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From the IHF Institute for Hormone and Fertility Research at the University of Hamburg, Hamburg, Germany
| Correspondence to: Dr Christiane Kirchhoff, IHF Institut für Hormonund Fortpflanzungsforschung an der Universität, Grandweg 64, D-22529 Hamburg, Germany (e-mail: kirchhoff{at}ihf.de). |
| Received for publication December 19, 2002; accepted for publication February 14, 2003. |
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Abstract |
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Key words: Canine epididymis, transporter, TSCOT homologue, apical membrane compartment
The epithelial cells lining the duct play an important role in establishing and maintaining the luminal fluid microenvironment. Knowledge about the membrane transport systems that may mediate epithelial uptake and efflux of the different organic and inorganic osmolytes, however, is still limited. Molecular cloning techniques have led to considerable progress recently. The organic-related cation transporter OCTN2 (CT1) is present in rat epididymis in a region-dependent manner and may be responsible for the transport of L-carnitine in this species (Rodriguez et al, 2002). A novel carnitine transporter, CT2, was identified in the apical membrane compartment of the human epididymis (Enomoto et al, 2002). The glutamate transporter EAAC1 mediating glutamate reabsorption was found in all regions of the mouse epididymis with high expression levels in the initial segment and cauda region (Wagenfeld et al, 2002). Moreover, rat efferent ducts and epididymis express various ion transport molecules that are also involved in kidney tubular functions, for example the Na+/H+ exchanger NHE3 (Bagnis et al, 2001; Zhou et al, 2001). Other epididymal transport molecules remain to be identified.
The primary structures of many transport proteins are now available (for
review, see Saier, 2000).
Although only very few have yet been studied by X-ray diffraction, it can be
deduced that solute transporters represent integral membrane proteins composed
of a variable number of highly hydrophobic transmembrane 
-helical
segments connected by intra- and extracellular loops (for review, see
Griffith and Sanson, 1998). Based on hydropathy profiling, many of them are predicted to adopt a
12-membrane–spanning domain structure with both N- and C-termini located
intracellularly. One of the largest groups of transport proteins adopting this
topology is the major-facilitator superfamily, including the sugar permeases
(Walmsley et al, 1998). In
addition to sugar transport, members of this superfamily play important roles
in antibiotic resistance, toxin secretion, and ion balance.
In higher organisms, nutrient capture and waste efflux appear to proceed in
a tissue-specific manner. We report here the cloning of a putative 12
transmembrane domain cotransporter highly enriched in the canine epididymis.
The corresponding canine epididymal 11 cDNA (CE11) was first obtained from an
epididymal cDNA library during the course of a differential screening (for
review, see Ivell et al, 1998;
Kirchhoff, 2002). Dogs were
chosen because they represent a key species in biomedical research and serve
as a large animal model for a number of complex human pathologies. The
+/–-screening approach enabled us to identify highly to moderately
abundant gene products involved in various aspects of epididymal physiology
(for review, see Kirchhoff,
2002). The present investigation describes the molecular
characterization of the putative CE11 cotransporter on the mRNA and protein
level. Prediction of multiple -helical transmembrane-spanning segments,
in situ transcript hybridization, Western blot analysis of epididymal membrane
proteins, and immunolocalization in the apical membrane compartment of the
canine epididymal duct epithelium suggested that CE11 represents an abundant
apical transport protein of the epididymis involved in the formation of the
specific luminal microenvironment.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Screening of Canine Epididymal cDNA Library
A canine epididymal cDNA library constructed in the Lambda UniZAP-XR vector
(Stratagene, LaJolla, Calif; Ellerbrock et
al, 1994) was screened employing a hybridization strategy
essentially as previously described
(Beiglböck et al, 1998).
Briefly, approximately 10 000 independent cDNA clones were plated at low
density (approximately 500 plaque-forming units per plate) and screened by a
multistep procedure employing 32P-radiolabeled single-stranded cDNA
pools from canine epididymis to provide positively hybridizing signals and
from liver, testis, and lung as negative controls. Bacteriophages from
epididymis-positive plaques were purified by successive dilution and the
inserted cDNA sequence recovered by in vivo excision as previously described
(Ellerbrock et al, 1994). The
resulting recombinant plasmid DNAs were amplified and purified, and replicate
cDNA clones were subjected to sequence analysis following standard
procedures.
RNA Preparation and Northern Blot Analysis
Total RNA from the various tissues was extracted into 15 to 20 volumes of
chaotropic solution as previously described
(Pera et al, 1996). Ten to 20
µg of total RNA per lane, depending on the type of experiment, were loaded
and separated by denaturing agarose gel electrophoresis. Equal loading was
ascertained by ethidium bromide staining of the gels prior to capillary RNA
transfer to Hybond N+ membranes (Amersham-Buchler, Braun-schweig, Germany). An
RNA size marker (Gibco-BRL, Eggenstein, Germany) was included in a number of
experiments, and mRNA lengths were estimated by comparison of electrophoretic
migration. Nonradioactive Northern hybridization employing digoxigenin (DIG)
labeled cDNA fragments was performed as previously described
(Pera et al, 1996;
Beiglböck et al,
1998).
Standard RT-PCR and Generation of Hybridization Probes
For standard reverse transcription-polymerase chain reaction (RT-PCR),
oligo(dT)-primed cDNA was synthesized from 5 µg of total epididymal RNA in
a 20 µL reaction using 200 U of Superscript II reverse transcriptase
(Gibco-BRL), 1 mM dNTP, and 0.5 µg oligo(dT)12–18.
Incubation was for 60 minutes at 45°C. PCR amplification was performed in
a 50 µL volume with 0.5 U Biotherm Taq-Polymerase (Genecraft, Münster,
Germany), 1 µL single-stranded cDNA, 200 nM dNTP, and 20 pmol each of
primers in the PCR buffer provided (Genecraft). Hypoxanthine-guanine
phosphoribosyltransferase (HPRT) cDNA control primers were
5'-CCTGCTGGATTACATTAAAGC-3' and
5'-GTCAAGGGCATATCCAACAAC-3'. Sequences of oligonucleotide primer
pairs and PCR conditions for CE11 were as shown in the Table. DIG-labeled cDNA
probes were prepared by PCR amplification from purified cDNA fragments
following the instructions of the supplier (Boehringer, Mannheim, Germany). A
DIG-labeled 766-bp Ce1 cDNA fragment
(Ellerbrock et al, 1994) was
employed as a control (Ce1 sense and antisense primer sequences in the
Table).
Generation of 5' Ends by Repeated Inverse PCR
cDNA synthesis with CE11 sequence-specific primers was performed in the
presence of betaine and trehalose as described by Spiess and Ivell
(2002). Sequences of primers
and specific PCR conditions are listed in the Table. Reverse transcription was
performed by temperature cycling between 55°C and 60°C in a thermal
cycler (PTC200, MJ Research, Watertown, Mass) following the cDNA synthesis
program by Carninci and Hayashizaki
(1999) with the modifications
described. Step 1 consisted of denaturation at 65°C for 10.5 minutes in 14
µL mixture A (5 µg canine epididymal total RNA, 500 ng of a CE11
sequence-specific primer and 12 µL of 5 M betaine). Following a hot start
after 10 minutes, 17 µL of mixture B (3 µL 10 x first-strand
buffer [Gibco]); 9 µL 2 M trehalose (Sigma, Deisenhofen, Germany); 3 µL
100mM DTT (Gibco); 1.5 µL 10 mM NTP (Genecraft); 1 µL RNase H (Gibco, 40
u/µL); and 1 µL Superscript II (200 u/µL [Gibco]) were added. Step 2
consisted of annealing at 61°C for 2 minutes. Step 3 was a negative ramp
at 0.2°C per second to 55°C (gradient annealing), and step 4 consisted
of 55°C for 5 minutes (complete annealing). Step 5 was a positive ramp at
0.1°C per second to 60°C, followed by 60°C for 2 minutes (step 6),
55°C for 2 minutes (step 7), 60°C for 2 minutes an additional 10 times
(step 8), 70°C for 10 minutes (step 9), 4°C for 10 minutes (step 10)
until the end (step 11). Second-strand synthesis, ring ligation, and inverse
PCR amplification was performed as previously described
(Gebhardt et al,
1999). 
PCR products
were isolated and ligated into the TA-cloning vectors pCRII (Invitrogen,
Heidelberg, Germany) or pGEM-T Easy (Promega, Mannheim, Germany). Plasmid DNA
was sequenced from both strands (MWG, Ebersberg, Germany), and sequence
analysis was performed using the Lasergene software package (DNASTAR Inc,
Madison, Wis).
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In Situ Transcript Hybridization
Epididymides from 2 small-sized dogs were fixed in Bouin solution for 6
hours immediately after castration, washed in 70% ethanol, and embedded in
paraffin wax. Five to 7 µm sections were prepared, deparaffinize, and
subjected to a nonradioactive hybridization procedure as previously described
(Beiglböck et al, 1998).
Specifically, CE11 sense and antisense cRNA probes were labeled by in vitro
transcription in the presence of digoxigenin-UTP. An
EcoRI/XhoI-cDNA fragment subcloned in p-Bluescript S/K
(Stratagene) was employed as a template that had been obtained from a 5'
terminally truncated CE11 cDNA and comprised approximately 310 nucleotides of
the open reading frame (nucleotide 912–1221; compare
Figure 1). Alkaline
phosphatase-conjugated anti-DIG antibody (Boehringer) and detection reactions
were performed following the instructions of the supplier. Negative controls
were performed in parallel on adjacent tissue sections using the
digoxigenin-labeled sense-strand cRNA. Sections were analyzed by bright-field
microscopy without counterstaining.
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Generation of Polyclonal Antibody
A 30–amino acid hydrophilic sequence was chosen from the central loop
of the predicted CE11 protein to raise polyclonal antibodies. The sequence of
the 30-mer peptide was NH2-CYRTLDPDHSDKQSVQGLHPPSPGKAKPRR-COOH; an
N-terminal cysteine was added for convenient coupling. Oligopeptides were
chemosynthetically obtained, conjugated to keyhole limpet hemocyanin (KLH) as
a carrier protein, and the KLH-peptide conjugates employed to immunize female
rabbits (Pineda-Antikörper Service, Berlin, Germany). Immune sera were
obtained after 60, 90, 120, and 135 days. Monospecific purification of
antipeptide antibodies was performed by affinity chromatography using the
30-mer oligopeptide as a ligand (Pineda-Antikörper-Service).
Membrane Preparation
Membranes of 2 canine epididymides were prepared essentially as previously
described by Obermann et al
(2003). Briefly, frozen
tissues were pulverized under liquid nitrogen in a dismembrator (Braun,
Melsungen, Germany) and suspended in 10x volume of ice-cold
homogenization buffer containing 50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM
dithiothreitol, and complete protease inhibitor as suggested by the supplier
(Roche Molecular Biochemicals, Mannheim, Germany). Suspensions were
homogenized by 10 strokes in a Potter-Elvehjem homogenizer and centrifuged to
separate debris (3000 x g, 5 minutes, 4°C). Supernatants
were ultracentrifuged at 100 000 x g, 30 minutes, 4°C.
Pellets containing the membrane fractions were resuspended in 50 mM Tris, pH
7.5, containing complete, and membrane suspensions were stored as aliquots at
–80°C. The corresponding supernatants were stored as cytosolic
fractions. Prior to electrophoresis, membrane suspensions were solubilized for
1 hour at ambient temperature in 20 mM Tris, pH 9.5, containing complete, 65
mM DTT, 7 M urea, 2 M thiourea, 4% CHAPS (3-[3-cholamidopropyl
dimethylammonio]-1-propanesulfonate), and 1% Triton X-100
(Quaranta et al, 2001).
Immediately before gel loading, samples were centrifuged to separate
nondissolved protein (3000 x g, 5 minutes, 4°C).
Western Blot Analysis
Proteins from solubilized membranes and corresponding cytosolic fractions
were denatured in Laemmli buffer (50 mM Tris-Cl, pH 7.5, 20 mM dithiothreitol,
2% SDS, 0.5% bromophenol blue, 10% glycerol). Five to 10 µL membrane
protein extract, depending on protein content, were loaded per lane, separated
on 15% Laemmli gels containing 7 M urea, and transferred to polyvinylidene
fluoride membranes (Amersham) in a discontinuous buffer system using a semidry
blotter (Phase, Lübeck, Germany). Immunodetection was carried out by
blocking in 1% blocking solution (Boehringer) for 1 hour followed by an
incubation with the affinity-purified polyclonal antibody (dilution 1:500)
against the chemosynthetic CE11 peptide (compare
Figure 1). Antibody binding was
recognized by a peroxidasecoupled goat anti-rabbit antibody (Sigma). For
chemoluminescence detection the CL-HRP substrate system (Pierce Chemical
Company, Rockford, Ill) was used at a dilution of 1:10 and exposed to x-ray
film (Fuji Photo, Tokyo, Japan). Specificity of antibody binding was shown by
competition with the chemosynthetic oligopeptide preincubating the anti-serum
with the peptide (20 µg/mL of diluted antibody, 100 µg of total CE11
peptide).
Immunohistochemistry
Cryosections (8 to 10 µm) of 2 dissected dog epididymides were postfixed
in 4% paraformaldehyde in PBS, followed by brief washings in PBS.
Immunoperoxidase staining and indirect immunofluorescence were performed as
previously described (Kirchhoff et al,
1996). The affinity-purified polyclonal CE11 antipeptide antibody
was employed at a concentration of approximately 2 µg/mL for Envision
immunoperoxidase processing (DAKO, Hamburg, Germany) and at a concentration of
approximately 5 µg/mL for indirect immunfluorescence employing Cy2
anti-rabbit antibody from goat (Jackson ImmunoResearch Laboratories,
Westgrove, Penn). Cytokeratin intermediate filaments of epididymal duct
epithelium were stained using rabbit polyclonal antibody Z622 (DAKO) at a
dilution of 1:1000.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Although the overall nucleotide sequence similarity between CE11 and TSCOT was high (approximately 80%), an RT-PCR approach based on the homologous human and mouse sequences did not yield the 5' end of CE11. An inverse RT-PCR protocol (Gebhardt et al, 1999) using specific CE11 cDNA synthesis primers (compare the Table) was repeatedly applied. After 4 cycles, the cDNA sequence comprised 2280 nucleotides (Figure 1), which is in good agreement with the length of the hybridizing mRNA band (see below). The full-length cDNA sequence (GenBank accession AY195876) contained an open reading frame of 481 codons, starting from a putative ATG initiation codon (nucleotide positions 74 to 76) and ending with a TGA stop codon (positions 1517 to 1519; Figure 1). Alignment with the predicted human and mouse TSCOT amino sequences showed a high degree of homology; however, 5' and 3' regions as well as the central most parts were divergent (Figure 2a).
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Characteristics of the Predicted CE11 Proteins
The protein predicted from the full-length CE11 cDNA comprised 481 amino
acid (Figure 2a) and appeared
to be extremely hydrophobic, with about half of the amino acids being nonpolar
and approximately 85% being uncharged. Using different prediction servers, no
typical signal peptide sequence was identified. Instead, 11 certain plus 1
weak transmembrane domains (Figure
2b) were predicted (Hofmann
and Stoffel, 1993). The weak transmembrane domain was located near
the N-terminus and could serve as a leader sequence for membrane integration.
Topology assignment using TMbase (Hofmann
and Stoffel, 1993) strongly matched that of a type 3b membrane
protein with 12 transmembrane -helices of 22 to 23 amino acids in
length and both, N- and C-termini, inside. The molecule appeared to consist of
2 halves of 6 -helices that were separated by a 58–amino acid
long central hydrophilic region (Figure
2b). A CE11 model as created by the Sosui server
(Hirokawa et al, 1998) is
shown in Figure
2,Figure 2.
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The protein property search (Propsearch, Hobohm and Sander, 1995) as well as a sequence pattern comparison employing Pfam (Bateman et al, 2002) suggested that CE11, like TSCOT, is related to the major facilitator superfamily, and more specifically to sugar (and other) transporters. Like in human and mouse TSCOT (Chen et al, 2000; Kim et al, 2000), an N-linked carbohydrate side chain was predicted in the first extracellular loop. Within this loop, however, the CE11 protein sequence was distinct by an insertion comprising a 10–amino acid long glycine-alanine (GA) repeat (amino acid 49 through 58), which was lacking in both TSCOT sequences.
Tissue Distribution of CE11 and TSCOT mRNAs
To study the tissue distribution of the CE11 mRNA, a Northern blot analysis
was performed employing total RNA extracts of different canine tissues. A
DIG-labeled 542-bp cDNA fragment (position 1013 to 1554) from the C-terminal
part of the CE11 open reading frame hybridized strongly with an approximately
2.4 kb mRNA of the epididymis but not of other canine tissues
(Figure 3a). Comparing
different parts of the canine epididymis, the CE11 mRNA was detected at
similar levels (not shown). EST database searches identified 2
CE11-overlapping EST clones from canine heart (GenBank accessions BU750104,
BU750103), suggesting a broader tissue distribution. Therefore, canine tissues
were screened for CE11 expression by the more sensitive technique of RTPCR. At
25 cycles, this analysis revealed a strong band in the epididymis and very
faint bands in other tissues, thus largely confirming our Northern blot
results (not shown). At 30 cycles, however, CE11 amplicons were obtained from
all canine tissues included in the study, albeit at different levels
(Figure 3b). Epididymis cDNA
still showed the strongest signal, followed by spleen and lymph nodes. In
addition to amplicons derived from full-length CE11 cDNA, our analysis
revealed smaller bands in most tissues with the exception of ovary. Subcloning
and sequencing showed that these bands corresponded to a shorter splice
variant (GenBank accession AY225149), which lacked 172 nucleotides
(nucleotides 1049 to 1220). The encoded protein isoform was predicted to
consist of 347 amino acids only (Figure
2a).
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Employing the same DIG-labeled 542-bp cDNA fragment (which comprised the most conserved part of the CE11 sequence) as a heterologous probe, we looked for CE11 species counterparts in other mammals. A DIG-labeled Ce1 probe, hybridizing with homologous mRNAs of all mammalian species, was employed as a positive control. At low stringency, CE11 cross-hybridizing mRNAs were observed in the epididymides of 2 ungulates (Sus and Equus) and the rabbit, but not in rodents or in the human (Figure 4). Considering the high sequence similarity between CE11 and TSCOT, this result was unexpected. In order to detect lower levels of TSCOT mRNA expression, different human and mouse tissues were screened by RT-PCR. In the mouse, clear TSCOT amplicons were obtained at 30 cycles from various tissues, including proximal and distal parts of the epididymis (Figure 5a). In the human epididymis, no amplicons were obtained at 30 cycles with different primer combinations, whereas human lung revealed a strong band with each of these pairs of primers (Figure 5b).
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Localization of the CE11 mRNA by In Situ Transcript
Hybridization
A 310-bp cDNA plasmid subclone was generated from the CE11 open reading
frame (see ``Materials and Methods'') and DIG-labeled, single-stranded
antisense and sense cRNA probes obtained from the linearized plasmid. The CE11
antisense probe specifically stained the epididymal duct epithelium, whereas
intertubular connective tissue and spermatozoa-containing lumen were
essentially negative (Figure
6a). Maximum staining intensity was observed within the principal
cells, most probably in the endoplasmic reticulum (ER). Employing the
corresponding sense probe on adjacent tissue sections, no epithelial staining
above the background was observed (Figure
6b). Comparing consecutive regions of the canine epididymis
(Figure 7) differential,
although equally strong patterns of epithelial labeling were observed in
caput, corpus, and cauda, most probably reflected changes in epithelial
morphology and ER location along the organ.
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Physical Analysis of CE11 Membrane Proteins
To verify the predicted CE11 protein(s) and to investigate their physical
nature, membrane suspensions were prepared from canine epididymides, proteins
solubilized (see ``Materials and Methods''), and separated by denaturing
SDS-PAGE for Western blot analysis, a continuous presence of protease
inhibitors excluding unspecific proteolysis. Polyclonal antibodies were raised
against a 30-mer chemosynthetic peptide corresponding to the central
hydrophilic region of CE11 (compare Figure
2,Figure 2).
Affinity-purified antibodies revealed a prominent immunopositive band in the
range of 35 kDa (Figure 8),
which did not react with the preimmune serum and was competed for by the
chemosynthetic CE11 oligopeptide (see ``Materials and Methods''). In most
experiments, this prominent protein band was accompanied by a second minor
band that migrated even faster. No such bands were observed in the
corresponding cytosolic protein fractions (not shown). The divergence between
the apparent masses of the immunopositive bands and the masses calculated from
the predicted CE11 peptide isoforms may be attributed to an abnormal
electrophoretic migration pattern of the membrane protein. Abnormal migration
during SDS-PAGE had previously been described for the recombinantly expressed
TSCOT protein (Kim et al,
2000). Localization of the CE11 protein within the canine
epididymis was achieved employing the affinity-purified antibodies in
immunoperoxidase staining and indirect immunofluorescence on postfixed
cryo-sections (Figure 9).
Preimmune serum was employed as a negative control. Both staining techniques
specifically detected a CE11-related antigen in the abluminal part of the
epithelium in most epididymal regions. Apical staining above the background
was observed, which was clearly distinct from the intracellular staining
pattern of epithelial keratins.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-helical domains, the 2 similar halves of 6 domains
separated by a large hydrophilic loop. The first transmembrane segment is
weak, which, however, is not unusual for sugar (and other) transporters
(Sato et al, 1998).
Despite a high degree of overall sequence homology to TSCOT, CE11 is
distinct by the sequence of its central hydrophilic region separating the two
6-transmembrane domain parts of the molecule. Another difference concerns the
insertion of a 10–amino acid GA repeat that is not found in human and
mouse TSCOT. The significance of this insertion as a part of the first
extracellular loop is unknown. However, a minimal octamer GA repeat with 3
alanines in different positions of I
B, was sufficient to prevent
the degradation of this protein (Sharipo
et al, 1998). It is assumed that the GA repeat interacts with an
as yet unidentified component of the ubiquitin-proteasome–dependent
degradation pathway. Thus the GA repeat in CE11 may likewise be able
specifically to stabilize the membrane protein and to reduce basal turnover. A
shorter splice variant was identified in various tissues of the dog. Similar
variants have not been previously reported for human or mouse TSCOT. It
appears that in the canine epididymis, both splice variants are translated and
transported to the plasma membrane since our Western blot analysis of
epididymal membrane proteins revealed 2 immunopositive bands.
In higher organisms, members of the various transporter families may be expressed in a tissue-specific manner (Griffith and Sanson, 1998). TSCOT mRNA is highly up-regulated in immunodeficient severe combined immunodeficiency disease mice and was assumed to be thymus-specific (Kim et al, 2000). However, we found this mRNA also in mouse epididymis, heart, kidney, and lung. In the dog, epididymal CE11 mRNA levels by far exceeded that of other tissues. Thymic tissue from the dog was not available, but may represent a major site of CE11 expression as well. In summary, our more sensitive RTPCR analysis suggests that CE11 is highly up-regulated in the canine epididymis, but similar to TSCOT has a broader tissue distribution. Human epididymides, on the other hand, did not seem to express detectable TSCOT mRNA levels. A reason for the differences between species is not known. Although it cannot be excluded that the human tissues derived from elderly patients showed abnormal expression, they may substitute for a putative CE11/TSCOT transporter function by the expression of a still unknown splice variant or a different member of the large family of transmembrane solute transporters.
As expected for highly hydrophobic membrane-spanning proteins, CE11 was difficult to solubilize and to demonstrate by Western blot analysis. This is indeed the first report on the endogenous membrane protein, although in vitro–synthesized TSCOT protein has previously been described (Kim et al, 2000). The detection of immunopositive bands in membrane preparations of the canine epididymis thus may reflect very high CE11 expression levels in this tissue. As already described for the in vitro–synthesized TSCOT protein (Kim et al, 2000), we encountered substantial electrophoretic migration anomalies during SDS-PAGE, which may be due to the hydrophobic nature of the CE11 proteins. The high numbers of hydrophobic domains in these proteins may result in the binding of an excess of SDS and/or incomplete unfolding during electrophoresis.
As a consequence of epithelial cell polarity, apical and basolateral membrane domains contain specific sets of transporters exhibiting a high degree of selectivity for the transported substances. In the epididymis, the apical membrane compartment is a primary site for the regulated entry and extrusion of various substances exchanged between the epithelial and luminal compartment. In addition to the more general functions of epithelial transport and protection, however, CE11 may also be involved in sperm transport, maturation, and storage. Immunohistochemical studies revealed that a CE11-related antigen is highly enriched in the apical membrane compartment. This staining pattern most probably resulted from the transport protein located in the epithelial ``brush border'' and orientated toward the duct lumen. Thus a CE11 transporter exposed at the interface between the luminal and epithelial compartment is in an ideal position to function in establishing and maintaining the specific luminal microenvironment.
The actual orientation of transmembrane regions and hydrophilic loops of members of the major facilitator superfamily, including CE11/TSCOT, is unknown. It is also not known which substrates are transported and which structural elements of the putative transporters may be involved in binding and translocation. In a future project, the use of specific antibodies raised against different short synthetic peptides corresponding to the various hydrophilic loops can be employed to map these loops to the extracellular (luminal) or cytosolic space. It might be expected that some of these antibodies also interfere with transport function and thereby identify functionally important amino acid residues of CE11. Based on these experimental data, it may be possible to construct a model of the transmembrane organization of the transporter, which then in turn can be used to predict its substrate specificity and sensitivity to inhibitors, which at the same time is an important prerequisite to possibly using this transporter as a drug carrier.
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Acknowledgments |
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Footnotes |
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References |
|---|
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bagnis C, Marsolais M, Biemesderfer D, Laprade R, Breton S.
Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis.
Am J Physiol Renal Physiol.2001; 280:F426
–F436.
Bateman A, Birney E, Cerruti L, et al. The Pfam protein families
database. Nucleic Acid Res.2002; 30:276
–280.
Beiglböck A, Pera I, Ellerbrock K, Kirchhoff C. Dog epididymis-specific mRNA encoding secretory glutathione peroxidase-like protein. J Reprod Fertil.1998; 112:357 –367.
Carninci P, Hayashizaki Y. High-efficiency full-length cDNA cloning. Methods Enzymol.1999; 303:19 –44.[Medline]
Chen C, Kim MG, Soo Lyu M, Kozak CA, Schwartz RH, Flomerfelt FA. Characterization of the mouse gene, human promoter and human cDNA of TSCOT reveals strong interspecies homology. Biochim Biophys Acta. 2000;1493:159 –169.[Medline]
Ellerbrock K, Pera I, Hartung S, Ivell R. Gene expression in the dog epididymis: a model for human epididymal function. Int J Androl. 1994;17:314 –323.[Medline]
Enomoto A, Wempe MF, Tsuchida H, et al. Molecular identification of
a novel carnitine transporter specific to human testis. Insights into the
mechanism of carnitine recognition. J Biol Chem.2002; 277:36262
–36271.
Gebhardt K, Ellerbrock K, Pera I, Ivell R, Kirchhoff C. Differential expression of novel abundant and highly regionalized mRNAs of the canine epididymis. J Reprod Fertil.1999; 116:391 –402.
Griffith JK, Sanson CE. The Transporter Facts Book. San Diego: Academic; 1998.
Hirokawa T, Boon-Chieng S, Mitaku S. SOSUI: classification and
secondary structure prediction system for membrane proteins.
Bioinformatics.1998; 14(4):378
–379.
Hobohm U, Sander C. A sequence property approach to searching protein databases. J Mol Biol.1995; 251:390 –399.[Medline]
Hofmann K, Stoffel W. TMbase—a database of membrane spanning proteins segments. Biol Chem.1993; 374:166 .
Ivell R, Pera I, Ellerbrock K, Beiglbock A, Gebhardt K, Osterhoff C, Kirchhoff C. The dog as a model system to study epididymal gene expression. J Reprod Fertil Suppl.1998; 53:33 –45.[Medline]
Kim MG, Flomerfelt FA, Lee KN, Chen C, Schwartz RH. A putative 12
transmembrane domain cotransporter expressed in thymic cortical epithelial
cells. J Immunol.2000; 164:3185
–3192.
Kirchhoff C. The dog as a model to study human epididymal function
at a molecular level. Mol Hum Reprod.2002; 8:695
–701.
Kirchhoff C, Osterhoff C, Young L. Molecular cloning and characterization of HE1, a major secretory protein of the human epididymis. Biol Reprod.1996; 54:847 –856.[Abstract]
National Institute of Health. Guiding Principles in the Care and Use of Laboratory Animals. Washington, DC: NIH. DHEW Publication 80–23.
Obermann H, Samalecos A, Osterhoff C, Schroder B, Heller R, Kirchhoff C. HE6, a two-subunit heptahelical receptor associated with apical membranes of efferent and epididymal duct epithelia. Mol Reprod Dev. 2003;64:13 –26.[Medline]
Pera I, Ivell R, Kirchhoff C. Body temperature (37°C) specifically down-regulates the messenger ribonucleic acid for the major sperm surface antigen CD52 in epididymal cell culture. Endocrinology.1996; 137:4451 –4459.[Abstract]
Quaranta S, Giuffrida MG, Cavaletto M, et al. Human proteome enhancement: high-recovery method and improved two-dimensional map of colostral fat globule membrane proteins. Electrophoresis.2001; 22:1810 –1818.[Medline]
Rodriguez CM, Labus JC, Hinton BT. Organic cation/carnitine
transporter, OCTN2, is differentially expressed in the adult rat epididymis.
Biol Reprod.2002; 67:314
–319.
Saalmann A, Münz S, Ellerbrock K, Ivell R, Kirchhoff C. Novel sperm-binding proteins of epididymal origin contain four fibronectin type II-modules. Mol Reprod Dev.2001; 58:88 –100.[Medline]
Saier MH Jr. Vectorial metabolism and the evolution of transport
systems. J Bacteriol.2000; 182:5029
–5035.
Sato M, Hresko R, Mueckler M. Testing the charge difference
hypothesis for the assembly of a eucaryotic multispanning membrane protein.
J Biol Chem.1998; 273:25203
–25208.
Sharipo A, Imreh M, Leonchiks A, Imreh S, Masucci MG. A minimal glycine-alanine repeat prevents the interaction of ubiquitinated I kappaB alpha with the proteasome: a new mechanism for selective inhibition of proteolysis. Nature Med.1998; 4:939 –944.[Medline]
Spiess AN, Ivell R. A highly efficient method for long-chain cDNA synthesis using trehalose and betaine. Anal Biochem.2002; 301:168 –174.[Medline]
Turner TT. Necessity's potion: inorganic ions and small organic molecules in the epididymal lumen. In: Robaire B, Hinton BT, eds. The Epididymis: From Molecules to Clinical Practice. New York: Kluwer Academic; 2002:131 –150.
Wagenfeld A, Yeung CH, Lehnert W, Nieschlag E, Cooper TG. Lack of
glutamate transporter EAAC1 in the epididymis of infertile c-ros receptor
tyrosine-kinase deficient mice. J Androl.2002; 23:772
–782.
Walmsley AR, Barrett MP, Bringaud F, Gould GW. Sugar transporters from bacteria, parasites and mammals: structure-activity relationships. Trends Biochem Sci.1998; 23:476 –481.[Medline]
Zhou Q, Clarke L, Nie R, et al. Estrogen action and male fertility:
roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in
reproductive tract function. Proc Natl Acad Sci U S A.2001; 98:14132
–14137.
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