/R spanning intron 4 of mouse Pcln1 was used for amplification sequence with accession no. AF333099. (fragment size, Ϸ1350 bp). Data vectors, which were based on two independent PCR analyses of the entire panel, with data arranged in Analysis of cDNA and Amino Acid Sequences the order specified for the Whitehead Institute/MIT Center for Ge- The final cDNA and amino acid sequences obtained were com- nome Research, Mouse EST RH Mapping Project, Public Data Re- pared with human and bovine PCLN1 by using the FASTA and lease 3 (April, 2000) were submitted to two-point maximum-likeli- Table 1. Oligonucleotide sequences used for PCR, rapid amplification of cDNA ends–PCR, and reverse transcriptase–PCR Primer Designation Sequence Product Length (bp) mmGSP 1 5Ј-GGGTAGTTCCTCTCAGGCCCAACATCT-3Ј 784 mmGSP 2 5Ј-CTGTGGATGTTTACGTCGAACGCTCCT-3Ј 485 rnGSP 1 5Ј-AGGAGCGTTCGACGTAAACATCCACAG-3Ј 814 rnGSP 2 5Ј-TTGACTGCGTGAAGTTCCTACCGGATG-3Ј 871 mm-Int1F-GW-1 5Ј-TCTTCTTCAGTACGCTGCCTGCTTCT-3Ј mm-Int1F-GW-2 5Ј-TTGGCCATATTCTCCACTGGGTTTTT-3Ј mm-Int1R-GW-1 5Ј-TATGGAGTCGTACTCATCGCAGGTTC-3Ј mm-Int1R-GW-2 5Ј-GAATCCCATCAAAAGCGTTTGTTACAC-3Ј mm-Int2-F 5Ј-ACCTGCGATGAGTACGACTCC-3ЈϷ3300 mm-Int2-R 5Ј-GATCATCAGTGCTCGAGTTACCAC-3Ј mm-Int3-F 5Ј-CTTTGTTGCAGGGACCACATTACTC-3ЈϷ3650 mm-Int3-R 5Ј-AGCATACCACACAGAACCGATGATT-3Ј mm-Int4-F 5Ј-CTCACCTGCTGTTTGTACCTCTTC-3ЈϷ1350 mm-Int4-R 5Ј-TGCAGTTGAATAGGGCTTCC-3Ј rn-PC-RT-F 5Ј-TTGTTGCAGGGACCGTATTACTCA-3Ј 248 rn-PC-RT-R 5Ј-GGGTAGTTCCTCTCAGGTCCAACA-3Ј rn-␤A-RT-F 5Ј-GAGTACAACCTCCTTGCAGCTC-3Ј 329 rn-␤A-RT-R 5Ј-TTGTAGAAAGTGTGGTGCCAAA-3Ј 2666 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2664–2672, 2001 hood analysis (http://www.genome.wi.mit.edu/cgi-bin/mouse_rh/ Solubilization of Rat Kidney Proteins rhmap-auto/rhmapper.cgi).

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