ARTICLE |
Correspondence to: Kojiro Wasano, Department of Anatomy and Cell Biology, Faculty of Medicine, Kyushu University, Fukuoka 812-0054, Japan..
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Summary |
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Galectin-4 (G4) is a member of a family of soluble galactoside-binding lectins found in various mammalian tissues. To determine the function of this protein in colorectal tissue, we separately produced the N- and C-terminal carbohydrate binding domains (CBD) of rat G4 as a recombinant glutathione S-transferase (GST) fusion protein (G4-N and G4-C) and examined the tissue binding site(s) of each CBD by light and electron microscopy (LM and EM). At the LM level, both fusion proteins stained the intercellular borders of the surface-lining epithelial cells of colorectal mucosa. At the EM level, two proteins recognized spatially close but distinct subcellular structures. G4-N stained electron-lucent flocculent substances freely located in the intercellular spaces, whereas G4-C bound to the lateral cell membranes demarcating the intercellular spaces. These findings suggest that colorectal G4 may be involved in crosslinking the lateral cell membranes of the surface-lining epithelial cells, thereby reinforcing epithelial integrity against mechanical stress exerted by the bowel lumen. (J Histochem Cytochem 47:7582, 1999)
Key Words: galectin-4, mammalian lectin, rat, colon, surface-lining epithelial cells, cell adhesion molecule, GST fusion protein, cytochemistry
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Introduction |
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A NUMBER of soluble galactoside binding lectins have been isolated from various mammalian tissues. All these proteins show well-conserved amino acid sequence in their carbohydrate binding domains (CBDs) and therefore are believed to constitute a distinct protein family recently designated galectins (
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Materials and Methods |
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Reagents
The reagents for SDS-PAGE and agarose gel electrophoresis were obtained from Bio-Rad (New York, NY). Restriction enzymes and DNA standards were from Fermentas (Vilinius, Lithuania). RT-PCR kits were purchased from Cetus (Emeryville, CA). The PCR cloning vector pGEM-T and T4 ligase were from Promega (Madison, WI). The expression vector pGEX-2T, goat anti-GST (Schistosoma japonicum origin) antibody, and GST purification kits equipped with glutathioneSepharose were purchased from Pharmacia (Uppsala, Sweden). XL-1 blue and BL21(DE3)pLysS competent cells were obtained from Stratagene (La Jolla, CA) and Novagen (Madison, WI), respectively. Unless stated otherwise, all other reagents of analytical grade were from Sigma (St Louis, MO).
DNA Cloning
All the manipulations of nucleic acids such as restriction, ligation, transformation, selection, cell culture, gel electrophoresis, and elution were done using standard protocols (
Expression and Isolation of recombinant N-CBD and C-CBD Proteins
The cDNA encoding N-CBD or C-CBD was released with EcoRI and BamHI restriction enzymes, gel-purified, and subcloned in between the same sites in the pGEX-2T expression vector containing a GST (Schistosoma japonicum origin) gene and a thrombin cleavage sequence upstream from the site. The host strain BL21(DE3)pLysS was transformed with the plasmid and inoculated on ampicillin-containing agar plates. The resulting colony was picked up in distilled water, boiled, and then colony PCR was done using the above primer sets. The PCR-positive bacteria were cultured in 200 ml SOC medium until absorbance at 600 nm reached 0.6. Protein production was induced by adding 0.4 mM IPTG at 30C for 2 hr. To isolate recombinant protein, the bacteria were cooled on ice, centrifuged into a pellet, and resuspended with 5 ml protein extraction solution (PES) (58 mM Na2HPO4, 18 mM KH2PO4, 75 mM NaCl, 2 mM EDTA, 2 mM benzamidine, 2 mM aminohexanoic acid, and 1 mM PMSF). For isolation of C-CBD protein, which contains two cysteine residues at amino acid positions 246 and 281, 0.4 mM iodoacetamide was added to the PES solution. The bacteria were lysed with an ultrasonicator (Tomy; Tokyo, Japan) at a maximal output twice for 10 sec. After centrifugation to remove cell debris, the clear supernatant was loaded into 1 x 3-cm lactosylSepharose. After washing with 100 ml PES, bound protein was eluted with PES containing 100 mM lactose. The eluted protein was concentrated with an Amicon ultrafiltration membrane Y-10, dialyzed against PES to remove lactose and stored at -80C. Crude extract from noninduced or IPTG-induced bacteria, and purified GST-fused N-CBD and C-CBD proteins (hereafter referred to as GST-N-CBD and GST-C-CBD) were analyzed by SDS-PAGE. Both fusion proteins were incubated with thrombin and the digestion products were analyzed by SDS-PAGE. Nonfused GST protein was expressed in bacteria containing pGEX-2T without any specific DNA insert, purified through glutathioneSepharose resin, and used for a cytochemical control experiment.
Histocytochemistry Using GST-N-CBD and GST-C-CBD
Ten Wistar rats (6 weeks old) were sacrificed with CO2 gas according to the guidelines of our institutional animal welfare committee. The ascending colon (1 cm distal from the ileocecal junction), the descending colon (1 cm distal from the colon passing between the distal end of the stomach and the cranial mesenteric root) (
For LM histochemistry, the tissue specimens were soaked overnight in 0.6 M sucrose and embedded into Tissue-Tek OCT compound (Miles; Elkhart, IN). Cryosections 5 µm thick were cut in a cryotome (American Optical; Buffalo, NY) and picked up onto poly-L-lysine-coated glass slides. The sections were covered by blocking solution (BS) containing 3% bovine serum albumin in ABS for 30 min to mask nonspecific protein binding sites. The sections were incubated with GST-N-CBD OR GST-C-CBD (0.1 µg/ml BS) for 30 min, washed with three changes of ABS for 5 min each, and then overlain with goat anti-GST antibody diluted 1000 times with BS for 30 min. After washing in ABS three times for 5 min each, the sections were stained with rabbit anti-goat IgG labeled with FITC diluted 500 times with BS for 30 min, washed as above, and examined under an epifluorescence microscope equipped with an automatic camera (Olympus; Tokyo, Japan).
For EM cytochemistry, tissue specimens were infused with 2.3 M sucrose in 0.1 M acetate buffer overnight, attached to aluminum stubs, and frozen in liquid nitrogen. Ultrathin frozen sections about 100 nm thick were cut with a dry type diamond knife in an Ultracut S equipped with an FCS cryoattachment (Leica; Vienna, Austria), and picked up onto collodion-coated nickel grids. All the staining steps were done by transferring the grids from a droplet to the next droplet with a platinum wire loop. The grids were inverted on a droplet of ABS to remove sucrose and transferred onto a BS droplet. The sections were stained and washed in the same way as for LM staining steps, except that rabbit anti-goat IgG antibody labeled with 15-nm colloidal gold (Bio-Cell; Cardiff, UK) was used instead of that labeled with FITC. The sections were finally stained with an aqueous 1% uranium salt solution for 10 sec and briefly coated with an aqueous 0.5% methylcellulose solution (
In control experiments, GST-N-CBD or GST-C-CBD was omitted or replaced with nonfused GST. GST-N-CBD or GST-C-CBD was preincubated with 50 mM thiodigalactoside (TDG) for 30 min, which is the most potent inhibitor of galactoside binding of galectins. Anti-GST or secondary antibody was omitted.
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Results |
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Preparation of Recombinant N-CBD and C-CBD as a GST Fusion Protein
After RT reaction using intestinal poly(A)RNA followed by PCR using two primer sets, we obtained about 480-BP and 460-BP products (Figure 1), which corresponded to the expected molecular size of cDNA encoding N-CBD and C-CBD, respectively.The DNA product was ligated into the expression vector containing a GST gene and a thrombin cleavage sequence upstream from the cloning site, and recombinant GST-N-CBD or GST-C-CBD induction was triggered with IPTG. SDS-PAGE analysis of a crude extract from noninduced and induced bacteria revealed IPTG-dependent induction of GST-N-CBD and GST-C-CBD protein at molecular weights of about 43 and 42.5 kD, respectively (Figure 2). The size of each fusion protein matched the sum of GST (26 kD) plus N-CBD (17 kD) or C-CBD (16.5 kD). Successful production of fusion protein was further confirmed by incubation with thrombin (Figure 2), which released a 17-kD band from GST-N-CBD and a 16.5-kD band from GST-C-CBD. Both fusion proteins were water-soluble, retained sugar-binding capability, and could be purified by binding to lactosylSepharose. For isolating GST-C-CBD that contains two cysteine residues, iodoacetamide was added to the extraction solution. In the absence of an alkylating agent, the sugar-binding capability of this domain is inactivated within several minutes after sonication, even when maintained at 4C, probably due to oxidation of the cysteine residue(s), as is the case with other types of galectins.
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GST Portion of the Fusion Protein as a Useful Cytochemical Marker
Two CBDs of G-4 were expressed as a GST fusion protein to examine their tissue binding site(s) by indirectly detecting the GST sequence as a cytochemical marker. GST is a ubiquitous enzyme that is involved in the transfer of sulfur from its donor (glutathione) to various kinds of acceptors and is therefore widely distributed within almost all kinds of tissues or cells. Therefore, before commencing our histochemical study, it was important to exclude the following possibilities: (a) whether the GST portion of the fusion protein reacts or binds to intrinsic glutathione remaining in cryosections, and (b) whether the anti-GST antibody, which was raised against GST of Schistosoma japonicum origin, crossreacts with intrinsic GST of rat origin. To check on these, several control experiments were performed. (a) GST-N-CBD or GST-C-CBD was omitted from the staining steps or replaced with plain nonfused GST. (b) The fusion protein was preincubated with TDG, the most potential inhibitor of the sugar-binding capability of galectins. (c) Anti-GST antibody or secondary antibody was omitted. All staining at both LM and EM levels vanished in all these control experiments (not shown), indicating that the staining was not an artifact caused by the interaction between intrinsic glutathione and the GST portion of the fusion protein, or between intrinsic rat GST and anti-Schistosoma japonicum GST antibody. This was also supported by the finding that GST-N-CBD and GST-C-CBD apparently recognized different subcellular structures as mentioned below.
Histocytochemistry Using GST-N-CBD and GST-C-CBD
For simplicity, the terms "GST-N-CBD and GST-C-CBD" are hereafter replaced by "G4-N and G4-C," because it is apparent from the above control experiments that the protein sequence responsible for the tissue binding of the recombinant protein is not its GST but its N-CBD or C-CBD portion.
Figure 3 shows fluorescence microscopic views of G4-N and G4-C binding sites in rat colorectal tissue. G4-N recognizes the intercellular borders of the surface-lining epithelial cells. On the other hand, G4-C binds not only to the same site as does G4-N but also to the apical balloon-like cytoplasm of goblet cells scattered throughout the colon mucosa, including the surface-lining and crypt epithelium. The staining pattern of the intercellular borders was different between G4-N and G4-C. The former stained granular structures mainly located in the lower half of the intercellular borders, whereas the latter labeled smooth linear structures extending throughout the intercellular borders from the apical to basal surface of the epithelium. No apparent labeling was seen on the apical or basal surface of the surface-lining epithelial cells with either G4-N or G4-C. However, in some areas of the sections, G4-C also decorated the luminal surface of some crypt cells (Figure 3D), but it is uncertain at the LM level whether this represents apical cell structure or secretions merely attached to the site. All the fluorescent structures completely vanished after the control stainings, as mentioned above.
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Figure 4 and Figure 5 show G4-N and G4-C binding sites at the EM level using a secondary antibody labeled with colloidal gold. Both G4-N and G4-C again recognize the intercellular spaces of the surface-lining epithelial cells. However, the labeling site was apparently distinct. G4-N recognized electron-lucent, flocculent substances found in the intercellular spaces (Figure 4). In contrast, G4-C intensely stained the lateral cell membranes, especially the membranes of long cell processes forming well-developed interdigitations (Figure 5). When the specimens were chopped and washed before cryosectioning, the staining density of G4-C was enhanced, in contrast to a significant decrease in G4-N staining. Although the reason for this enhancement of G4-C staining after the treatment is unknown, it appears probable that the procedure enabled G4-C to access its ligand(s) by removing the G4-N-positive substances filling the intercellular spaces. After staining with either fusion protein, no apparent labeling was observed on any cellcell junction, basal cell membrane, or basal lamina (Figure 4 and Figure 5). The secretory granule contents of goblet cells were almost entirely lost during the preparation for ultracryosectioning, and therefore it was impossible to determine what structure is responsible for the positive G4-C staining seen in the luminal surface or apical cytoplasm of the cells. In the control experiments, all labeling completely vanished (not shown).
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Discussion |
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We have previously demonstrated the immunocytochemical localization of G-4 in esophageal epithelium (
To circumvent this technical problem, we separately cloned cDNA encoding N-CBD and C-CBD of G-4 and expressed each of them as a recombinant GST fusion protein. The resulting fusion proteins were used to clarify the endogenous ligand(s) on both LM and EM sections of rat colorectal tissue. In all parts of the colon from the ascending colon to the rectum, the intercellular borders of the surface-lining epithelial cells were intensely stained with both G4-N and G4-C. Because even at the LM level the binding pattern of G4-N appeared to be different from that of G4-C, we investigated the binding site(s) in more detail with EM using colloidal gold as a cytochemical marker. The binding sites of G4-N and G4-C were strikingly different. The former recognized flocculent substances located in the intercellular spaces, whereas the latter closely attached to the lateral cell membranes of the cells. This finding indicates the following possibilities. (a) Two CBDs within a G-4 molecule recognize distinct ligands, i.e., ligand located in the intercellular spaces and ligand associated with (or anchored to) the lateral cell membranes. (b) The opposite lateral cell membranes can be cross-bridged with two G-4 molecules via these two ligands. (c) By crosslinking lateral cell membranes in this way, G-4 may be involved in cellcell adhesion of the colorectal surface-lining epithelial cells. This idea is further supported by the following findings. First, the architecture of the G-4 molecule, consisting of two structurally distinct domains with different sugar specificities (
We were unable to detect any significant labeling on the basal cell membrane or basal lamina with either G4-N or G4-C. This result is inconsistent with the observation (
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