Na+-dependent neutral amino acid transporter ATB0 is a rabbit epithelial cell brush-border protein

Nelly E. Avissar1, Charlotte K. Ryan2, Vadivel Ganapathy3, and Harry C. Sax1

Departments of 1 Surgery and 2 Pathology, University of Rochester Medical Center, Rochester, New York 14642; and 3 Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

System B0 activity accounts for the majority of intestinal and kidney luminal neutral amino acid absorption. An amino acid transport system, called ATB0 (also known as ASCT2), with functional characteristics similar to those of system B0, has been recently cloned. We generated polyclonal antibodies to human and rabbit ATB0 COOH-terminal peptides and used Western blot analysis to detect ATB0 protein in rabbit tissues, rabbit ileal brush-border membrane vesicles (BBMV), and HeLa cells transfected with plasmids containing ATB0 cDNAs. Immunohistochemistry was used to localize ATB0 in rabbit kidney and intestine. In Western blots of rabbit tissues, ATB0 was a broad smear of 78- to 85-kDa proteins. In transfected HeLa cells, ATB0 appeared as a smear consisting of 57- to 65-kDa proteins. The highest expression was found in the kidney. ATB0 was enriched in rabbit ileal BBMV and in HeLa cells transfected with ATB0 cDNAs. In the kidney and in the intestine, ATB0 was confined to the brush-border membrane (BBM) of the proximal tubular cell and of the enterocyte, respectively. Tissue and intracellular distribution of ATB0 protein parallels that of system B0 activity. ATB0 protein could be the transporter responsible for system B0 in the BBM of epithelial cells.

intestine; kidney; system B0 protein; transfected HeLa cells; C2BBe1; immunohistochemistry


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AMINO ACIDS ARE ABSORBED across the intestinal and renal epithelial cells by a multitude of transport systems that are expressed differentially in the brush-border membrane (BBM) vs. the basolateral membrane of these cells (10, 11). These systems are L, y+, y+L, b0,+, A, ASC, B0, B0,+, and X<UP><SUB>AG</SUB><SUP>−</SUP></UP>. They can be differentiated functionally by their substrate specificity and driving forces. Among these, systems L, y+, and b0,+ are Na+ independent, system y+L is Na+ independent for cationic amino acids and Na+ dependent for neutral amino acids, systems A, ASC, B0, and X<UP><SUB>AG</SUB><SUP>−</SUP></UP> are Na+ dependent, and system B0,+ is Na+ and Cl- dependent. Recent cloning studies have shown that many of these transport systems actually consist of two or more subtypes (8, 10, 11, 23, 26, 36). The BBM of the intestinal and renal epithelial cells has been shown to possess systems L, b0,+, ASC, B0, B0,+, and X<UP><SUB>AG</SUB><SUP>−</SUP></UP>. In contrast, the basolateral membrane of these cells possesses systems L, y+L, and A (8, 10, 11, 23, 26).

The focus of the present study is system B0. It is a broad-spectrum neutral amino acid transporter energized by a transmembrane Na+ gradient. It was originally called system NBB (neutral brush border) (32, 33) and later renamed system B0 (9). Under normal conditions, system B0 is responsible for the major portion of the luminal transport of neutral amino acids into enterocytes and kidney proximal tubular cells (15, 23, 27). We have found that system B0 activity is downregulated in rabbit residual gut after massive enterectomy and that combined epidermal growth factor and growth hormone treatment reverse this downregulation (13, 28). Specific cDNAs, which confer system B0 activity in oocyte and mammalian cell expression systems, have been cloned from rabbit intestine and human cell lines, including the human intestinal cell line Caco-2 (15, 16). The amino acid sequences of rabbit and human ATB0s are highly homologous to those of rat and mouse ASCT2 and are partially homologous to ASCT1 and to members of the glutamate transporter family (4, 11, 19, 26, 35). When expressed in heterologous systems, ATB0 induces Na+-dependent transport of neutral amino acids. The substrate specificity of the induced amino acid transport system is similar to that of system B0 described in the rabbit intestine and in Caco-2 cells (27, 32, 33). Even though ATB0 has been cloned from the small intestine and Caco-2 cells, the subcellular location of this transport protein has not been determined. System B0 is known to be restricted to the intestinal and renal BBM (8, 10, 11, 21, 26, 34). If ATB0 is also located in the intestinal and renal BBM, it will provide additional supporting evidence for the identity of ATB0 with system B0. To address this issue, we produced and characterized specific polyclonal anti-peptide antibodies against rabbit and human ATB0 proteins. Using the antibodies against the rabbit ATB0, we have determined the subcellular localization of the protein in the rabbit intestine and kidney. These studies show that ATB0 is a BBM protein and that the tissue distribution and abundance of ATB0 is similar to that of system B0.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of antibodies to rabbit and human ATB0 protein. Anti-ATB0 antibodies were custom generated by quality-controlled biochemicals (BioSource International, Hopkinton, MA). Two sequences, each from the COOH-terminal end of the rabbit or the human ATB0 proteins, were chosen. These sequences are partially homologous to ASCT2 but not to ASCT1. They are hydrophilic, modeled to extend into the cytoplasma, and were predicted to successfully generate antibodies. Two ATB0 peptides with an inclusion of a COOH-terminal cysteine (not found in the original sequence) were synthesized and purified. The sequence of the rabbit ATB0 peptide is Ac-YVDRTEQRGSEPELTQVC-NH2, and the human ATB0 peptide is Ac-YVDRTESRSTEPELIQVC-NH2 (in bold: sequence differences). The peptides were purified and conjugated to keyhole limpet hemocyanin and subsequently to tetanus toxoid. Conjugated rabbit ATB0 peptide was injected into a goat, and conjugated human ATB0 peptide was injected into a rabbit. Animals were boosted and bled five times, and the serum was titrated against the corresponding peptide. Serums (50 ml) with a high titer (>= 1/40,000) were affinity purified on the corresponding peptide affinity columns. Goat anti-rabbit ATB0 antibody concentration was 2.59 mg/ml, and rabbit anti-human ATB0 antibody concentration was 1.48 mg/ml.

Transfection of HeLa cells with ATB0 cDNAs. The cDNAs were functionally expressed in HeLa cells using the vaccinia virus expression system (1) as described previously (15). Each ATB0 cDNA was cloned into pSPORT in such an orientation that it would be controlled by the plasmid T7 promoter in the plasmid. Subconfluent HeLa cells in 24-well culture plates were transfected first with a recombinant vaccinia virus (VTF7-3) encoding T7 RNA polymerase and then transfected with the pSPORT-cDNA construct or the empty vector in the presence of Lipofectin (Life Technologies, Grand Island, NY). After 12 h, the cells were resuspended in 5 mM Tris · HCl buffer, pH 7.5, with protease inhibitors [1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 µg/ml of aprotinin, pepstatin, and leupeptin], subjected to one cycle of freeze-thaw, and lysed by passing five times through a 25-gauge needle. The membranes were collected by centrifugation at 21,000 g and resuspended in the lysis buffer. Transport measurements performed previously on transfected cultures showed a significant increase in B0 transport activity (15, 16).

Preparation of cultured human C2BBe1 cells for Western blot analysis. C2BBe1, a brush border-expressing subclone of Caco-2 (American Type Culture Collection no. CRL-2102), was grown to subconfluence in 90% Dulbecco's modified Eagle's medium with 4 mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 1 mg/ml sodium pyruvate, 0.01 mg/ml human transferrin, and 10% heat-inactivated fetal bovine serum.

C2BBe1 cells were lysed in a modified RIPA buffer [10 mM Tris, 150 mM NaCl, 0.5% Nonidet P-40, 1% Triton X-100, and 1 mM EDTA, pH 7.4, with protease and phosphatase inhibitors (PPI: 1 mM activated sodium orthovanadate, sodium fluoride, and PMSF; and 10 µg/ml of aprotinin, pepstatin, and leupeptin)].

Preparation of tissues for Western blot analysis. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Rochester. Non-gut tissues from healthy control New Zealand White rabbits were immediately frozen in liquid nitrogen. Intestinal and colon tissues were obtained, and mucosa was scraped and frozen. Crude extracts and brush-border membrane vesicles (BBMV) were prepared from mucosa by the magnesium precipitation technique as described previously (28), with addition of PPI to all the buffers.

Deglycosylation of proteins in crude extract from ileal mucosa. Glycoprotein deglycosylation kit (Calbiochem-Novabiochem, San Diego, CA) was used to deglycosylate the proteins in ileal mucosa crude extract. The procedure used was essentially as described in the manufacturer's data sheet with the addition of protease inhibitors (10 µg/ml of leupeptin, aprotinin, and pepstatin). Deglycosylation of native proteins for 24 h at 37°C with all five deglycosylation enzymes included in the kit was applied.

Western blot analysis. Crude extract of tissues or cells and samples of membranes were brought up to 0.06 M Tris · HCl, 10% glycerol, 0.1 M dithiothreitol, 1% SDS, and 0.0005% bromphenol blue and (without boiling) analyzed by 7.5% SDS-PAGE according to the method of Laemmli (17). Proteins were electotransferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked by incubation for 1 h at room temperature with 3% nonfat dry milk in 13 mM Tris and 150 mM NaCl, pH 7.5, containing 0.1% Tween 20 (TTBS). The membranes were incubated overnight at 4°C without and with goat anti-rabbit ATB0 or rabbit anti-human ATB0 antibodies (diluted 1:1,000 in the blocking solution). Membranes that were incubated without a primary antibody showed no staining (data not shown).

For negative controls, membranes were incubated with the primary antibodies in the presence or absence of 20× molar excess of the corresponding rabbit or human ATB0 peptide. To rule out nonspecific inhibition of the antibody by the specific peptides, the membranes were also incubated with the primary antibody in the presence of a nonspecific peptide from the COOH-terminal end of the mouse cationic amino acid transporter 2 (mCAT2). This peptide (KSVMQANDHHQR), a gift from Dr. Carol J. MacLeod, has no sequence homology to ATB0 peptides. PVDF membranes were then washed extensively with TTBS and incubated with horseradish peroxidase (HRP)-conjugated donkey anti-goat or donkey anti-rabbit antibodies (1:10,000) in blocking solution for 1 h at room temperature. Excess secondary antibody was washed with TTBS, and bound secondary antibody was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were exposed to X-OMAT AR film (Kodak, Rochester, NY). Benchmark standards (Life Technologies) were used to determine apparent molecular weight.

Immunohistochemical analysis of ATB0 in rabbit kidney and intestine. Fresh kidney and ileal tissues were fixed in neutral-buffered Formalin overnight and embedded in paraffin. Embedded tissues were cut into 4- to 5-µm sections and mounted on Plus slides. Sections were deparaffinized in xylene, rehydrated in 100, 95, and 70% ethanol, and rinsed in PBS. The sections were quenched with 3% hydrogen peroxide for 6 min and washed with running water and PBS. For antigen unmasking with heat retrieval, the sections were submerged in 1 mM citrate buffer, pH 6.0, in a microwave pressure cooker (1.5 kW, power level 5) for 30 min and then cooled with running distilled water rinse for 15 min. The slides were first blocked using avidin biotin-blocking kit according to the manufacturer's instructions (Dako, Carpendina, CA) and then blocked with 4% donkey serum in PBS for 20 min at room temperature. The blocked sections were washed with PBS and incubated for 1 h at room temperature with 1:800 dilution of goat anti-rabbit ATB0 in PBS plus 1% BSA with or without 20× molar excess of rabbit ATB0 or mCAT2 peptides. After being extensively washed in PBS, biotinylated donkey anti-goat (1:200 in PBS; Jackson ImmunoReasearch Laboratories, West Grove, PA) was added for 30 min at room temperature. The slides were washed and treated with streptavidin-HRP (Jackson ImmunoReasearch Laboratories) for 30 min, rinsed with PBS, and developed with 3-amino-9-ethylcarbazole chromogen (ScyTek, Logan, UT) for 5 min. Slides were counterstained in Mayer's modified hematoxylin blue in 0.3% ammonia. Control staining was performed in an identical way but without the primary antibodies or with the primary antibody in the presence of 20× molar excess of the specific rabbit ATB0 peptide or the nonspecific mCAT2 peptide. ATB0 protein in ileum could be detected only in frozen sections.

Ileal tissue was submerged in optimum cutting temperature compound (Sakura Finetek USA, Torrance, CA) and frozen in liquid nitrogen. Tissue sections were fixed for 5 min in acetone, washed in PBS, and processed for immunostaining with anti-rabbit ATB0 antibody at a 1:400 dilution as described for the paraffin-fixed sections.

Periodic acid-Schiff and anti-human cytokeratin staining. Immunohistochemical staining for rabbit kidney cytokeratin AE1/AE3 (a broad-spectrum cytokeratin), using the Vector-Jackson biotin and streptavidin detection method and staining by periodic acid-Schiff, was performed by standard procedures (2, 22).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Western blot analysis of ATB0-transfected HeLa cells. The custom-prepared polyclonal antibodies against the rabbit and human ATB0 peptides had a titer of >1/40,000 when tested against the corresponding peptides (data not shown). To determine whether the antibodies react with ATB0 protein, we transfected HeLa cells with plasmid alone or with a plasmid carrying either rabbit or human ATB0 cDNA. The membrane proteins were analyzed by Western blotting. Rabbit kidney crude extract was applied to one of the lanes because of a high inherent system B0 activity and ATB0 mRNA (15, 16, 34). With anti-rabbit ATB0 antibody, no protein band was detected in HeLa cells transfected with plasmid alone (Fig. 1A, left, lane 2), indicating that this antibody does not recognize endogenous human ATB0. Cells transiently transfected with plasmid that contained rabbit ATB0 and harvested 12 h later showed a broad protein band of 57-65 kDa (Fig. 1A, left, lane 3). In cells transfected with plasmid carrying human ATB0, a very light band was detected by the anti-rabbit ATB0 antibody, indicating only minor cross-reactivity between the human ATB0 and the anti-rabbit ATB0 antibody (Fig. 1A, left, lane 4). In rabbit kidney tissue, the antibody detected a broad diffuse band containing several distinct bands of apparent molecular weight of 75-94 kDa (Fig. 1A, left, lane 1). The bands were specific to ATB0 because concurrent incubation of the membranes in the presence of the antibody and rabbit ATB0 peptide entirely eliminated them (Fig. 1A, right, lanes 1-4).


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Fig. 1.   Western blots of ATB0 protein in rabbit kidney and in HeLa cells transfected with plasmids with and without rabbit or human ATB0 cDNAs. Hela cells were transfected with plasmid alone (A and B, lane 2) or with a plasmid containing rabbit ATB0 cDNA (A and B, lane 3) or human ATB0 (A and B, lane 4) cDNA. After 12 h, cells were harvested and crude membranes were prepared and subjected to Western blot analysis (160 µg protein/lane). For comparison, crude extract of rabbit kidney (A, lane 1) was also applied (160 µg protein/lane). Benchmark standards were used to determine apparent molecular weight. A: membranes were probed with anti-rabbit ATB0 antibody with (right) and without (left) rabbit ATB0 peptide or (B) with anti-human ATB0 antibody with (right) and without (left) human ATB0 peptide.

With anti-human ATB0 antibody, a band of apparent molecular weight of 57-63 kDa was detected in the cells transfected with plasmid alone or with plasmid that contained human or rabbit ATB0 cDNA. The band was enriched in cells transfected with plasmid plus rabbit or human ATB0 cDNA compared with cells transfected with plasmid alone (Fig. 1B, left, lanes 2-4). The expression of the protein, which was induced by transfection, was reduced by simultaneous incubation of the antibody with the human ATB0 peptide (Fig. 1B, right, lanes 3 and 4), but the endogenous band was not reduced (Fig. 1B, right, lanes 2-4). Therefore, the anti-human ATB0 antibody recognized induced human and rabbit ATB0 proteins and perhaps an endogenous ATB0 protein in HeLa cells. Previous experiments have shown that these transfections caused an increase in system B0 activity (15, 16).

Western blot analysis of ATB0 in rabbit tissues and in human C2BBe1 cells. To determine whether ATB0 protein expression parallels the previously reported system B0 activity along the jejunal-ileal axis in the intestine, we harvested the total length of rabbit intestinal tissue. Crude extracts were prepared from mucosa of duodenum, jejunum, midgut, ileum, and colon as well as from whole pancreas and stomach. They were then subjected to Western blot analysis. ATB0 protein in the duodenum, jejunum, midgut, and ileum appeared as an 85- to 88-kDa band. In the colon, pancreas, and stomach there was an additional smear of lower molecular bands that most likely presented proteolysis (Fig. 2A). All the bands were reduced or eliminated in the presence of the competing peptide (Fig. 2B). In the gut, ATB0 protein expression was highest in colon and was higher in ileum than in jejunum. The lowest expression in the gut was detected in the duodenum. Pancreas and stomach had at least as high an expression of ATB0 as the colon. To determine whether ATB0 protein is an intestinal BBM protein, we subjected crude extracts and BBM vesicles from rabbit ileal mucosa to Western blot analysis. ATB0 appears as a diffuse band with a median apparent molecular weight of 85 kDa. The 85-kDa band is enriched in ileal BBM vesicles (Fig. 2C, IV) compared with its amount in the crude extract (Fig. 2C, I), indicating that ATB0 is a BBM protein. The strong median band is eliminated by simultaneous incubation of the membrane with the antibody and the specific peptide (Fig. 2D).


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Fig. 2.   Western blots of ATB0 protein in rabbit gastrointestinal tissues and in human colon carcinoma cell line C2BBe1. Duodenum (D), jejunum (J), midgut (MG), ileum (I), colon (C), pancreas (P), and stomach (S) were harvested from healthy rabbits. Crude extracts were prepared from scraped rabbit mucosa (D, J, MG, I, and C), whole rabbit tissues (P and S), and human C2BBe1 cells (C2). Brush-border membrane (BBM) vesicles were prepared from ileal crude extracts (IV). Proteins in the lysates (160 µg/lane) were subjected to Western blot analysis with anti-rabbit ATB0 antibody (A and C), anti-human ATB0 antibody (E), anti-rabbit ATB0 in the presence of 20× molar excess of rabbit ATB0 peptide (B and D), or with anti-human ATB0 antibody in the presence of 20× molar excess of human ATB0 peptide (F). Apparent molecular weights according to benchmark standards is shown.

Using the rabbit anti-human ATB0, we performed Western blot analysis to detect ATB0 protein in crude extracts of the human brush border-expressing colon carcinoma cell line, C2BBe1. Of the two main bands detected (Fig. 2E), the 94-kDa band was eliminated, and the 78-kDa band was greatly reduced by simultaneous incubation with excess human ATB0 peptide (Fig. 2F). However, because the anti-human ATB0 antibody detected several other bands, this antibody was not as specific for human ATB0 as the anti-rabbit antibody for rabbit ATB0. Compared with HeLa cells, in C2BBe1 cells, an endogenous 57- to 65-kDa band was not detected.

To determine whether the tissue distribution and abundance of ATB0 is similar to the tissue distribution and abundance of system B0, ATB0 protein expression in kidney and liver, compared with its expression in ileal mucosa, was analyzed by Western blotting. As shown in Fig. 2, ATB0 in the ileum appeared as a band of 85 kDa (Fig. 3A). This band was eliminated by incubation of the membrane with the antibody in the presence of excess rabbit ATB0 peptide but not in the presence of excess of the nonspecific peptide, mCAT2 (Fig. 3, B and C). Although a broad band of median 85 kDa was detected in the liver, it was not reduced by incubation with ATB0 peptide, indicating that it might not be ATB0 protein. A 120-kDa band, which was eliminated by incubation with the antibody in the presence of the peptide, was present in the liver extract (Fig. 3). This band is probably a protein that cross-reacts with the antibody, but is of a molecular weight too high to be an ATB0 protein. ATB0 protein in the kidney is composed of a broad diffuse band of several distinct bands of 80-90 kDa, as shown in Fig. 1. The band in the kidney is markedly reduced by preincubation with the specific ATB0 peptide but not with the nonspecific mCAT2 peptide (Fig. 4). The abundance of ATB0 in the kidney is much higher than in the ileum.


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Fig. 3.   Western blots of ATB0 protein in rabbit tissues. Ileum (I), liver (L), and kidney (K) were harvested from healthy rabbits. Crude extracts were prepared from ileal mucosa and from whole liver and kidney tissues. Proteins were subjected to Western blot analysis with anti-rabbit ATB0 antibody alone (A), with the antibody in the presence of the competing rabbit ATB0 peptide (B), or with the antibody in the presence of a nonspecific peptide, mouse CAT2 peptide (C). Eighty micrograms of protein were applied per lane.



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Fig. 4.   Western blots of rabbit ileal mucosa crude extracts before and after deglycosylation. Ileal mucosa crude extract was subjected to N- and O-deglycosylation procedure for 24 h at 37°C as described in MATERIALS AND METHODS with (lane 2) and without (lane 1) deglycosylating enzymes. The samples were subjected to Western blot analysis with anti-rabbit ATB0 antibody. Benchmark standards were used to determine the apparent molecular weight. The arrows point to ATB0 band before and after deglycosylation.

Western blot analysis of rabbit ileal mucosa crude extract before and after deglycosylation. The expected molecular weight of ATB0 according to amino acid sequence is 57 kDa. The apparent molecular weight obtained in Western blot analysis of rabbit tissues was 85 kDa.

To determine whether the difference in expected and obtained apparent molecular weight is due to extensive glycosylation of the protein in the tissue, we subjected crude extract from rabbit ileal mucosa to simultaneous incubation with all 5 N- and O-deglycosylation enzymes of the Calbiochem kit. After a 24-h incubation at 37°C, the apparent molecular weight of the ATB0 band was reduced by deglycosylation from 85 kDa (Fig. 4, lane 1) to 78 kDa (Fig. 4, lane 2).

Immunohistochemistry of ATB0 protein in rabbit kidney and ileum. To localize ATB0 protein to specific cells and organelles within the rabbit kidney and ileum, we used immunohistochemical methods. Because the antibody to human ATB0 was not monospecific, we did not use it in immunohistochemical studies. In the rabbit kidney, ATB0 was found only in regions where proximal tubular cells are present, i.e., in the inner and outer cortex but not in the medulla (Fig. 5, B and F vs. J). In these regions, ATB0 was present only in proximal tubular cells and was confined to the BBM (Fig. 5, B and F, closed arrows). In another section from the same kidney, similar structures were stained with PAS, which is known to stain the BBM of proximal tubular cells (3) (Fig. 5K, closed arrows), indicating that ATB0 is confined to the BBM of the proximal tubular cells in the kidney. ATB0 was not found in distal tubular cells or collecting duct epithelia (Fig. 5, B and F, open arrows). Cytokeratin AE1/AE3 is present in distal tubules, collecting ducts, and the loop of Henle, but not in proximal tubular cells (5, 6, 12). In another section of the same kidney, cells with similar morphology to the ones that were not stained with ATB0 were stained for cytokeratin AE1/AE3 (Fig. 5L, open arrows). This reinforces that only proximal tubular cells contained ATB0. Several controls were used to ensure specificity of the antibodies. Sections probed without anti-rabbit ATB0 showed some background staining on the basolateral side of all cells (Fig. 5, A, E, and I). The same background staining was also present in sections that were incubated with anti-rabbit ATB0 antibody in the presence of 20× molar excess of the ATB0 peptide against which the antibody was raised (Fig. 5, C and G). Incubations of sections with anti-rabbit ATB0 antibody in the presence of a nonspecific peptide (mCAT2) did not eliminate the specific staining of the BBM in cells resembling proximal tubular cells (Fig. 5, D and H). Together, these results indicate that ATB0 is confined to the BBM of the proximal tubular cells in the rabbit kidney.


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Fig. 5.   Immunohistochemical analysis of ATB0 in Formalin-fixed rabbit kidney. Rabbit kidney was fixed in buffered Formalin, embedded in paraffin, sectioned, and subjected to immunohistochemical analysis with (B-D, F-H, and J) and without (A, E, and I) anti-rabbit ATB0 antibody. Sections were incubated with anti-rabbit ATB0 antibody in the presence of 20× molar excess of rabbit ATB0 peptide (C and G) or mCAT2 peptide (D and H). Sections from the same kidney were stained with periodic acid-Schiff (PAS; K) and with anti-human cytokeratin AE1/AE3 (a broad-spectrum anticytokeratin antibody) (L). Outer cortex: A-D, L, and K; inner cortex: E-H; medulla: I and J. Closed arrows point to the BBM of the proximal tubular cells stained by anti-rabbit ATB0 antibody and by PAS. Open arrows point to distal tubular cells stained by anti-human cytokeratin AE1/AE3, but not by anti-rabbit ATB0 antibody. Magnification, ×220.

We were not able to detect a specific staining in the rabbit gut tissue with anti-rabbit ATB0 antibodies when the ileum was fixed with Formalin. However, when ileal tissue was snap-frozen, sectioned, fixed with acetone, and then immunoprobed for ATB0, the BBM of the ileal enterocytes were stained (Fig. 6B). The stain was present in crypt cells but with decreased intensity (Fig. 6D). The specific stain was eliminated when the sections were incubated without the primary antibody (Fig. 6A) or when the sections were incubated with anti-rabbit ATB0 antibody in the presence of 20× molar excess of the ATB0 peptide against which the antibody was raised (Fig. 6C). Therefore, ATB0 in the ileum is confined mainly to the BBM of the enterocytes.


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Fig. 6.   Immunohistochemical analysis of ATB0 in frozen rabbit ileal sections. Rabbit ileum was snap-frozen, fixed with acetone, sectioned, and subjected to immunohistochemical analysis with (B-D) and without (A) anti-rabbit ATB0 antibody. The section in C was incubated with anti-rabbit ATB0 antibody in the presence of 20× molar excess of rabbit ATB0 peptide. Villus cells (A-C); crypt cells (D). Arrows point to ATB0 on the BBM of the enterocytes and crypt cells. Magnification, ×220.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Most neutral amino acids enter the portal circulation from the intestinal lumen and are reabsorbed from the kidney lumen by system B0 (8, 24, 26). We hypothesized that ATB0, a recently cloned amino acid transporter that confers a B0-like transport activity upon transfection to oocytes and mammalian cells (15, 16), could be the main enterocyte and the kidney proximal tubular cell system B0 transporter. The qualitative and quantitative tissue distribution and the intracellular localization in the kidney and enterocyte of ATB0 protein described here support the hypothesis that it may serve as the main B0 transporter protein.

To enable us to look at ATB0 on the protein level, we generated antibodies to ATB0 peptides. The antibodies were generated against rabbit and human ATB0 cytoplasmic COOH-terminal peptides, which have sequence homology to each other (15, 16), to mouse testis ASCT2 (35), to mouse adipocyte amino acid transporter (19), and to rat astroglial ASCT2 (4), but not to any other known protein. In addition to sharing between 76 and 83% sequence identity, the above-mentioned proteins have identical amino acid transport activity (4, 15, 16, 35). It has been suggested that ATB0 and ASCT2 are species-specific variants of the same protein (11, 26). The antibody against rabbit ATB0 detected only trace amounts of human ATB0, but the anti-human ATB0 detected rabbit ATB0 (Fig. 1). Therefore, it is possible that each of these antibodies might cross-react with tissue-specific variants of ATB0 or ASCT2. Nevertheless, the proteins detected all have broad-spectrum neutral amino acid transport activity that could not be differentiated on the basis of activity measurements.

In the ATB0-cDNA transiently transfected HeLa cells, the antibodies detected bands (Fig. 1) with apparent molecular weight (57-65 kDa) that approximates the calculated molecular weight for ATB0 and ASCT2 proteins from their amino acid sequence (57 and 65 kDa) (4, 15, 16, 19, 35). On the other hand, in rabbit tissue, the protein band detected by anti-rabbit ATB0 antibody has an average apparent molecular weight of 85 kDa (Figs. 1-4). ATB0 and ASCT2 have two N-glycosylation sites and two protein kinase C phosphorylation sites (4, 15, 16, 19, 35). In the tissues, but not in the transiently transfected cells, the proteins probably have been posttranslationally processed. Enzymatic N- and O-deglycosylation reduced the apparent molecular weight of the ATB0 band in rabbit ileal crude extract to 78 kDa (Fig. 4). However, it is not known whether the deglycosylation was complete, because the protein could contain oligosaccharides resistant to enzymatic deglycosylation. Other contributors to the difference in the expected vs. the apparent molecular weight could be alternative splicing of ATB0 mRNA, the degree of phosphorylation (tissues were harvested in a buffer containing phosphatase inhibitors), residual structure (that could delay the mobility of the protein), and ubiquitination. For example, deglycosylation of growth hormone receptor cannot account for the discrepancy between the expected molecular weight of 70 kDa and the apparent molecular weight of 130 kDa (18).

The antibody-detected ATB0 proteins, which were synthesized from the transfected cDNAs or the endogenous ATB0, were competed out in the presence of 20× molar excess of the specific peptides, but not in the presence of the nonspecific peptide (Figs. 1-3). This indicates that the bands detected are indeed ATB0. The endogenous bands detected in HeLa cells by anti-human ATB0 and in rabbit liver by anti-rabbit ATB0 did not decline with simultaneous incubation of the antibodies and excess of the specific peptides. We cannot rule out the possibility that, in some tissues, there are other proteins that cross-react with the antibodies. On the other hand, these proteins could be ATB0 that for some unknown reason have higher affinity to the antibody than to the peptides.

Both B0 transport activity and ATB0 mRNA were reported to be severalfold higher in the rabbit kidney than in the rabbit small intestine (16, 32). In parallel, the amount of ATB0 protein is severalfold higher in the kidney than in the ileum (Fig. 3). B0 activity in the intestine is higher in the colon and ileum than in the jejunum and duodenum (7, 14, 25). A similar gradient along the jejunal-ileal axis is present for ATB0 protein. ATB0 protein level was higher in the colon and ileum than in the jejunum and duodenum (Fig. 2). To the best of our knowledge, the distribution of B0 activity along the villus-crypt axis has not been determined. The fact that ATB0 protein level is higher in the villus tip than in the crypt indicates that B0 activity might follow the same gradient along the villus-crypt axis. ATB0 and ASCT2 mRNA are high in the pancreas and in the kidney and are severalfold higher in the colon than in the small intestine (15, 35). This is in agreement with the level of ATB0 protein in these tissues (Figs. 2 and 3). Hepatocytes are devoid of B0 activity and ATB0 mRNA, but B0 activity and ATB0 mRNA are present in liver endothelial cells (20). Our results concerning the presence of ATB0 protein in the liver are inconclusive because a band with apparent molecular weight identical to ATB0 in other tissues was present but was not competed out by the specific ATB0 peptide (Fig. 3). In addition, we were not able to get ATB0 staining in either Formalin-fixed or frozen liver sections (data not shown).

B0 activity is confined to the BBM of the kidney proximal tubular cell and to the BBM of the enterocyte (21, 34). ATB0 protein was enriched in ileal BBMV (Fig. 2) and was confined to the BBM of the kidney proximal tubular cell and the BBM of the ileal enterocyte (Figs. 5 and 6). Therefore, ATB0 tissue distribution, the comparative amounts of ATB0 among the various tissues, and the intracellular localization of ATB0 protein to the BBM of the enterocyte and the kidney proximal tubular cell all support our hypothesis that ATB0 may be the main system B0 transporter. However, there is still a possibility that the cloned ATB0/ASCT2 is not system B0. Only future investigations may resolve this issue.

Neutral amino acids play a central role in catabolic illness. Glutamine is the major oxidative fuel of the enterocyte (31). Branch-chained neutral amino acids have an anabolic role in protein synthesis regulation during stress states (29). The main uptake system of these amino acids from the intestinal lumen and the main reabsorption system from the kidney lumen is B0 (8, 24, 26). Hartnup disorder, which is characterized by a pellagra-like light-sensitive rash, cerebellar ataxia, emotional instability, and a broad-spectrum neutral amino acid aminoaciduria, has been suggested to result from a defect in B0 transport activity (26, 30). We have generated antibodies against ATB0 protein and showed by Western blot analysis and immunohistochemistry that it is most probably the major B0 system transporter. The antibodies can serve as a tool to determine ATB0 protein in pathological states and to follow its regulation in response to treatment.


    ACKNOWLEDGEMENTS

We thank Patricia Bourne for expert technical assistance with the immunohistochemical studies.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2RO1-DK-47989-06-A1 (to H. C. Sax).

Address for reprint requests and other correspondence: N. E. Avissar, Dept. of Surgery, Univ. of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642-8410 (E-mail: nelly_avissar{at}urmc.rochester.edu).

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 13 September 2000; accepted in final form 9 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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