(Received for publication, November 6, 1995; and in revised form, January 17, 1996)
From the
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.
Discovery of the aquaporin family of water transporters provided
a molecular explanation for osmotically driven water transport (P) (
)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.
Figure 2:
Osmotic water permeability of oocytes
expressing human and rat AQP5. Oocytes were injected with 5 ng
of indicated cRNAs or 50 nl of water. Oocyte swelling was determined as
described under ``Materials and Methods.'' Depicted are mean
values ± S.D. of 4-6 oocytes (stippled bars),
oocytes incubated for 5 min in 1 mM HgCl (black bars), or oocytes incubated for 5 min in 1 mM HgCl
and subsequently incubated for 30 min in 5 mM
-mercaptoethanol (open
bars).
Figure 1: Comparative 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 domains are indicated by single bold lines (TM 1-TM 6). Polypeptide sequences within connecting loops A-E are double-underlined. Identical amino acids are joined with straight lines, while dissimilar amino acids are connected with dots or colons based on polarity and charge.
Transmembrane water flow by human AQP5 was evaluated by expression
in Xenopus oocytes and measurement of osmotic induced
swelling. Oocytes injected with 5 ng of AQP5 cRNA or 50 nl of
water were incubated for 3 days at 18 °C. Oocytes were then
transferred from 200 to 70 mosM modified Barth's
solution, and increases in volume were detected by videomicroscopy. The
human AQP5 oocytes exhibited a 20-fold increase in P when compared to the water-injected oocytes; this increase in P
was blocked by treatment with 1 mM HgCl
, and the block was reversed by incubation with
-mercaptoethanol (Fig. 2). Thus, the human AQP5 cDNA encodes an aquaporin which is functionally equivalent to the
rat homolog.
Figure 3:
Restriction map and exon-intron
organization of human AQP5. The 7.4-kb EcoRI-SalI fragment of genomic DNA was used to
determine the sites of exons 1-4 using P-labeled
probes prepared from the human AQP5 cDNA (see ``Materials
and Methods''). Black rectangles represent coding
regions, and open rectangles represent untranslated regions
established by cDNA and genomic sequencing (see text). S = SalI, B = BamHI, A = AccI, H = HindIII, K = KpnI, X = XhoI, E = EcoRI.
Nucleotide sequencing with primers corresponding to the human AQP5 cDNA revealed the exon-intron boundaries (Fig. 4). Exons 1-4 corresponded to amino acids 1-121, 122-176, 177-204, and 205-265. Nucleotide sequences of these exons were identical to sequences obtained from the human submaxillary cDNA. Exon-intron class 0 boundaries were identified for all four exons. Using exon-specific sense and antisense oligonucleotide primers, introns 1-3 were determined by polymerase chain reaction to be 1.2, 0.5, and 0.9 kb (Fig. 3). Sequencing of 500 bp of the 3`-flanking sequence of the human AQP5 genomic clone revealed a polyadenylation consensus sequence 490 bp from the last amino acid of AQP5 (Fig. 4).
Figure 4: Exon-intron boundaries of the human AQP5 gene. Nucleotide sequences surrounding the coding regions of each exon (numbered black boxes) along with corresponding 5` splice-donor and 3` splice-acceptor regions were determined by dideoxynucleotide sequencing using oligonucleotide primers generated from the AQP5 cDNA. Partial 5`- and 3`-untranslated sequences with a polyadenylation consensus sequence (underlined) are represented.
Analysis of the 5`-flanking region of the human AQP5 gene was undertaken to determine the site of transcription initiation and identify possible regulatory elements (Fig. 5A). Sequence obtained from the genomic clone was identical with that found in the 5`-UTR of the human AQP5 cDNA. Using human lung mRNA as a template, a single transcription start site was identified 518 bp upstream from the translation start site of the AQP5 gene (Fig. 5B). This site was 5 bp upstream of the 5` terminus of the cDNA clone isolated. A major band of identical size was identified using human submaxillary RNA (data not shown). RNase protection using antisense RNA to the DNA region of interest confirmed the presence of a major band of the same approximate size as that seen with primer extension (data not shown). Several common response elements (Fig. 5A) were identified upstream of the transcription initiation site (Prestridge, 1991; Ghosh, 1990). A defined TATA consensus was not identified within the 406 bp of 5`-flanking sequence upstream of the transcription initiation site (Fig. 5A).
Figure 5: Analysis of 5`-flanking region and primer extension of human AQP5 gene. A, nucleotide sequence of 5`-flanking region. Nucleotide sequences of genomic DNA were obtained by dideoxynucleotide sequencing. The site of transcription initiation is indicated by an arrow and designated +1. Characteristic Sp1 and AP-2 sites are underlined. The first three codons of the AQP5 cDNA are in bold italicized letters, and corresponding amino acid designations are below each codon. B, identification of transcription initiation by primer extension. A synthetic primer designated S1 (see ``Materials and Methods'') was end-labeled and hybridized to 10 µg of total RNA from human lung overnight at 42 °C. Primer extension was performed as described under ``Materials and Methods,'' and the isolated products were treated with RNase A (5 µg, U. S. Biochemical Corp.). The final product (PE) was analyzed on a 10% polyacrylamide gel alongside sequencing products obtained by using a SalI-AccI genomic subclone (see Fig. 3) as a template and S1 as a sequencing primer to generate antisense DNA sequence.
Genomic Southern analyses were performed to determine if AQP5 exists as a single copy gene. Human leukocyte DNA from two unrelated individuals was digested with restriction enzymes and hybridized at high stringency with the coding region to the human AQP5 cDNA (Fig. 6). Identical hybridization patterns were found in the DNA from the two individuals. Based on the restriction map for the AQP5 genomic clone (Fig. 3), the anticipated sizes of BamHI-digested DNA are 1.7 and 2.9 kb, which is consistent with the hybridization pattern observed from the genomic Southern (Fig. 6). Moreover, digestion of the original phage clone with EcoRI yielded a fragment of approximately 7.7 kb (not shown) similar to that seen with human DNA (Fig. 6). Like the other aquaporins, AQP5 exists as a single copy gene (Moon et al., 1993; Pisano and Chepelinsky, 1991; Uchida et al., 1994; Yang et al., 1995).
Figure 6:
Genomic Southern analysis with human AQP5 cDNA. Ten µg of DNA from two unrelated individuals
was digested with BamHI, EcoRI, or HindIII.
Southern analysis was undertaken with a P-labeled probe
corresponding to the coding region of human AQP5.
Figure 7: Chromosomal localization of human AQP5 genomic locus. A, representative photograph demonstrating chromosomal spread from normal male lymphocytes hybridized with the 7.4-kb human AQP5 genomic clone. In situ hybridization of banded metaphase chromosomes localized all signals to the long arm of chromosome 12 where 25 of 27 signals were located on band 13. B, ideogram of human chromosome 12 showing localization of signals to 12q12-13. Each dot represents a paired signal seen on metaphase chromosomes. Signals clearly located on a single band are shown to the right. Signals which could not be localized to a single band are shown to the left.
Figure 8:
Aqp5 maps to the distal region of
mouse chromosome 15 by interspecies backcross analysis. Top,
segregation patterns of Aqp5 and flanking genes in 104
backcross animals typed for all loci. More than 104 animals were typed
for individual pairs of loci (see text). Each column represents the
chromosome identified in the backcross progeny that was inherited from
the (C57BL/6J M. spretus) F
parent. The shaded boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome
is listed at the bottom of each column. A partial chromosome
15 linkage map showing the location of Aqp5 in relation to
linked genes is represented at the bottom of the figure.
Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of known loci in
human chromosomes are shown to the right.
The interspecies map of mouse chromosome 15 was compared to a composite linkage map that reports map locations of many uncloned mouse mutations (Mouse Genome Data Base, a computerized data base maintained in The Jackson Laboratory, Bar Harbor, ME). Aqp5 mapped to a region of the composite map that did not include any known mouse mutations with a phenotype consistent with defects in the tissues of Aqp5 expression (not shown). Nevertheless, the distal region of mouse chromosome 15 shares homology with human chromosomes 22q and 12q (Fig. 7). In particular, Wnt1 has been placed on human 12q13. The tight linkage between Wnt1 and Aqp5 suggests that Aqp5 resides on 12q, which was confirmed by our in situ hybridization (Fig. 7).
These studies have defined the genomic organization of human AQP5. The AQP5 gene is structurally similar to MIP (AQP0), AQP1, and AQP2 (Pisano and Chepelinsky, 1991; Moon et al., 1993; Uchida et al., 1994). The lack of a defined TATA consensus within the region of the transcription initiation site of AQP5 is the first example of a TATA-less promoter in the aquaporin gene family. Transcription begins approximately 30 bp downstream of the TATA box in most eukaryotic genes (reviewed by Sawadogo and Sentenac, 1990). A growing number of eukaryotic genes lack a typical TATA box (reviewed by Roeder(1991) and Weis and Reinberg(1992)), but some of these genes contain an initiator element (Inr) that serves to organize the various transcription factors at the site of transcription initiation (Smale and Baltimore, 1989). Analysis of the AQP5 promoter failed to reveal any close sequence similarities with previously recognized Inr families (Weis and Reinberg, 1992), although there is currently no clear Inr consensus sequence (O'Shea-Greenfield and Smale, 1992). Studies directed at defining the active promoter regions of the AQP5 gene are currently underway to clarify this issue. The recognition of developmental expression patterns, identification of possible pharmacological modulation of expression, and definition of cellular and subcellular sites of expression may provide additional questions which may be answered by studies of AQP5 gene regulation.
Precise chromosomal localizations of candidate genes may permit linkage to mutant phenotypes. AQP1 has been mapped to human chromosome 7p14 (Moon et al., 1993). The genes for MIP (AQP0) and AQP2 have both been localized to human chromosome 12q13 (Saito et al., 1995). Although the chromosomal localization site for the AQP3 gene was reported to be 7q36 (Inase et al., 1995), this has been corrected to 9p12-21 (Mulders et al., 1995b). Nevertheless, localization of AQP5 to human chromosome 12q13 is well supported: (i) fluorescence in situ hybridizations with large genomic DNA probes is highly reliable; (ii) AQP5 colocalized with Wnt1 to the distal arm of mouse chromosome 15 which corresponds to human chromosome 12q; (iii) colocalization of AQP5 to the same human chromosomal region as MIP (AQP0) and AQP2 identifies 12q13 as the site of an aquaporin gene cluster. To date, no obvious mutations in mice or humans suggesting AQP5 dysfunction have been mapped to the AQP5 gene locus. It is likely that AQP5 participates in the generation of pulmonary secretions, saliva, and tears, as well as prevention of corneal edema. Thus, definition of the AQP5 gene structure may aid in the identification of the role of AQP5 in normal physiology and may possibly reveal clinical disorders related to this protein.
The chromosomal clustering of MIP (AQP0), AQP2, and AQP5 may reflect similar modes of regulation of the proteins, since AQP5 is most closely related to AQP0 and AQP2 at the amino acid level, and preliminary evidence indicates that phosphorylation is involved in the regulation of both MIP and AQP2 (Kuwahara et al., 1995; Ehring et al., 1991). AQP1 and AQP3 are not part of this gene cluster and are believed to be constitutively activated (reviewed by Agre et al.(1995)). The conserved cAMP-protein kinase A consensus sequences within rat and human AQP5 is similar to the consensus present in the vasopressin-regulated AQP2, suggesting that AQP5 may be under neurohormonal control. Moreover, AQP5 is expressed in salivary glands and lacrimal glands where rapid fluid shifts occur in response to adrenergic stimuli (reviewed by Nauntofte(1992)). The mechanism by which AQP2 is targeted to the cell surface in response to phosphorylation has been the subject of intense recent investigation (reviewed by Agre et al.(1995)). Studies designed to determine the mechanism of AQP5 protein regulation as well as AQP5 gene expression are currently underway.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U46566[GenBank]-U46569[GenBank].