ARTICLE |
Correspondence to: William S. Sly, Edward A. Doisy Dept. of Biochemistry and Molecular Biology, Saint Louis U. School of Medicine, 1402 S. Grand Blvd., St Louis, MO 63104.
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Summary |
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MN/CA IX is a recently discovered member of the carbonic anhydrase (CA) gene family that has been identified in the plasma membranes of certain tumor and epithelial cells and found to promote cell proliferation when transfected into NIH3T3 cells. This study presents localization of MN/CA IX in human gut and compares its distribution to those of CA I, II, and IV, which are known to be expressed in the intestinal epithelium. The specificity of the monoclonal antibody for MN/CA IX was confirmed by Western blots and immunostaining of COS-7 cells transfected with MN/CA IX cDNA. Immunohistochemical stainings of human gut revealed prominent polarized staining for MN/CA IX in the basolateral surfaces of the enterocytes of duodenum and jejunum, the reaction being most intense in the crypts. A moderate reaction was also seen in the crypts of ileal mucosa, whereas the staining became generally weaker in the large intestine. The results indicate isozyme-specific regulation of MN/CA IX expression along the cranialcaudal axis of the human gut and place the protein at the sites of rapid cell proliferation. The unique localization of MN/CA IX on the basolateral surfaces of proliferating crypt enterocytes suggests that it might serve as a ligand or a receptor for another protein that regulates intercellular communication or cell proliferation. Furthermore, MN/CA IX has a completely conserved active site domain of CAs suggesting that it could also participate in carbon dioxide/bicarbonate homeostasis. (J Histochem Cytochem 46:497504, 1998)
Key Words: carbonic anhydrase, gastrointestinal, gut, human, immunohistochemistry, intestine, plasma membrane
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Introduction |
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The carbonic anhydrases (CAs) participate in a variety of physiological processes that involve pH regulation, CO2 and HCO3- transport, and water and electrolyte balance (
MN/CA IX was first recognized as the novel tumor-associated antigen, MN, a transmembrane protein that was detected in several human carcinomas and in normal gastric mucosa (
The major aim of this study was to immunolocalize MN/CA IX in different segments of the human intestine and to determine whether its membrane localization suggests a relationship to sites of cell proliferation. The availability of reagents for the other isozymes enabled us to contrast the localization of MN/CA IX with those of other CAs that have been reported to be present in gut but never completely evaluated in parallel.
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Materials and Methods |
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Antibodies Against CA Isozymes
Murine monoclonal antibody (MAb) M75 recognizing the N-terminal domain of MN/CA IX protein has been previously described by
Transfection of COS-7 Cells
The cDNA of human MN/CA IX (
SDS-PAGE and Western Blotting
All the reagents for SDS-PAGE were from Bio-Rad Laboratories (Richmond, CA) or Sigma (St Louis, MO). The electrophoreses were performed in a Mini-Protean electrophoresis unit (Bio-Rad Laboratories) under reducing conditions according to
Immunocytochemistry
The COS-7 cells transfected with MN/CA IX cDNA were fixed with 4% neutral-buffered formaldehyde for 15 min. Then they were rinsed with PBS and subjected to immunofluorescence staining that consisted of the following steps: (a) pretreatment of the cells with 1:10 diluted cow colostral whey in PBS for 40 min and rinsing in PBS; (b) incubation for 1 hr in the M75 hybridoma medium diluted 1:10 in 0.1% bovine serum albumin in PBS (BSAPBS); and (c) incubation with 1:100 diluted fluorescein-conjugated goat anti-mouse IgG antibodies (Sigma) in 0.1% BSAPBS. The cells were washed three times for 10 min after the incubation steps. All incubations and washings were done in the presence of 0.05% saponin. The cells were viewed with a conventional Zeiss Axioplan epifluorescence microscope (Oberkochen, Germany) and a confocal laser scanning microscope (Zeiss Axiovert 135 microscope combined with an LSM 410 CLSM system). The specimens were excited with a laser beam at a wavelength of 488 nm and the emission light was focused through a pinhole aperture. The full field was scanned in square image formats of 512 x 512 pixels and built-in software was used to reconstruct the images obtained from the confocal sections.
Samples of human duodenum (n = 2), jejunum (n = 2), ileum (n = 3), cecum (n = 3), ascending colon (n = 3), transverse colon (n = 3), descending colon (n = 3), sigmoid colon (n = 3), and rectum (n = 2) were obtained together with routine histopathological specimens taken during surgical operations for gastric carcinoma (n = 2), rectal carcinoma (n = 1), colon polyposis (n = 2), or colon inertia (n = 1). The procedures had the approval of the ethics committee of Oulu University Hospital and the research was carried out according to the provisions of the Declaration of Helsinki. Each tissue sample was divided into several small pieces about 5 mm thick. The specimens were fixed in Carnoy's fluid (absolute ethanol + chloroform + glacial acetic acid 6:3:1) for 6 hr or in 4% paraformaldehyde in PBS for 18 hr. All fixations were carried out at 4C. The samples were then dehydrated, embedded in paraffin in a vacuum oven at 58C, and sections of 5 µm were placed on gelatin-coated microscope slides. Carnoy's fluid preserved best the antigenicity of CA I, II, and MN/CA IX, whereas the staining reaction for CA IV was best preserved after paraformaldehyde fixation, which also was found previously to be the most suitable fixative for CA VI (
The CA isozymes were immunostained by the biotinstreptavidin complex method, employing the following steps: (a) pretreatment of the sections with undiluted cow colostral whey for 40 min and rinsing in PBS; (b) incubation for 1 hr with the primary antiserum diluted 1:100 (CA I, II, IV, and VI) or the M75 hybridoma medium diluted 1:10 in 1% BSAPBS; (c) treatment with cow colostral whey for 40 min and rinsing in PBS; (d) incubation for 1 hr with biotinylated swine anti-rabbit IgG (Dakopatts; Glostrup, Denmark) or rabbit anti-mouse IgG (Dakopatts) diluted 1:300 in 1% BSAPBS; (e) treatment with cow colostral whey for 5 min and rinsing in PBS; (f) incubation for 30 min with peroxidase-conjugated streptavidin (Dakopatts) diluted 1:500 in PBS; and (g) incubation for 2 min in DAB solution containing 9 mg 3,3'-diaminobenzidine tetrahydrochloride (Fluka; Buchs, Switzerland) in 15 ml PBS + 10 µl 30% H2O2. The sections were washed three times for 10 min in PBS after incubation Steps b, d, and f. All the incubations and washings were carried out at RT, and the sections were finally mounted in Permount (Fisher Scientific; Fair Lawn, NJ). The stained sections were examined and photographed with a Leitz Aristoplan microscope (Wetzlar, Germany).
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Results |
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Specificity of Anti-MN/CA IX Antibody
The monoclonal anti-MN/CA IX antibody, which identified a strong 58-kD and a weak 54-kD polypeptide band on Western blots of COS-7 cells transfected with MN/CA IX cDNA, showed no crossreactivity with other CAs (CA I, II, and IV) which are known to be expressed in the human gut (
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Immunohistochemistry of MN/CA IX in the Gut
MN/CA IX showed a unique staining pattern in human gut. First, it was highly expressed in duodenum and jejunum (as in stomach) and was distinctly less expressed in more distal segments of the gut. Second, the intestinal staining for MN/CA IX was most intense in the crypts. Third, the positive signal for MN/CA IX was confined to the basolateral plasma membranes in all stained enterocytes.
Table 1 and Figure 3 show the immunohistochemical localization of MN/CA IX in different segments of the gut. Figure 3A (duodenum) and 3B (jejunum) show the intense staining in the crypt enterocytes, whereas the epithelial cells in upper portions and tips of the villi showed only a weak immunoreaction. MN/CA IX was also present in enterocytes of human ileum (Figure 3C), although the positive reaction was much weaker than in duodenum and jejunum. In the large intestine, the basolateral surfaces of the crypt enterocytes showed positive staining for MN/CA IX, with a gradual change in staining intensity in a distal direction. The reaction was still moderate in cecum (Figure 3D) and ascending colon (Figure 3E), whereas the transverse (Figure 3F) and descending parts (Figure 3G) showed considerably weaker signals. Only sporadic enterocytes in sigmoid colon and rectum exhibited weak basolateral staining (Figure 3H).
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Immunohistochemistry of CA I, II, and IV in the Gut
CA I showed three distinct sites of expression in the gut. In jejunum and ileum, weak but definite cytoplasmic staining for CA I was localized to the epithelial cells of crypts of Lieberkühn, whereas no staining was seen in the villi (Figure 4A). In the large intestine, prominent reactions for CA I were seen in the non-goblet epithelial cells of the surface epithelial cuff, but the staining gradually decreased towards the base of the gland epithelium (Figure 4D). The endothelial cells of the subepithelial capillaries were the third major site of CA I expression (Figure 4A, and Figure 4D). This is consistent with previous reports showing that CA I is expressed in the capillary endothelium of gallbladder (
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In the small intestine, CA II was localized in the villi of duodenum and jejunum and the staining decreased towards the base of the crypts (Figure 4B). In the ileum, only sporadic non-goblet epithelial cells showed positive signal for CA II (data not shown), whereas the staining again became intense in the non-goblet surface epithelial cells of the large intestine (Figure 4E).
The membrane-associated CA IV showed intense immunoreaction in two different cell types: the endothelial cells of the subepithelial capillaries (Figure 4C) and the epithelial cells of the large intestine (Figure 4F). The epithelial brush border membrane showed intense immunoreaction in all segments of the large intestine. The reaction was strongest in the luminal part of the mucosa.
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Discussion |
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MN/CA IX is a novel member of the -CA gene family and is the only known integral membrane protein with CA activity (unpublished observations). Another membrane-associated CA isozyme, CA IV, is not considered an integral membrane protein because the mature protein is anchored to the plasma membrane through a glycosyl phosphatidylinositol linkage (
The present studies demonstrated that the distribution of MN/CA IX in the gut has unique features. First, its subcellular localization is restricted to the basolateral surfaces of epithelial cells. Second, its cellular distribution is restricted to the crypt enterocytes, which is not seen with any other CA. Third, its regional expression is distinctive compared to other CAs, being most intense in duodenum and jejunum, and decreasing distally from moderate expression in crypts of ileum, cecum, and ascending colon to only weak and sporadic expression in distal large intestine.
Restriction of MN/CA IX to the epithelial cells with the greatest proliferative capacity is consistent with its proposed role in cell proliferation (
As a CA-homologous transmembrane protein, MN/CA IX has some structural similarity with recently described receptor-type protein tyrosine phosphatases (RPTPs) ß and , which are members of a distinct group of phosphatases that have CA-homologous regions in their extracellular domains (
showed about 3050% amino acid identity with CAs (
have only one of the three conserved histidine residues required to bind to zinc ion in catalytically active CAs. Therefore, it is unlikely that they have any CA activity. Recent studies have indicated that the CA-like domain of RPTPß binds to contactin, a neuronal cell recognition molecule (
, MN/CA IX has a completely conserved active site domain that suggests that it could also participate in carbon dioxide/bicarbonate homeostasis. It will be of interest to search for ligands and/or receptors for MN/CA IX and to explore its functional role in the regulation of carbon dioxide homeostasis and in the growth and development of intestinal cells.
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Acknowledgments |
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Supported by grants DK40163 and GM34182 from the U.S. Public Health Service to WSS, from the Sigrid Juselius Foundation to SP, from the Maud Kuistila Foundation and Academy of Finland to AKP, and from Chiron Diagnostics Corporation and the Slovak Scientific Grant Agency to SP and JP.
Received for publication June 30, 1997; accepted October 30, 1997.
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