Department of Anatomy II, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
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ABSTRACT |
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Previous experiments have shown that an amino acid transport system B0+ transporter in cultured colonic epithelial cells mediates amino acid absorption. Here we describe the cloning and functional characterization of a system B0+ transporter selectively expressed in the colon. Using the combination of an expressed sequence tag database search and RT-PCR approaches, we cloned a mouse colonic amino acid transporter, designated mCATB0+. Northern blot analysis revealed that mCATB0+ was selectively expressed in the large intestine. In situ hybridization showed the mCATB0+ mRNA to be localized in absorptive epithelial cells. When expressed in Xenopus oocytes, mCATB0+ exhibited a Na+-dependent stereoselective uptake and a broad specificity for neutral and cationic amino acids, which is characteristic of amino acid transport system B0+. In vivo [3H]glycine uptake assay demonstrated that a system B0+-like transporter protein was expressed on the apical surface of the colonic absorptive cells. Our data suggest that a mouse colonic amino acid transporter mCATB0+ may absorb amino acids from the intestinal contents in the colon.
cDNA cloning; absorptive epithelial cell
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INTRODUCTION |
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THE COLON IS THE FINAL SITE not only for fluid and electrolyte conservation but also for absorption of nutrients in the gastrointestinal system. For example, in patients with short bowel syndrome, functional adaptations such as increased colonic absorption of nutrients and minerals can be observed (10). Recent research has elucidated various cellular and molecular mechanisms for water and electrolyte transport in colon. Amiloride-sensitive sodium channels located in the apical cell membrane, through which sodium ions are taken into the epithelial cells, are a case in point (2, 12). By contrast, little is known about the molecular mechanisms that regulate the colonic absorption of nutrients, including essential amino acids.
Amino acid transport across the plasma membrane is mediated by
transporters, which are distinguished primarily by substrate selectivity and ionic dependence (11). In the rabbit small
intestine, transport of glycine, lysine, and -alanine across the
brush-border membrane is mediated by a system B0+
transporter (8), which is characterized by
sodium-dependent uptake and broad specificity, with high affinity for
neutral and cationic amino acids (11). In cultured colonic
epithelial cells (Caco-2), neutral amino acids are absorbed via the
system B0+ transporter and system ASC transporter (a
sodium-dependent alanine/serine/cysteine transporter), and these amino
acids are then released from the cells in an intracellular
concentration-dependent manner (1). System B0+
is widely distributed on epithelial cells and mediates amino acid
transport on the apical surface (11).
In this study, we describe the molecular cloning and functional expression of a system B0+ transporter selectively expressed in the colon. In addition, we also provide evidence that a system B0+-like amino acid transporter is expressed at the protein level in the apical membrane of the colonic absorptive cells, absorbing amino acids from the intestinal contents in the colon. Our data will be helpful in understanding the molecular mechanisms that underlie amino acid absorption in the colon, which may be crucial in patients with short bowel and severe malabsorption in the remaining small intestine.
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METHODS |
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Molecular cloning and DNA sequencing.
Comparison of the glycine transporter protein sequence
(15) with the database of expressed sequence tags (EST)
identified one cDNA sequence from total mouse embryos (GenBank
accession no. AI006618). This clone showed a considerable similarity to
the putative transmembrane domain I-III of sodium-dependent transporters (9). The insert cDNA of the EST clone was
subcloned into pBluescript II SK() vector (Stratagene), and
sequencing was carried out on both strands by the dideoxy chain
termination method by using successive synthetic oligonucleotides.
Northern blot analysis. Twenty micrograms of total RNA isolated from various adult mouse organs were separated by electrophoresis on 1% agarose-formaldehyde gels and blotted onto Hybond-N+ membranes (Amersham Pharmacia). The procedures were basically the same as those described previously (18). The blots were hybridized with a random-primed 32P-labeled fragment of the mouse colonic amino acid transporter B0+ (mCATB0+) cDNA corresponding to nucleotides 767-1782. The hybridization signals were analyzed with a bioimaging analyzer BAS 2500 (FUJIX).
In situ hybridization.
Localization of the mCATB0+ mRNA in embryos at embryonic
day 18 and in adult mouse colon was determined by in situ
hybridization. Mouse embryos were also purchased from JAPAN SLC. The
procedures were basically the same as those described previously
(18). Fresh-frozen sections (6-µm thick) were cut on a
cryostat. To prepare riboprobes, a 1,016-bp mCATB0+ cDNA
insert (bases 767-1782) subcloned in the pBluescript II SK()
vector was used. The specificity of hybridization signals was confirmed
by a control study with the sense cRNA probe.
Functional characterization by Xenopus oocyte expression.
The coding sequence of mCATB0+ was subcloned by using
modified pBluescript (pBsMXT; Stratagene). The multiple cloning site of pBsMXT was flanked by Xenopus -globin 5'- and
3'-untranslated regions to promote stable mRNA expression in oocytes.
The procedures for two-electrode voltage-clamp recording were basically
the same as those described previously (4). Current
(I) as a function of substrate concentration ([S])
was fitted by least squares to I =
Imax · [S]/(Km
+ [S]), where Imax is the maximal current
and Km is the transport constant.
Glycine uptake by mouse colonic epithelial cells in vivo. Male ddy mice (at 10 wk of age) were fasted overnight before the experiments. Anesthesia was induced by an intraperitoneal injection of chloral hydrate (42 mg/kg). After the anesthesia was achieved, a modified Hanks' balanced salt solution (HBSS) containing D-glucose (19 mM) and [3H]glycine (20 µM), with or without 20 mM unlabeled various L-amino acids, was administered onto the luminal surface of the colon by intestinal infusion. Subsequently, 60 min after the administration, the colons (~7 cm in length) were dissected away, cut open longitudinally, and washed in the ice-cold HBSS three times. After the wash, they were digested with proteinase K, and the incorporated radioactivity was counted in a Beckman scintillation counter. Data were analyzed by using the unpaired Student's t-test. The level of significance was set at P < 0.01.
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RESULTS |
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Cloning of the full-length mCATB0+ cDNA.
Virtual screening of the dbEST database of the National Center for
Biotechnology Information (NCBI) with probes corresponding to the
glycine transporter sequence led to the identification of a system
B0+ transporter. Since this EST clone was synthesized by
using a PCR-based method (13), the insert cDNA might
contain some mutations. To address this problem, we recloned the same
transporter from adult mouse colon, and subsequent sequence analysis
revealed one point mutation at position 1449 of the EST clone (A
replaced by G). We confirmed that the cDNA was 1,923 bp long with an
open reading frame of 1,914 bp encoding a protein of 638 amino acids (Fig. 1), and the corresponding gene was
designated mCATB0+. Hydropathy analysis (5) of
the primary amino acid sequence of the predicted protein showed the
presence of 12 putative transmembrane domains. The predicted
mCATB0+ protein lacks an identifiable signal sequence,
which is consistent with a cytoplasmic location of the NH2
terminus. Seven potential N-linked glycosylation sites are located at
positions 155, 163, 174, 185, 193, and 198 in the putative second
extracellular loop and at position 298 in the putative third
extracellular loop. There are three potential sites for protein kinase
C-dependent phosphorylation. Recently, Sloan and Mager
(14) reported the cloning of the human amino acid
transporter system B0+ (hATB0+), which shows
88% amino acid identity with mCATB0+. They
also submitted the sequence for mouse ATB0+
(mATB0+) to GenBank. Only four amino acids are
different between mCATB0+ and mATB0+. As
described in DISCUSSION, however, mCATB0+
and hATB0+ showed quite different tissue distribution
patterns, raising the sufficient possibility that mCATB0+
is not a mouse counterpart to hATB0+.
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Localization of mCATB0+ by Northern blot
analysis and in situ hybridization.
Tissue-specific expression of mCATB0+ mRNA was examined by
Northern blot analysis (Fig. 2). In the
adult mouse, mCATB0+ mRNA (~4.0 kb long) was abundantly
and selectively expressed in the large intestine. Barely detectable
signals were observed in the lung. No signals were found in other
tissues such as brain, heart, liver, small intestine, kidney, spleen,
testis, and skeletal muscle. The level of mCATB0+ mRNA in
the lung was far lower than that in the large intestine, indicating
that mCATB0+ is almost specifically expressed in the large
intestine.
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Oocyte expression of mCATB0+.
To assess the transport activity, two-electrode voltage-clamp recording
was performed. Voltage-clamp measurements showed
concentration-dependent inward currents in response to glycine
(Km =142.1 ± 19.9 µM) (Fig. 4, A-C). The inward
current elicited by glycine superfusion was not seen when lithium was
substituted for sodium, indicating that the transport activity was
sodium dependent. No detectable currents were observed in
water-injected oocytes under the same extracellular conditions.
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Glycine uptake in the native mouse colon.
The results from our in situ hybridization and electrophysiological
experiments suggest that the native mouse colon is able to absorb amino
acids from the intestinal contents via the amino acid transporter
mCATB0+. To examine whether mCATB0+ protein is
expressed on the apical surface of the absorptive cells, we directly
administered [3H]glycine, with or without competitive
inhibitors, into the colonic lumen by intestinal infusion (Fig.
6). Administration of 20 µM [3H]glycine to the luminal surface of the colon resulted
in glycine influx into the absorptive epithelium [total uptake:
136.2 ± 9.6 pmol · animal1 · h
1
(mean ± SE of 3 separate experiments)]. By contrast, the
[3H]glycine influx was reduced to 84.1 ± 3.8 pmol · animal
1 · h
1
(n = 3) in the presence of 20 mM unlabeled glycine.
Although the 1,000-fold amounts of unlabeled glycine failed to inhibit the total [3H]glycine uptake completely, the difference
was statistically significant (P < 0.01), indicating
that there are specific glycine uptake systems in the colonic
absorptive cells. The component of specific glycine uptake was
completely inhibited in the presence of 20 mM unlabeled
L-histidine (a cationic amino acid), whereas coadministration of unlabeled L-aspartate (a negatively
charged amino acid) did not display any inhibitory effects. {The
[3H]glycine uptake in the presence of 20 mM unlabeled
L-histidine and L-aspartate were 41.9 ± 4.4 and 121.0 ± 18.2 pmol · animal
1 · h
1
(n = 3 each), respectively.} These results suggest
that the transporter that displays high affinity for neutral and
cationic amino acids is expressed in the apical membrane of the colonic
absorptive cells.
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DISCUSSION |
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We have described here the isolation and characterization of a sodium-dependent amino acid transporter abundantly and selectively expressed in colonic absorptive epithelial cells. On the basis of amino acid sequence homology, mCATB0+ is a member of the family of sodium/chloride-dependent transporters with a characteristic membrane topology of 12 transmembrane helices and potential N-linked glycosylation sites in the putative second extracellular loop (9, 11). The sequence of mCATB0+ shows 39 and 31% identities with human glycine transporter GlyT2 (7) and human L-proline transporter (13), respectively, and shows 88% identity with hATB0+ (14). A common structural feature of mCATB0+ and hATB0+ is a potential N-linked glycosylation site in the putative third extracellular loop that has not been found in other family members, including orphan transporters (9).
When expressed in Xenopus oocytes, mCATB0+ demonstrated a sodium-dependent stereoselective uptake and a broad specificity for neutral and cationic amino acids, which is characteristic of amino acid transport system B0+. mCATB0+ prefers hydrophobic L-amino acids with a large R group and has very low affinity for L-proline, which differs from other nonpolar amino acids in that it has a cyclic structure. Despite these findings, however, mCATB0+ shows an apparent affinity for several D-amino acids. Together with other structural features, a potential N-linked glycosylation site in the putative third extracellular loop may play a key role in accepting such a wide range of amino acid substrates.
Although the transport functions of mCATB0+ and hATB0+ are similar, the tissue distribution patterns of the two transporters are quite different. hATB0+ was expressed abundantly in lung, trachea, and salivary gland and weakly in colon (14), whereas mCATB0+ was expressed intensely in colon and faintly in lung. In human bronchial epithelial cells, hATB0+ on the apical surface may play a significant role in removal of amino acids to maintain a low amino acid concentration in the airway surface liquid (3). Similarly, mCATB0+ in the colon may be involved in the absorption of amino acids from the intestinal contents. Chen et al. (1) reported a system B0+ amino acid transporter on the apical surface in human colonic epithelial cells (Caco-2) involved in amino acid absorption. In Caco-2 cells, the apical uptake of amino acids is dependent on a combination of transport system B0+ and ASC, and the basolateral uptake is more dependent on the system L, sodium-independent system mainly for bulky side-chain amino acids (11). To examine the involvement of mCATB0+ in amino acid absorption in the colon, we investigated the localization of mCATB0+ mRNA in absorptive epithelial cells. Our in situ hybridization experiments showed dense hybridization signals for mCATB0+ localized in absorptive epithelial cells. These findings suggest that mCATB0+ actively absorbs neutral and cationic amino acids into the absorptive cells by using a electrochemical potential gradient, which is generated by high sodium concentrations in the intestinal contents.
The remarkable differences in the tissue distribution patterns of mCATB0+ and hATB0+ raise the sufficient possibility that mCATB0+ is not a mouse homologue of hATB0+. There may be a human homologue of mCATB0+ specifically expressed in the colon. The ability of the normal human small intestine to absorb amino acids is highly efficient, with only small amounts of amino acids reaching the colon. Therefore, it is also possible that hATB0+ in the colon is usually expressed at low levels and that the expression of hATB0+ mRNA can be markedly upregulated in a variety of pathological circumstances as the need for colonic absorption of amino acids arises. Further investigations are needed to examine these hypotheses.
In this study, we have also investigated whether a system B0+ transporter is expressed at the protein level and is functional on the apical surface of the colonic absorptive cells. Figure 6 clearly shows that, despite large numbers of microorganisms that can take up amino acids and metabolize them rapidly (6), the native tissue (mouse colon) can absorb the neutral amino acid glycine and the cationic amino acid L-histidine with high affinity but cannot absorb the negatively charged amino acid L-aspartate via the same pathway, a substrate specificity similar to that described for transport system B0+. The results also showed that the 1,000-fold amounts of unlabeled glycine failed to inhibit the total [3H]glycine uptake completely in vivo (the uptake was found to be ~60% of the original), whereas heterologous expression of the mCATB0+ protein in Xenopus oocytes displayed almost complete inhibition of total [3H]glycine uptake in the presence of cold glycine under the similar treatment conditions of solutions (Fig. 5). The high background radioactivity value of the glycine uptake assay in vivo was probably due to high viscosity of mucins and complex tertiary structures of the colonic epithelium. Alternatively, the colon may have another high Km pathway for glycine uptake, which would not be expected to be as sensitive to competition by unlabeled glycine. Together with the findings of the system B0+ transporter in human enterocyte-like Caco-2 cells and the localization of mCATB0+ mRNA, our in vivo glycine uptake assay suggests that mCATB0+ protein is expressed on the apical surface of the colonic absorptive cells, absorbing amino acids from the intestinal contents in the colon.
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ACKNOWLEDGEMENTS |
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We thank K. Tanaka and K. Kajita for their skillful technical assistance.
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FOOTNOTES |
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The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EBI Data Bank with accession no. AB033285.
Address for reprint requests and other correspondence: S. Ugawa, Dept. of Anatomy II, Nagoya City Univ. Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan (E-mail: ugawa{at}med.nagoya-cu.ac.jp).
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.
Received 5 December 2000; accepted in final form 23 March 2001.
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