(Received for publication, January 24, 1997, and in revised form, June 3, 1997)
From the A new member of the aquaporin (AQP) family has
been identified from rat testis. This gene, referred as aquaporin 7 (AQP7), encodes a 269-amino acid protein that contained the conserved NPA motifs of MIP family proteins. AQP7 has the amino acid sequence homology with other aquaporins (~30%), and it is highest with AQP3
(48%), suggesting that both AQP3 and AQP7 belong to a subfamily in the
MIP family. Injection of AQP7-cRNA into Xenopus oocytes expressed a 26-kDa protein detected by immunoblotting. The expression of AQP7 in oocytes stimulated the osmotic water permeability by 10-fold
which was not inhibited by 0.3 mM mercury chloride. The Arrhenius activation energy for the stimulated water permeability was
low (2.1 kcal/mol). AQP7 also facilitated glycerol and urea transport
by 5- and 9-fold, respectively. The activation energy for glycerol was
also low (5.3 kcal/mol after the correction of the endogenous glycerol
permeability of oocytes). Northern blot analysis revealed a
1.5-kilobase pair transcript expressed abundantly in testis. In
situ hybridization of testis revealed the expression of AQP7 at
late spermatids in seminiferous tubules. The immunohistochemistry of
testis localized the AQP7 expression at late spermatids and at maturing
sperms. AQP7 may play an important role in sperm function.
Recent studies have identified several water channels (aquaporins)
that belong to the MIP family (reviewed in Ref. 1). The MIP family
proteins are widely expressed in almost all organisms. In
Escherichia coli, there are two MIP family proteins; one is a glycerol facilitator
(GlpF)1 (2), and the other is
a water channel (AQP Z) (3). A yeast (Saccharomyces
cerevisiae) has four MIP family proteins in its genome. Plants
have many MIP family proteins. For example, there are more than 12 MIP
family proteins in Arabidopsis thaliana (4). In mammals,
more than eight members have been reported; most of them transport
water and those have been named "aquaporins." Seven members of
aquaporins have been reported in rat and human (1). AQP0 (originally
named MIP26) is present exclusively at lens epithelium. AQP1 is present
in many tissues including red blood cells, kidney, eye, lung, choroid
plexus, bile duct, and vascular endothelium. AQP2 is solely present at
the apical membrane of kidney collecting duct cells. AQP3 and AQP4 are
colocalized at the basolateral membranes in some tissues such as
kidney, colon, and trachea. AQP3 is present in urinary bladder, skin,
and sclera of eye, and AQP4 is present in stomach, skeletal muscle,
spinal chord, brain, and retina. AQP5 is present at the apical
membranes of exocrine tissues. AQP6 (originally named WCH3 or hKID) is
present only in kidney (5).
Aquaporins are usually found in the selected tissues where water
movements are abundant and/or physiologically important (6). Unexpectedly, a high water permeability of human and ram sperm has been
reported (7). It is also reported that the high water permeability of
human sperm is mercury-resistant and not mediated by AQP1 (8). The
molecular basis for this high water permeability is unknown. The sperm
water channel may be veterinary important for the cryopreservation of
sperms. Here we report the cloning and the functional expression of a
new aquaporin (AQP7) from rat testis.
One microgram of rat testis total
RNA was reverse-transcribed and used for PCR with 5 µM
set of degenerative primers as previously reported (9): sense strand,
5 An oligo(dT)-primed rat
testis cDNA library in EcoRI
fragment (1.3 kb) of AQP7 cDNA (containing open reading frame and
the untranslated sequences) was blunt-end-ligated into the
BglII site of a pSP64T-derived BlueScript vector containing 5 Two
polyclonal anti-AQP7 antibodies were raised in rabbits against a
synthesized N-terminal peptide (MAGSVLENIQSVLQK) and a C-terminal
peptide (GLIHAGIPPQGS), respectively. The enzyme-linked immunosorbent
assay titers of each antiserum were 13,200 for N-terminal antibody and
15,200 for C-terminal antibody. The oocytes membranes were isolated as
described previously (10). The membrane fraction was resuspended in a
loading buffer containing 3% SDS, 65 mM Tris-HCl, 10%
glycerol, and 5% 2-mercaptoethanol. After being heated at 70 °C for
10 min, the solubilized proteins were separated by SDS-polyacrylamide gel electrophoresis. Membrane proteins from 3 oocytes were applied in
each lane. The proteins were transferred to Immobioln-P filter (Millipore) using a semi-dry system. The filters were incubated for
1 h with the above polyclonal antibodies (100-fold dilution). The
filters were further incubated for 1 h with
125I-protein A solution, followed by autoradiography.
Water permeability was
measured as described previously (9). In brief, oocytes were
transferred from 200 to 70 mOsm of modified Barth's buffer at
25 °C, and oocyte swelling was monitored by video microscopy. The
coefficient of osmotic water permeability (Pf,
µm/s) was calculated from the initial slope of oocyte swelling as
previously reported (9). The Arrhenius activation energy was calculated
by measuring Pf at 5 and 25 °C.
The oocytes were incubated
in Barth's solution either with [14C]glycerol (specific
activity, 5.88 GBq/mmol; Amersham Corp.) or [14C]urea
(specific activity, 2.02 GBq/mmol; Amersham Corp.) at room temperature
for 2-10 min. The oocytes were then rapidly washed four times with
ice-cold Barth's solution. The individual oocytes were lysed in 200 µl of 10% SDS solution overnight for liquid scintillation counting.
The Arrhenius activation energy for glycerol was calculated by
measuring Pgly at 10, 20, and 30 °C.
A rat multiple tissue Northern blot
(CLONTECH) was hybridized under high
stringency condition with a 1.3-kb AQP7 cDNA labeled with
[ Cryosections (10 µm) were cut from rat testis from 12-week-old Harlan Sprague Dawley
strain rats, fixed with 4% formaldehyde in phosphate-buffered saline,
and treated with 0.25% acetic anhydride, 0.1 M
triethanolamine HCl (pH 8.0) for 10 min. Digoxigenin-labeled antisense
and sense riboprobes were made with T7 or T3 RNA polymerase from 0.5-kb
cDNA C-terminal fragment of AQP7 by DIG RNA labeling kit
(Boehringer Mannheim) following the manufacturer's instruction. DIG
nucleic acid detection kit (Boehringer Mannheim) was used for the
detection of AQP7 RNA in the tissues. In brief, sections were
hybridized with digoxigenin-labeled RNA probes (0.5 ng/ml) for 24 h at 37 °C. After the blocking buffer treatment, sections were
incubated in anti-DIG-AP conjugate for 5 h at room temperature. Following the incubation in detection buffer and subsequent
color-substrate solution, sections were immersed in quenching buffer,
and photographed.
The testis from
Wistar rats (9-month-old) was fixed with Bouin's fixative. The testis
was embedded in paraffin. The sections of 8 µm were stained with a
polyclonal antibody against the C terminus of AQP7 in 1:3000 dilution
after blocking with 5% normal goat serum and 3% bovine serum albumin.
Subsequently, the sections were treated with anti-rabbit IgG conjugated
with peroxidase (Sigma). Following the diaminobenzidine reaction and
the counter staining with hematoxylin, the sections were mounted with
Permafluor (Lipshaw, Pittsburgh, PA).
We used the two highly conserved NPA boxes of MIP family
proteins for designing a set of degenerative oligonucleotide PCR primers. Searches for new aquaporins from rat testis cDNA using these primers led to the identification of a new clone. We screened a
rat testis cDNA
We analyzed AQP7 protein
expressed in the Xenopus oocyte by injection of rat AQP7
cRNA. The immunoblot of oocytes membranes revealed a protein with an
apparent molecular mass of 26 kDa (arrows) detected by both
C-terminal and N-terminal antibodies (Fig.
2). The size is slightly smaller than
that expected by calculated molecular mass (29 kDa). The glycosylation
band seemed to be absent, which is consistent with the absence of
consensus glycosylation site in AQP7.
We examined the
function of AQP7 when expressed in Xenopus oocytes. The
osmotic water permeability coefficient (Pf) of
AQP7-cRNA (5 ng)-injected oocytes was 10 times higher than Pf of water-injected oocytes at 25 °C (Fig.
3A) (186 ± 15 µm/s;
n = 12 versus 17 ± 2 µm/s;
n = 12, mean ± S.E.). The induction was
comparable to the levels observed in other aquaporins. The previous
study on human sperm water permeability revealed that 50 µM mercury chloride did not affect its water permeability (8). The toxicity of mercury chloride to sperm precluded further study
with higher concentration of mercury chloride. In this study of the
heterologous expression system, we examined the effect of much higher
concentrations of mercury chloride. 0.3 mM
HgCl2 did not affect the increase of Pf
(Fig. 3A) (179 ± 15 µm/s; n = 12).
To examine the temperature dependence of Pf, Pf values at 5 and 25 °C were measured
(n = 12 each). The determined activation energy from
the Arrhenius equation of Pf was 2.1 kcal/mol, a
value in the range expected for a water channel.
As AQP3 transport glycerol (9, 11, 12) and urea (9, 13), we examined
the glycerol and urea uptake in AQP7-expressing oocytes. Oocytes were
incubated in the presence of 165 µM
[14C]glycerol, and intracellular radioactivity was
measured after 2 min. AQP7 cRNA (20 ng) injection stimulated glycerol
uptake by 5-fold (Fig. 3B) (1020 ± 76 cpm;
n = 8 versus 202 ± 23;
n = 4) (the calculated Pgly was
18.9 × 10 Northern blot
analysis revealed that AQP7 mRNA (1.5 kb) was expressed most
abundantly in testis (Fig. 4). The size
of the bands of heart and kidney (2.4 and 1.35 kb) seemed to be
different from that of testis. Much weaker bands were detected in
skeletal muscle and brain. The molecular identity of these bands
remains to be clarified. In situ hybridization of testis
showed that the positive staining was observed only when antisense
probe was used (Fig. 5A).
Signals were localized at the inner surface of seminiferous tubules and
not detected in interstitial tissues including Leydig cells. In
seminiferous tubules, the positive cells are probably the cells of late
stages of spermatogenesis, i.e. late spermatids (Fig.
5C). Immunohistochemistry of testis also revealed the
expression of AQP7 at the late stages of spermatogenesis, from late to
maturing spermatids (Fig. 6A).
The high expression was observed at the plasma membrane of late
spermatids.
We have cloned a new water channel (AQP7) from rat testis. AQP7
has the sequence and functional similarity to AQP3. Thus, AQP3 and AQP7
comprise a new subfamily in aquaporins. On the other hand, AQP0, -1, -2, -4, -5, and -6 have amino acid homology with each other on the
order of 40-50% and comprise the other subfamily. The latter group
seems to be more selective in the permeation of water, and the former
group seems to be less selective and also permeable to glycerol.
However, the nature and the precise localization of water and/or
glycerol pore(s) within each group are not yet determined. The
discovery of another member of the AQP3 group will facilitate the
comparison of sequence-related functional differences between two
groups.
The water permeability of aquaporins is inhibited by mercury chloride
except for AQP4 (14) and plant aquaporin RD28 (15). The
mercury-sensitive Cys is located just in front of the second NPA box in
AQP1, AQP2, AQP5, and AQP6 indicating that this is a functionally
important region of the protein (10). AQP4 and RD28 do not have such
Cys in their amino acid sequences. AQP7 is also insensitive to mercury
chloride and does not have such Cys near the second NPA box. Although
there are three Cys (Cys-86, -169, -195) in deduced amino acid sequence
of AQP7, they may not be localized near the AQP7 aqueous pore.
The previous studies on water permeability of sperms of human, ram,
fowl, and bull showed their high water permeability with low activation
energy, which is insensitive to mercury chloride (7, 8, 16). The
character of the sperm water channel agrees well with that of AQP7, and
the immunohistochemical study revealed that AQP7 is present at sperms.
The physiological role of such a high water permeability in sperm is
not clear at present. In E. coli, its glycerol transporter
(GlpF) is localized in the operon with glycerol kinase (2), and the
functional coupling between GlpF and glycerol kinase has been shown
(17). Interestingly, testis has a unique glycerol kinase encoded by a
different gene from the systemic one (18). It is possible that
testis-specific glycerol kinase and AQP7 are functionally coupled and
that the major function of AQP7 is glycerol transport rather than water transport. As glycerol has been used as an almost universally effective
cryoprotectant for sperm, the glycerol permeability of sperm may be
important as a determinant of optimal cooling rate. Whether the
activity of AQP7 is critical for sperm cryopreservation remains to be
clarified.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB000507. We thank Kouji Takahashi, Hiroyuki Ooshima,
Masanobu Kawasaki, and Kiyohide Fushimi for helpful discussions.
Second Department of Internal Medicine and
Department of Urology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Reverse-transcribed PCR
-CCGAATTCYTNAAYCCNGCNGTNAC-3
, and antisense strand,
5
-CCGGATCCAARTCNCKNGCNGGRTT-3
(the abbreviation recommended by the
IUPAC-IUB). The primers were derived from the consensus amino acid
sequences of the MIP family (4) (Leu-Asn-Pro-Ala-Val-Thr and
Asn-Pro-Ala-Arg-Asp-Phe, respectively). The PCR was conducted in the
following profile: 94 °C for 1 min, 46 °C for 1 min, and 72 °C
for 3 min for 30 cycles. The PCR products were cut with EcoRI and BamHI on both ends, ligated into
EcoRI- and BamHI-cut pSPORT (Life Technologies,
Inc.), and sequenced.
ZAP (Stratagene) was screened under a
stringent condition (6 × SSPE, 5 × Denhardt's solution,
0.2% SDS, 100 µg/ml salmon sperm DNA, 50% formamide at 42 °C)
with a PCR clone labeled with [
-32P]dCTP (random
priming; Amersham Corp.). A positive clone (AQP7) was isolated and
in vitro excised as described in the Stratagene protocol and
sequenced by the dideoxy chain termination method (Sequenase version 2;
U. S. Biochemical Corp.).
- and 3
-untranslated sequences of
-globin gene of
Xenopus (pXBG-ev1; a generous gift from Dr. Peter Agre).
Capped cRNA was synthesized using T3 RNA polymerase after a digestion
with BamHI to linearize the plasmids. The defoliculated
Xenopus oocytes were injected with 50 nl of water or of AQP7
cRNA and incubated at 18 °C for 48 h in modified Barth's
buffer.
-32P]dCTP. Each lanes has 2 µg of
poly(A)+ RNA from rat tissues. The filter was washed under
high stringency conditions.
Cloning of the cDNA and Analysis of the Amino Acid Sequence of
AQP7
Zap library with this PCR clone as a probe. We
isolated 16 partially overlapping clones. A cDNA clone of 1.3 kb,
AQP7 (Fig. 1A), was chosen for
further study. The translation initiation site was assigned to the
first ATG triplet that is downstream of nonsense codons found in-frame
and similar to a good Kozak initiation of the translation site
(ACCATGG). cDNA consists of a 5
-untranslated region of
260 base pairs and 3
-untranslated region of 199 base pairs followed by
a poly(A) tail. An open reading frame encodes a protein of 269 amino
acids with a relative molecular mass calculated as 28,876 kDa.
Hydropathy analysis predicts six transmembrane regions with N terminus
and C terminus localized in the cytosol similar to other MIP family
members (Fig. 1B). The C terminus, however, is exceptionally
short with little hydrophilic residues. The human AQP7 also had an
identical stop site.2 The 3
noncoding sequence contains a consensus polyadenylation signal (double
underlined) with poly(A) tail. No potential N-linked glycosylation site (NX(S/T)) nor the consensus protein
kinase A phosphorylation site is present in the predicted amino acid sequence of AQP7. The cytoplasmic second loop contains a potential phosphorylation site by protein kinase C (residue Thr-174). Searching the protein data base revealed highest amino acid sequence identity with AQP3 (48%) and lesser identity with other MIP family proteins including E. coli GlpF (38%), AQP Z (36%), AQP1 (33%),
AQP2 (36%), AQP4 (30%), AQP5 (35%), and AQP-
TIP (34%). For
comparison, AQP3 and GlpF were aligned with AQP7 in Fig. 1C.
Two highly conserved areas (NPA boxes) are evident as previously
indicated. However, the second NPA box of AQP7 is not NPA, but it is
NPS. Such a modified NPA box is only found in yeast FPS1 in which its
first NPA box is NPS (reviewed in Ref. 4). The second NPA box of human
AQP7 was also NPS.2 The previous phylogenic comparison
between AQP3 and 8 MIP family proteins revealed that AQP3 is most
related to GlpF and developed in a different branch from other water
channels (9). AQP7 belongs to this branch.
Fig. 1.
Sequence analysis of testis AQP7.
A, nucleotide sequence and deduced amino acid sequence of
the clone isolated from a rat testis cDNA library. Probable
transmembrane domains are underlined. A polyadenylation
consensus is double-underlined. B,
hydropathy analysis of deduced amino acid sequence using a 13-residue
window (19). The average local hydrophobicity at each residue was
plotted on the vertical axis and the residue number on the horizontal
axis. C, alignment of the amino acid sequences of E. coli GlpF (2), rat AQP3 (13), and rat AQP7. Gaps are
inserted to maximize matching. White letters in black boxes denote the amino acid residues conserved at least two of them. The predicted transmembrane domains of AQP7 are
underlined. The conserved NPA motifs are
overlined.
[View Larger Version of this Image (65K GIF file)]
Fig. 2.
Western blot analysis of oocyte membrane
proteins probed with polyclonal antibodies against AQP7. Oocytes
were injected with water (cRNA()) or 5 ng of cRNA of AQP7
(cRNA(+)). Membranes prepared from three oocytes were loaded
in each lane. The blots were probed with anti-AQP7 antiserum against
the C-terminal peptide (A) or against the N-terminal peptide
(B). The specific bands for AQP7 cRNA injection were
indicated by arrows. The positions of the molecular markers
(kDa) are indicated.
[View Larger Version of this Image (31K GIF file)]
Fig. 3.
Functional expression of AQP7 in
Xenopus oocytes. A, osmotic water permeability
(Pf) of oocytes injected with 50 nl of water or 5 ng
of AQP7 cRNA. Bars show mean ± S.E. of five
determinations of oocytes. Hg indicates that the assay was performed after 5 min incubation in 0.3 mM mercury
chloride. B, time course of [14C]glycerol
uptake into oocytes injected with water () or 20 ng of AQP7 cRNA
(
). C, time course of [14C]urea uptake into
oocytes injected with water (
) or 20 ng of AQP7 cRNA (
).
D, glycerol permeability (Pgly) of
oocytes expressing AQP7 at different temperatures. Each point
represents means of 7-8 measurements. Pgly was
measured in water-injected oocytes (
) and in AQP7-cRNA-injected
oocytes (×) at 4, 20, and 20 °C. To estimate
AQP7-dependent Pgly (
),
Pgly of control oocytes was subtracted from
Pgly of AQP7 oocytes at each temperature.
AQP7-dependent Pgly was calculated to be 5.3 kcal/mol from the fitted line.
[View Larger Version of this Image (24K GIF file)]
6 cm/s), which was comparable with the
previous reports of glycerol uptake by AQP3 (9, 13). Urea uptake was
also stimulated with AQP7 expression (Fig. 3C). The
incubation in 22.6 µM [14C]urea for 5 min
resulted in the increase of urea uptake through AQP7 by 9-fold
(824 ± 238 cpm; n = 5 versus 93 ± 4; n = 6) and for 10 min by 16-fold (1717 ± 107 cpm; n = 4 versus 107 ± 5;
n = 9) (the calculated Purea was
12.0 × 10
6 cm/s). The degree of stimulation of urea
uptake by AQP7 is much higher than that of AQP3 (9, 11, 13). The
activation energy for glycerol permeability was calculated by measuring
Pgly at different temperatures. However, the
result was complicated by the endogenous glycerol permeability of
oocytes. AQP7-dependent Pgly was
obtained by subtracting Pgly of control oocytes
from Pgly of oocytes expressing AQP7 at each
temperature (Fig. 3D). The activation energy for
AQP7-dependent Pgly was calculated
to be 5.3 kcal/mol. As both activation energies for water and glycerol are low, water and glycerol permeate AQP7 by channel mechanism and may
share the same pathway.
Fig. 4.
Northern blot analysis of AQP7. 2 µg
of poly(A)+ RNA from various rat tissues
(CLONTECH) was hybridized with
32P-labeled probe of AQP7 cDNA (Fig. 1A).
The positions of the RNA markers (kb) are indicated.
[View Larger Version of this Image (34K GIF file)]
Fig. 5.
The detection of AQP7 mRNA by in
situ hybridization in rat testis. A, antisense probe
staining is apparent in round spermatids. B, serial section
hybridized with sense probe demonstrated no staining. C, the
view at higher magnification revealed the message was localized to late
spermatids in seminiferous tubules.
[View Larger Version of this Image (122K GIF file)]
Fig. 6.
Immunohistochemistry of rat testis with
anti-AQP7 antibody. A, the cells of the later stages of
spermatogenesis, from late to maturing spermatids, were selectively
stained by the antiserum against C-terminal peptide of AQP7.
B, the staining with the preimmune serum was negative. The
nuclei were stained by hematoxylin.
[View Larger Version of this Image (149K GIF file)]
*
This work was supported by a grant-in-aid from the Ministry
of Education, Science and Culture (Japan) and a grant from The Salt
Science Research Foundation.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence and reprint requests should be
addressed: Second Dept. of Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan. Fax: 81-3-5803-0132; E-mail: kishibashi.med2{at}med.tmd.ac.jp.
1
The abbreviations used are: GlpF, glycerol
facilitator; AQP, aquaporin; PCR, polymerase chain reaction; kb,
kilobase pair(s).
2
K. Ishibashi, unpublished observations.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.