ARTICLE |
Correspondence to: Kojiro Wasano, Dept. of Anatomy and Cell Biology, Faculty of Medicine, Kyushu Univ., Fukuoka 812, Japan.
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Summary |
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Rat gastrointestinal (GI) tract is rich source of galectins, a family of mammalian galactoside-binding lectins. To determine which tissue component is the relevant glycoconjugate ligand for the galectins, we produced recombinant galectin-1 and surveyed its binding sites on tissue sections of rat GI tract. Mucin and epithelial surface glycocalyces of both gastric and intestinal mucosa were intensely stained. This finding raises the possibility that some GI tract galectins known to be secreted by the epithelia may recognize these glycoconjugates and crosslink them into a macromolecular mass. This galectin-ligand complex may play a role in protecting the epithelial surface against luminal contents such as gastric acid, digestive enzymes, and foreign organisms. (J Histochem Cytochem 45:275-283, 1997)
Key Words: galectins, rat, gastrointestinal tract, recombinant protein, glycoconjugates, mucosal defense, mucin, glycocalyx
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Introduction |
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Galectins are a family of galactoside-binding lectins found in a variety of mammalian tissues. To date, eight different types of galectins that exhibit significant sequence similarity in their galactoside recognition domains have been characterized and numbered sequentially (
To test the possibility that galectins recognize any cell and/or ECM component other than laminin, we produced recombinant rat galectin-1 and surveyed its binding site on tissue sections of rat GI tract that are known to be rich not only in glycoconjugates but also in galectins themselves (
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Materials and Methods |
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Reagents
The reagents used for SDS-polyacrylamide and agarose gel electrophoresis were obtained from Bio-Rad Japan (Tokyo, Japan). Sepharose 4B was from Pharmacia Biotec (Tokyo, Japan). Restriction enzymes and DNA standards were from Fermentas (Vilnius, Lithuania). Unless otherwise noted, all other reagents of analytical grade were purchased from Sigma (St Louis, MO).
DNA Cloning and Expression of Recombinant Galectin-1
Poly(A) RNA was obtained from frozen rat lungs (Rockland; Gilbertsville, PA) using an mRNA isolation kit (Stratagene; La Jolla, CA). Reverse transcription was done with avian myeloblastosis virus reverse transcriptase (Cetus; Emeryville, CA) using 1-2.5 µM of anti-sense downstream primer 5'-CTGGaTccCTTCACTCAAAGGCCACACAC-3' (nucleotide position 399-418 of cDNA of rat galectin-1) at 60C for 20 min. The BamHI site incorporated into the primer for the purpose of subcloning is underlined and the bases changed for generating the site are shown in lower-case letters. After incubation at 95C for 5 min to stop the reaction, DNA polymerase chain reaction (PCR) amplification was performed using a PCR kit (Cetus) containing taq DNA polymerase in the conditions suggested by manufacturer's instructions, except that 1-2.5 µM sense upstream primer 5'-AAcCATGGCCTGTGGTCTGGTCGCCAGC-3' (nucleotide position from -4 to 24 of cDNA of rat galectin-1; the NcoI site incorporated into the primer is underlined and the base changed is shown in lower-case letters) was added to the reaction mixture. PCR products were gel-purified and then ligated into pCR-script SK(+) (Stratagene) vector. Host bacteria (XL-1 blue strain; Stratagene) harboring recombinant plasmids were inoculated onto agar plates containing 0.4 mM isothiopropyl ß-D-thiogalactopyranoside (IPTG) and 40 µg/ml of 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-Gal). The white colony was picked up with a sterile toothpick and cultured in 50 ml SOC medium at 37C overnight. Recombinant plasmids were isolated by the SDS-NaOH method. DNA sequencing was carried out on both strands using universal T3 and T7 primers with a non-isotopic Sequenase Images kit (US Biochemical; Cleveland, OH).
A DNA fragment was cut out from the clone-positive pCR-script vector using NcoI and BamHI restriction enzymes, gel-purified and subcloned in between the same sites in a pET 11d expression vector (Novagen; Madison, WI). A host bacterial strain BL21(DE3) (Novagen) was transformed by the vector and inoculated onto agar plates containing ampicillin. Several colonies were separately picked up into distilled water, boiled, and then colony PCR was done using the gene-specific primer set to ensure successful ligation of the target insert into the expression vector. The insert-positive bacterial clone was separately cultured in 50 ml SOC medium overnight, centrifuged, resuspended in 1 ml SOC medium containing 8% glycerol, numbered, and stored at -80C. Fifty µl from the bacterial stock solution was inoculated into 5 ml SOC medium and then into 200 ml SOC medium at 37C until the absorbance at 600 nm reached about 0.6. Protein production was induced by adding 5 mM IPTG to the culture medium for 2 hr. The bacteria were incubated at 30C during IPTG induction to keep the recombinant protein in a soluble form according to the manufacturer's bulletin. The bacteria were then cooled on ice, collected by centrifugation, and the pellet was resuspended in 20 ml protein extraction solution (PES) containing 58 mM Na2HPO4,18 mM KH2PO4,75 mM NaCl, 2 mM EDTA, and 2 mM PMSF. After one cycle of freeze-thawing, 4 mM 5-iodoacetamido-fluorescein (5-IAF) (Molecular Probes; Eugene, OR) for the labeling experiments or 4 mM iodoacetamide for the control experiment was added into the solution and then the bacteria were lysed with an ultrasound sonificator (Tomy-Seiko; Tokyo, Japan) at maximal power twice for 10 sec. Cell debris was removed by centrifugation at 18,000 x g for 20 min. The soluble extract was loaded into a column containing 50 ml lactose-coupled Sepharose 4B resin and the unbound fraction was extensively washed with PES until the resin became free of the yellowish color of 5-IAF, except for a yellowish band bound to a narrow area (2-3 mm in height) just beneath the surface of the resin. Bound protein was eluted with 20 ml PES containing 100 mM lactose. The eluted protein was dialyzed against 200 ml PES twice for 12 hr at 4C, concentrated using Amicon Y-10 ultrafiltration membranes, and then stored at -80C in the dark. Crude extract from IPTG-induced BL21 and affinity-purified recombinant galectin-1 (hereafter referred to as r-galectin) were analyzed with SDS-PAGE.
Histocytochemistry Using Fluorescein-labeled r-Galectin
Ten Wistar rats (6-8 weeks old) of both sexes were sacrificed with CO2 according to the guidelines of our institutional animal welfare committee. Fundic and pyloric areas were obtained from the stomach. The duodenum (just below the pyloric ring), jejunum (1 cm distal to the duodeno-jejunal junction), and ileum (1 cm proximal to the ileocecal junction) were dissected from each animal and opened. The luminal contents were washed away with jet stream of acetate buffered saline (ABS; 10 mM sodium acetate, 150 mM NaCl, pH 7.4) through a 20-gauge needle attached to a 20-ml plastic injector. The tissues were then cut into small cubes, fixed in 4% paraformaldehyde plus 2.5% glutaraldehyde in 0.1 M acetate buffer for 4 hr, and incubated in 10 mM glycine in the same buffer for 1 hr to quench residual aldehydes. Of the several fixation schedules tested, this schedule was the best one for preserving general tissue structure and galectin binding to cryosections.
For light microscopic histochemistry, the tissue specimens were soaked in 0.6 M sucrose in 0.1 M acetate buffer overnight and embedded in M-1 Embedding Matrix (Lipshaw; Pittsburgh, PA). Cryosections 5 µm were cut in a cryotome (Microm; Walldorf, Germany) and placed on poly-L-lysine-coated glass slides. The sections were covered with blocking solution (BS) containing 10 mM sodium phosphate (pH 7.4), 150 mM NaCl, and 3% bovine serum albumin for 30 min to mask nonspecific protein binding sites. After removal of BS with tissue papers by tilting the slides, the sections were incubated with fluorescein-labeled r-galectin diluted (1 µg/ml BS) with BS for 30 min, washed in PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) three times for 5 min each, and then examined under an epifluorescence microscope (Olympus; Tokyo, Japan).
For electron microscopic cytochemistry, the tissues were infiltrated stepwise in increasing concentrations of sucrose (from 0.6 to 2.3 M sucrose) in 0.1 M acetate buffer (pH 7.4). The tissue blocks were attached to aluminum stubs and snap-frozen in liquid nitrogen. Ultrathin frozen sections 90-100 nm thick were cut with a diamond knife in an Ultracut S microtome equipped with an FC4E cryoattachment (Reichert; Vienna, Austria), and picked up on collodion-coated nickel grids by means of a droplet of 2.3 M sucrose in a platinum loop. The grids were inverted onto a droplet of distilled water to remove sucrose and transferred to a BS droplet for 30 min. The grids were then incubated on a droplet containing fluorescein-labeled r-galectin diluted (0.1 µg/ml BS) with BS for 30 min, washed on a droplet of PBS three times for 15 min each, and then stained with rabbit anti-fluorescein antibody (Molecular Probes) diluted 200 times with BS for 30 min. After washing on a droplet of PBS three times for 15 min each, the grids were floated on a droplet of goat anti-rabbit IgG antibody labeled with 10 nm colloidal gold (Bio-Cell; Cardiff, UK), which was diluted 200 times with BS for 30 min, then washed on ABS three times for 5 min each to remove phosphate ions, and stained on a droplet of 1% aqueous uranyl acetate solution for 30 sec. After brief coating on a droplet of aqueous 1.5% methylcellulose solution, the grids were inverted onto a filter paper, air-dried at 60C for 1 hr, and examined under a 1200EX electron microscope (JEOL; Tokyo, Japan).
In control experiments, fluorescein labeled r-galectin was preincubated with 10 mM thiodigalactopyranoside (TDG) for 30 min, anti-fluorescein antibody was omitted from the staining steps, or non-labeled r-galectin (treated with non-labeled iodoacetamide) was used.
Statistical Analysis
Fundus, jejunum, and ileum were obtained from three rats. Each GI tract site was cut into three tissue blocks and processed for cryosectioning as above. Differences in the degree of r-galectin binding to mucin granules of each GI tract site were compared by counting the number of gold particles per unit area of granules. The electron-dense core region of fundic mucin granules, which was devoid of gold labeling, was excluded from counting. To assess intestinal mucin labeling, goblet cells located in the middle one third of villous processes were examined by careful trimming of the tissue blocks during cryosectioning. At least 0.25 µm2 x 4 areas within mucin granules of 10 fundic surface mucous cells or intestinal goblet cells were examined per tissue block per animal. Data were expressed as the mean ± SEM and were analyzed with Fisher's least significant difference (LSD) method. The numbers of gold particles attached to microvilli of enterocytes located in the middle third of villous processes of jejunum and ileum were counted in the same way as above and were analyzed by Student's t-test.
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Results |
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Preparation of Recombinant Galectin-1
By reverse transcription of lung poly(A) RNA followed by PCR amplification using two primers flanking the entire coding sequence of rat galectin-1, we obtained about a 420-base pair (BP) DNA product (not shown) that matched the expected molecular mass of rat galectin-1 DNA. The DNA product was ligated into an expression vector and recombinant protein production was triggered with IPTG. We found a protein band migrating at molecular weight of 14.5 kD in the crude extract of bacteria (Figure 1). The extract was then passed through a lactose-coupled resin column. The lactose-eluted fraction contained a single protein band at a molecular weight of 14.5 kD (Figure 1), indicating that biologically active r-galectin was successfully produced. Because the r-galectin was proved to be a good histochemical probe (see below), no further detailed characterization of the protein was performed.
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For use as a histochemical probe, r-galectin was irreversibly alkylated with 5-IAF (or non-labeled iodoacetamide), because this procedure stabilizes the protein against oxidative inactivation of the sugar-binding activity. Under these conditions, the protein can be stored for at least 3 months (
Binding Sites of r-Galectin in Gastrointestinal Tract
Fluorescence microscopic examination revealed that r-galectin recognizes distinct cell populations and cellular sites. In the fundic glands of the stomach, the apical cytoplasm of surface mucous cells and the mucous neck cells were intensely stained (Figure 2). Large, scattered oval cells located in the middle portion of the glands were also stained. In the pyloric glands, the apical cytoplasm of surface mucous cells was intensely labeled (Figure 3). In the small intestine (Figure 7 and Figure 8), the epithelial brush border was intensely stained. The staining appeared to be much denser on the surface of villi than on that of crypts. Scattered under this brush border were goblet cell granules which were also stained with r-galectin. Secretory granules of Paneth cells and Brunner's gland cells were barely stained. When r-galectin was preincubated with TDG, all staining was completely abolished (not shown).
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In ultracryosections, the secretory granules of both surface mucous cells (Figure 4) and mucous neck cells of the fundic glands were intensely labeled. Although the mucosa was thoroughly washed with a jet stream of physiological saline before fixation, apparent labeling was observed on the apical plasma membrane of the surface mucous cells (Figure 5). The cell surface and the intracytoplasmic canaliculi of parietal cells were also labeled (Figure 6), indicating that the large oval cells seen in the fundic glands are parietal cells. The secretory granules and surface membranes of the pyloric surface mucous cells were also labeled (not shown). The microvilli of enterocytes from duodenum to ileum were intensely labeled with r-galectin (Figure 9). The secretory granules of intestinal goblet cells were also labeled (Figure 10), but the staining density appeared to be much sparser compared with that of the mucin granules of gastric glands and the microvilli of adjacent enterocytes. Incubation of r-galectin with TDG before staining (Figure 11) completely abolished immunogold labeling in all r-galectin-reactive GI tract sites.
Statistical analysis (Figure 12) of the labeling density of fundic and intestinal mucin granules verified that mucin of the fundic surface mucous cells (150.3 ± 15.92/µm2) showed significantly (p<0.001) higher affinity to galectin-1 than did goblet cell mucin of the jejunum (28.9 ± 1.95/µm2) and ileum (30.3 ± 2.13/µm2). There was no significant difference in the labeling density of goblet cell mucin (above values) and apical microvilli (jejunum 95.8 ± 3.45/µm2 vs ileum 102.9 ± 4.75/µm2) between the two distinct intestinal regions examined.
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Discussion |
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To examine galectin-binding sites in GI tract tissues, we chose galectin-1 as a cytochemical probe among several kinds of GI tract galectins for the following reasons. (a) This protein undergoes little post-translational modification, except for the N-terminal methionine cleavage and acetylation (
Are the mucin and ECSG natural ligands for galectin-1? Galectin-1 has been shown to be localized in muscle tissues (
The intracellular canaliculi of parietal cells were also stained with r-galectin. In the portion of the fundic glands where the parietal cells are located, there are neither r-galectin-positive secretory cells nor secretion adjacent to the cells. Therefore, the labeling appears to be caused by this cell type's own ECSG. Although the specialized intracellular structure of the parietal cells is known to be involved in acid secretion, it is unclear whether the r-galectin-positive ECSG is related to such function.
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