The Human Aquaporin-5 Gene

Abstract

The cDNA for the fifth mammalian aquaporin (AQP5) was isolated from rat, and expression was demonstrated in rat salivary and lacrimal glands, cornea, and lung (Raina, S., Preston, G. M., Guggino, W. B., and Agre, P.(1995) J. Biol. Chem. 270, 1908-1912). Here we report the isolation and characterization of the human AQP5 cDNA and gene. The AQP5 cDNA from a human submaxillary gland library contains a 795-base pair open reading frame encoding a 265-amino acid protein. The deduced amino acid sequences of human and rat AQP5 are 91% identical with 6 substitutions in the 22-amino acid COOH-terminal domain. Expression of human AQP5 in Xenopus oocytes conferred mercurial-sensitive osmotic water permeability (P) equivalent to other aquaporins. The human AQP5 structural gene resides within a 7.4-kilobase SalI-EcoRI fragment with four exons corresponding to amino acids 1-121, 122-176, 177-204, and 205-265 separated by introns of 1.2, 0.5, and 0.9 kilobases. A transcription initiation site was identified 518 base pairs upstream of the initiating methionine. Genomic Southern analysis indicated that AQP5 is a single copy gene which localized to human chromosome 12q13; this coincides with the chromosomal locations of the homologous human genes MIP and AQP2, thus confirming 12q13 as the site of an aquaporin gene cluster. The mouse gene localized to distal chromosome 15. This information may permit molecular characterization of AQP5 expression during normal development and in clinical disorders. The cDNA for the fifth mammalian aquaporin (AQP5) was isolated from rat, and expression was demonstrated in rat salivary and lacrimal glands, cornea, and lung (Raina, S., Preston, G. M., Guggino, W. B., and Agre, P.(1995) J. Biol. Chem. 270, 1908-1912). Here we report the isolation and characterization of the human AQP5 cDNA and gene. The AQP5 cDNA from a human submaxillary gland library contains a 795-base pair open reading frame encoding a 265-amino acid protein. The deduced amino acid sequences of human and rat AQP5 are 91% identical with 6 substitutions in the 22-amino acid COOH-terminal domain. Expression of human AQP5 in Xenopus oocytes conferred mercurial-sensitive osmotic water permeability (P) equivalent to other aquaporins. The human AQP5 structural gene resides within a 7.4-kilobase SalI-EcoRI fragment with four exons corresponding to amino acids 1-121, 122-176, 177-204, and 205-265 separated by introns of 1.2, 0.5, and 0.9 kilobases. A transcription initiation site was identified 518 base pairs upstream of the initiating methionine. Genomic Southern analysis indicated that AQP5 is a single copy gene which localized to human chromosome 12q13; this coincides with the chromosomal locations of the homologous human genes MIP and AQP2, thus confirming 12q13 as the site of an aquaporin gene cluster. The mouse gene localized to distal chromosome 15. This information may permit molecular characterization of AQP5 expression during normal development and in clinical disorders. INTRODUCTIONDiscovery of the aquaporin family of water transporters provided a molecular explanation for osmotically driven water transport (P) 1The abbreviations used are: Pcoefficient of osmotic water permeabilityUTRuntranslated regionRFLPrestriction fragment length polymorphismbpbase pair(s)kbkilobase(s)PIPES1,4-piperazinediethanesulfonic acid.across cell membranes of mammalian and plant tissues (reviewed by Chrispeels and Agre(1994)). When expressed in Xenopus oocytes, aquaporins confer large increases in P without conducting small molecules or ions. AQP1 is present in red cells, renal proximal tubules, lung, and other tissues (reviewed by Agre et al. (1993)). The major intrinsic protein of lens (MIP, AQP0) was recently confirmed as a weak water channel (Mulders et al., 1995a). cDNAs encoding AQP2 through AQP5 were isolated by homology cloning (reviewed by Knepper(1994)). In response to vasopressin, AQP2 (Fushimi et al., 1993) is targeted to the apical surface of renal collecting duct principal cells (Nielsen et al., 1993). AQP3 is located at the basolateral membranes of renal collecting ducts (Ishibashi et al., 1994, Ma et al., 1994; Echevarria et al., 1994). AQP4 is the major water channel in brain (Jung et al., 1994; Hasegawa et al., 1994). AQP5 is expressed in rat salivary and lacrimal glands, corneal epithelium, and lung (Raina et al., 1995).The human genes for several aquaporins have recently been characterized (Moon et al., 1993; Uchida et al., 1994; Pisano and Chepelinsky, 1991; Inase et al., 1995; Mulders et al., 1995b; Yang et al., 1995). Identification of the Colton blood group antigens on the first exofacial loop of AQP1 led to the identification of three Colton-null individuals who had different mutations in AQP1, but total lack of AQP1 is not associated with an apparent phenotype (Smith et al., 1994; Preston et al., 1994). In contrast, a subset of patients with nephrogenic diabetes insipidus have mutations of the AQP2 gene resulting in a lack of clinical response to vasopressin (Deen et al., 1994; van Lieburg et al., 1994). Mice with mutations in MIP (AQP0) suffer from congenital cataracts (Shiels and Bassnett, 1996). Disease relevance of AQP3 and AQP4 remains to be determined. These studies reported here were undertaken to elucidate the gene structure and chromosomal localization of AQP5 as initial steps needed to understand regulation of the gene and to identify possible linkages to human disease.MATERIALS AND METHODSIsolation of Salivary cDNA ClonesThe 1018-bp rat AQP5 cDNA containing the coding region plus 109-bp 5′-UTR and 114-bp 3′-UTR (Raina et al., 1995) was labeled with [α-P]dCTP (Amersham) by random DNA labeling (Boehringer Mannheim). This probe was used to screen 1.5 × 105 plaques of an adult human submaxillary gland gt11 cDNA library (Clontech) under conditions of moderate stringency (0.5 × SSC, 0.1% SDS, 55°C). Two positive plaques were purified. DNA inserts were released with EcoRI, gel-purified, and ligated into pBS II KS(-) (Stratagene). Both strands were sequenced by the dideoxynucleotide termination method (U. S. Biochemical Corp.). A 1348-bp cDNA containing the entire coding region was utilized for all future studies.Functional Expression in OocytesAn 850-bp SfiI-HindIII fragment of the human AQP5 cDNA containing the entire open reading frame along with 15-bp 5′-UTR and 40-bp 3′-UTR was ligated into the BglII site of the Xenopus expression construct pXβG, which contains the HindIII-PstI insert of pSP64T in pBS II KS(+) (Preston et al., 1992). Capped cRNA was synthesized in vitro after digestion with XbaI (Preston et al., 1993). Oocyte swelling was measured after cRNA expression (Raina et al., 1995).Isolation of Genomic DNA ClonesA human genomic DNA library in EMBL-3 was generously provided by Dr. Jeremy Nathans, Johns Hopkins University (Wang et al., 1992). The P-labeled rat AQP5 cDNA probe was used to screen 5 × 105 plaques under conditions of moderate stringency. DNA from plaque-purified recombinants was digested with EcoRI and SalI, gel-purified, and ligated into pBS II KS(-).Restriction Mapping and Determination of Exon-Intron BoundariesThe 7.4-kb EcoRI-SalI genomic clone was digested with restriction enzymes; DNA fragments were analyzed by Southern analysis with P-labeled cDNA probes obtained from the 1348-bp human submaxillary cDNA clone. Nucleotide sequences corresponding to 5′- and 3′-UTR were determined using oligonucleotide primers to sense and antisense strands. Exon-intron boundaries were identified using oligonucleotide primers designed from coding regions of the human AQP5 cDNA. Exon 1-intron 1 (sense, 310-326), 5′-ATCCTCTACGGTGTGGC-3′; intron 1-exon 2 (antisense, 389-405), 5′-CTCCACCACCATGGCCT-3′; exon 2-intron 2 (sense, 493-509), 5′-ATTGGCCTGTCTGTCAC-3′; exon 3-intron 2 (antisense, 585-601), 5′-GGCTGAACCGATTCATG-3′; exon 3-intron 3 (sense, 565-581), 5′-TCTTTTGGCCCTGCGGT-3′; exon 4-intron 3 (antisense, 683-699), 5′-ACTCAGGCTCAGGGAGT-3′. Sizes of introns 1-3 were determined by polymerase chain reaction using the intron flanking primers (30 cycles: 1 min 94°C; 1 min 47°C; 2 min 72°C). Polymerase chain reaction products were electrophoresed into a 0.8% agarose gel with 1-kb DNA ladder standards.Primer ExtensionAn oligonucleotide primer (designated S1) complementary to nucleotides +94 to +71 was end-labeled with [-P]ATP, and 1 × 106 cpm were hybridized to 10 μg of total human lung or submaxillary RNA at 60°C in 40 mM PIPES (pH 6.4), 1 mM EDTA, 400 mM NaCl, 80% formamide for 12 h. Following ethanol precipitation of hybridized products, primer extension was performed in 50 mM Tris-Cl (pH 7.6), 60 mM KCl, 10 mM MgCl, 2.5 mM dNTP, 1 mM dithiothreitol, 1 unit/μl RNase inhibitor, and 250 units Moloney murine leukemia reverse transcriptase (Life Technologies, Inc.) for 2 h at 37°C. Synthesized products were analyzed on a 10% polyacrylamide sequencing gel alongside an adjacent sequencing ladder generated using the same oligo as a primer and a 600-bp SalI-AccI genomic fragment as a template.Genomic Southern AnalysisTen micrograms of human genomic DNA from leukocytes of two unrelated individuals was digested with EcoRI, BamHI, or HindIII, electrophoresed into a 0.8% agarose gel, and transferred to a nylon membrane (Genescreen Plus, DuPont NEN). Southern analyses were performed using the human AQP5 cDNA as a probe (Moon et al., 1993).Chromosomal Localization and in Situ HybridizationThe plasmid containing the 7.4-kb genomic clone of human AQP5 (Fig. 2) was nick-translated with biotin-14 dATP (Life Technologies, Inc.); 41% incorporation was determined by 3H incorporation. Chromosomal spreads of normal male lymphocytes cultured with BrUrd (Bhatt et al., 1988) were used for fluorescence in situ hybridization (Moon et al., 1993) and were photographed with color slide film (Kodak Ektachrome 400HC).Interspecies Mouse Backcross MappingMating of F females (C57BL/6J × Mus spretus) and males (C57BL/6J) generated 205 N mice which were used to map the Aqp5 locus (Copeland and Jenkins, 1991). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, and Southern transfer to Hybond N (Amersham) nylon membranes were described (Jenkins et al., 1982). The 1442-bp EcoRI-XhoI fragment of rat AQP5 cDNA (Raina et al., 1995) was P-labeled with dCTP by nick translation (Boehringer Mannheim) and used to probe blots washed to a final stringency of 0.5 × SSCP, 0.1% SDS, 65°C. An 11.0-kb fragment was detected in SphI-digested C57BL/6J DNA and a 9.4-kb fragment in M. spretus DNA. Presence or absence of the 9.4-kb SphI M. spretus-specific fragment was followed in backcross mice. Description of the probes and RFLPs for the loci linked to Aqp5 including platelet-derived growth factor, endothelial cell (Pdgfec), wingless-related Moloney murine tumor virus integration site 1 (Wnt1), and retinoic acid receptor (Rarg) were reported (Brannan et al., 1992). Recombination distances were calculated by computer program SPRETUS MADNESS (Green, 1981). Gene order was determined by minimizing the number of recombination events required to explain the allele distribution pattern. References for the human map positions of cited loci are from GDB (Genome Data Base), a computerized data base of human linkage information maintained by the William H. Welch Medical Library, Johns Hopkins University (Baltimore, MD).RESULTSIsolation and Analysis of Human AQP5 cDNAThe rat AQP5 cDNA was used to isolate a recombinant from a human submaxillary gland cDNA library. The 1348-bp insert contained an open reading frame encoding a 265-amino acid protein. Further sequencing revealed the presence of 513 bp of 5′-UTR and only 40 bp of 3′-UTR. No other ATG translation initiation signals were identified within the 5′-UTR; the 3′-UTR did not contain a polyadenylation consensus sequence. Human and rat AQP5 cDNAs were compared by GAP analysis program (Fig. 1). The deduced amino acid sequences were 91% identical. Like all members of the aquaporin family, tandem repeats of the NPA motif (Asn-Pro-Ala) were present in rat and human cDNAs. The consensus for cAMP-protein kinase A phosphorylation (SRRTS) present in rat AQP5 was also in the human cDNA. Greatest divergence between the rat and human cDNAs was found within the 22-residue carboxyl-terminal domain where six substitutions were identified.Figure 1Comparative alignment of deduced amino acid sequences of human and rat AQP5 by GAP program analysis. Conserved NPA and cAMP-protein kinase motifs are enclosed in rectangles. Presumed bilayer-spanning are indicated by single sequences within are amino acids are with amino acids are with or on and water by human AQP5 was by expression in Xenopus oocytes and of osmotic with 5 of AQP5 cRNA or 50 of water were for 3 at were transferred from to and increases in were detected by The human AQP5 oocytes a in P compared to the this in P was by with 1 mM and the was by with (Fig. the human AQP5 cDNA an aquaporin which is equivalent to the rat of the Human AQP5 Genomic different genomic were isolated from a human genomic DNA library using the rat cDNA as a molecular The insert from isolated was released with EcoRI and and The restriction map for AQP5 (Fig. was confirmed by digestion with single restriction or of followed by Southern analysis not map and of human The 7.4-kb EcoRI-SalI fragment of genomic DNA was used to the of exons using P-labeled probes from the human AQP5 cDNA and coding and open regions by cDNA and genomic sequencing SalI, BamHI, A HindIII, sequencing with primers corresponding to the human AQP5 cDNA revealed the boundaries (Fig. to amino acids 1-121, 122-176, 177-204, and Nucleotide sequences of exons were identical to sequences obtained from the human submaxillary cDNA. Exon-intron boundaries were identified for all four sense and antisense oligonucleotide introns 1-3 were determined by polymerase chain reaction to be 1.2, 0.5, and 0.9 (Fig. of bp of the of the human AQP5 genomic clone revealed a polyadenylation consensus bp from the amino acid of AQP5 (Fig. boundaries of the human AQP5 gene. Nucleotide sequences the coding regions of exon along with corresponding and regions were determined by dideoxynucleotide sequencing using oligonucleotide primers generated from the AQP5 cDNA. 5′- and sequences with a polyadenylation consensus are of the region of the human AQP5 gene was undertaken to the site of transcription initiation and identify possible (Fig. obtained from the genomic clone was identical with that found in the 5′-UTR of the human AQP5 cDNA. human lung as a a single transcription site was identified 518 bp upstream from the translation site of the AQP5 gene (Fig. This site was 5 bp upstream of the of the cDNA clone A major of identical was identified using human submaxillary RNA not RNase using antisense RNA to the DNA region of confirmed the presence of a major of the same as that with primer extension not response (Fig. were identified upstream of the transcription initiation site 1991; A consensus was not identified within the bp of upstream of the transcription initiation site (Fig. of region and primer extension of human AQP5 gene. of Nucleotide sequences of genomic DNA were obtained by dideoxynucleotide The site of transcription initiation is indicated by an and and are The first three of the AQP5 cDNA are in and corresponding amino acid are identification of transcription initiation by primer A primer and was end-labeled and hybridized to 10 μg of total RNA from human lung at extension was performed as described under and and the isolated products were with RNase A S. Biochemical Corp.). The final was analyzed on a 10% polyacrylamide gel alongside sequencing products obtained by using a SalI-AccI genomic as a and as a sequencing primer to antisense DNA Southern analyses were performed to AQP5 as a single copy gene. Human DNA from two unrelated individuals was digested with restriction and hybridized at stringency with the coding region to the human AQP5 cDNA (Fig. hybridization were found in the DNA from the two on the restriction map for the AQP5 genomic clone (Fig. the of DNA are and which is with the hybridization from the genomic Southern (Fig. digestion of the clone with EcoRI a fragment of to that with human DNA (Fig. Like the other AQP5 as a single copy gene (Moon et al., 1993; Pisano and Chepelinsky, 1991; Uchida et al., 1994; Yang et al., Southern analysis with human AQP5 cDNA. μg of DNA from two unrelated individuals was digested with BamHI, EcoRI, or Southern analysis was undertaken with a P-labeled probe corresponding to the coding region of human Chromosomal Localization of AQP5 7.4-kb genomic clone of the human AQP5 gene was used as a molecular probe for in situ hybridization with chromosomal spreads from normal male lymphocytes (Fig. Analysis of cells that cells had at pair of signals of a single When were photographed on color slide signals were signals were located on the of a chromosome the chromosome and of the the hybridized were by fluorescence plus and with the color to (Fig. signals were analyzed after and were located on chromosome and localization of human AQP5 genomic chromosomal from normal male lymphocytes hybridized with the 7.4-kb human AQP5 genomic clone. In situ hybridization of localized all signals to the of chromosome 12 where of signals were located on of human chromosome 12 localization of signals to a on located on a single are to the which not be localized to a single are to the Chromosomal Localization of Aqp5 studies may for analysis of the of aquaporin gene Mouse chromosomal localization of Aqp5 was determined by backcross analysis using from of × M. spretus) × This backcross been for loci that are all and the chromosome (Copeland and Jenkins, 1991). C57BL/6J and M. spretus were digested with several and analyzed by Southern hybridization for RFLPs using the rat AQP5 cDNA The 9.4-kb SphI for M. spretus was used to of the Aqp5 locus in backcross mice. The identified Aqp5 in the distal region of mouse chromosome linked to and mice were analyzed for as in the analysis (Fig. to mice were for pairs of locus was analyzed in for recombination using the of the total of mice recombinant to the total number of mice analyzed for pair of loci and the gene order are: that and AQP5 were in in that the two loci are within of other to the distal region of mouse chromosome by backcross analysis. of Aqp5 and flanking genes in backcross for all were for pairs of loci the chromosome identified in the backcross that was from the (C57BL/6J × M. spretus) F The the presence of a C57BL/6J and the presence of a M. spretus The number of of chromosome is at the of A chromosome linkage map the of Aqp5 in to linked genes is at the of the Recombination distances between loci in are to the of the and the positions of loci in human are to the map of mouse chromosome was compared to a linkage map that map locations of mouse mutations Data a computerized data base maintained in The Aqp5 to a region of the map that did not mouse mutations with a phenotype with in the tissues of Aqp5 expression the distal region of mouse chromosome homology with human and (Fig. In been on human The linkage between and Aqp5 that Aqp5 resides on which was confirmed by in situ hybridization (Fig. studies have the genomic of human The AQP5 gene is to MIP AQP1, and AQP2 and Chepelinsky, 1991; et al., 1993; Uchida et al., 1994). The lack of a consensus within the region of the transcription initiation site of AQP5 is the first of a in the aquaporin gene bp of the in genes (reviewed by and A number of genes lack a (reviewed by and and but of genes contain an that to the transcription at the site of transcription initiation and Analysis of the AQP5 to with and is consensus and 1992). at the regions of the AQP5 gene are to this The of expression identification of possible of and of and of expression may which may be by studies of AQP5 gene chromosomal of genes may permit linkage to AQP1 been to human chromosome (Moon et al., 1993). The genes for MIP (AQP0) and AQP2 have been localized to human chromosome 12q13 et al., 1995). the chromosomal localization site for the AQP3 gene was reported to be et al., this been to (Mulders et al., localization of AQP5 to human chromosome 12q13 is fluorescence in situ with large genomic DNA probes is AQP5 with to the distal of mouse chromosome which to human chromosome of AQP5 to the same human chromosomal region as MIP (AQP0) and AQP2 12q13 as the site of an aquaporin gene cluster. mutations in mice or AQP5 have been to the AQP5 gene is that AQP5 in the of and as as of corneal of the AQP5 gene structure may in the identification of the of AQP5 in normal and may clinical to this chromosomal of MIP AQP2, and AQP5 may of regulation of the AQP5 is to and AQP2 at the amino acid and that phosphorylation is in the regulation of MIP and AQP2 et al., 1995; et al., 1991). AQP1 and AQP3 are not of this gene and are to be (reviewed by Agre et The cAMP-protein kinase A consensus sequences within rat and human AQP5 is to the consensus present in the AQP2, that AQP5 may be under AQP5 is expressed in salivary and lacrimal where in response to (reviewed by The by which AQP2 is targeted to the cell surface in response to phosphorylation been the of (reviewed by Agre et designed to the of AQP5 protein regulation as as AQP5 gene expression are INTRODUCTIONDiscovery of the aquaporin family of water transporters provided a molecular explanation for osmotically driven water transport (P) 1The abbreviations used are: Pcoefficient of osmotic water permeabilityUTRuntranslated regionRFLPrestriction fragment length polymorphismbpbase pair(s)kbkilobase(s)PIPES1,4-piperazinediethanesulfonic acid.across cell membranes of mammalian and plant tissues (reviewed by Chrispeels and Agre(1994)). When expressed in Xenopus oocytes, aquaporins confer large increases in P without conducting small molecules or ions. AQP1 is present in red cells, renal proximal tubules, lung, and other tissues (reviewed by Agre et al. (1993)). The major intrinsic protein of lens (MIP, AQP0) was recently confirmed as a weak water channel (Mulders et al., 1995a). cDNAs encoding AQP2 through AQP5 were isolated by homology cloning (reviewed by Knepper(1994)). In response to vasopressin, AQP2 (Fushimi et al., 1993) is targeted to the apical surface of renal collecting duct principal cells (Nielsen et al., 1993). AQP3 is located at the basolateral membranes of renal collecting ducts (Ishibashi et al., 1994, Ma et al., 1994; Echevarria et al., 1994). AQP4 is the major water channel in brain (Jung et al., 1994; Hasegawa et al., 1994). AQP5 is expressed in rat salivary and lacrimal glands, corneal epithelium, and lung (Raina et al., 1995).The human genes for several aquaporins have recently been characterized (Moon et al., 1993; Uchida et al., 1994; Pisano and Chepelinsky, 1991; Inase et al., 1995; Mulders et al., 1995b; Yang et al., 1995). Identification of the Colton blood group antigens on the first exofacial loop of AQP1 led to the identification of three Colton-null individuals who had different mutations in AQP1, but total lack of AQP1 is not associated with an apparent phenotype (Smith et al., 1994; Preston et al., 1994). In contrast, a subset of patients with nephrogenic diabetes insipidus have mutations of the AQP2 gene resulting in a lack of clinical response to vasopressin (Deen et al., 1994; van Lieburg et al., 1994). Mice with mutations in MIP (AQP0) suffer from congenital cataracts (Shiels and Bassnett, 1996). Disease relevance of AQP3 and AQP4 remains to be determined. These studies reported here were undertaken to elucidate the gene structure and chromosomal localization of AQP5 as initial steps needed to understand regulation of the gene and to identify possible linkages to human