ARTICLE |
Correspondence to: Hiroshi Kimura, Dept. of Forensic Medicine, Kurume Univ. School of Medicine, Kurume 830-0011, Japan.
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Summary |
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We have investigated by immunochemistry the distribution of H Type 3/4 chains of the ABO histo-blood group system in human submandibular gland using a monoclonal anti-H MBr1 antibody specific for H Type 3/4 chains, and have found the expression of H Type 3/4 chains was mainly in the serous cells. Serous cells from secretors were stained by MBr1 but not by anti-A and anti-B antibodies, whereas serous cells from nonsecretors exhibited a negative reaction with MBr1. Mucous cells were not stained by MBr1. Only a few striated duct cells showed a weak reaction with anti-H MBr1. These results suggested that the H Type 3/4 chains were distributed predominantly in the serous cells of the human submandibular gland and that secretor Type (1,2)fucosyltransferase (Se enzyme) controlled the synthesis of H Type 3/4 chains in vivo. Saliva also contained H Type 3/4 chains, which were controlled by the secretor gene (FUT2). The differences in the distributions of H Type 1, H Type 2, and H Type 3/4 chains of the ABO histo blood group system in the submandibular gland are discussed. (J Histochem Cytochem 47:889894, 1999)
Key Words: ABO, MBr1, H type 3 chain, H type 4 chain, secretor gene (FUT2), submandibular gland
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Introduction |
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Because carbohydrate chains are the ABO histo- blood group determinants, their expression is under control of the genes that encode specific glycosyltransferases (1-R; Type 4, Galß1-3GalNAcß1-R) (
In this study, we used MAb anti-H MBr1, which is defined as a breast cancer-associated antigen and is specific for H Type 3/4 antigens (
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Materials and Methods |
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The ABO histo-blood group phenotypes of tissue donors were determined on red blood cells using the conventional hemagglutination method with mouse MAbs (Kokusai; Kobe, Japan) against histo-blood group antigens A and B (
Two anti-H MAbs, MBr1 and 1E3, were used in the present study. MAb MBr1, specific for H Type 3/4 structures (Fuc1-2Galß1-3GalNAc-) (
1-2Galß disaccharide, using a series of synthetic oligosaccharides (
Submandibular glands from individuals with different ABO histo-blood groups were obtained from autopsy cases (five secretors and four nonsecretors from O blood group individuals and four secretors from A and four secretors from B blood group individuals). Deparaffinized sections of 10% formalin-fixed, paraffin-embedded submandibular glands were immunostained using the streptavidinbiotin complex immunoperoxidase method as described previously (
A dot ELISA was carried out as described previously (
To examine whether the H Type 3/4 antigens were synthesized by the H enzyme or the Se enzyme, the wild-type allele of the FUT1 for H enzyme or the FUT2 for Se enzyme was subcloned into a mammalian expression vector pRc/CMV (Invitrogen; San Diego, CA) (pRc/CMV-H or pRc/CMV-Se), as described previously (
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Results |
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The murine MAb MBr1, raised against the human breast cancer cell line MCF-7, recognized a saccharide epitope on human breast, ovary, and lung carcinomas (1-2Galß1-3GalNAcß1-3Gal
1-4Galß1-4Glcß1-Cer) in MCF-7 cells (
1-2Galß1-3GalNAc, H Type 3/4) on glycolipids and on glycoproteins, irrespective of the anomeric structure of the internal GalNAc residue (
1-2Galß1-3GalNAcß1 (H Type 4 chain).
Previous studies have revealed the presence of Type 3 chain-based ABH antigens in saliva and ovarian cyst fluids (
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Next, we immunohistochemically investigated the human submandibular gland using anti-H MBr1 and anti-H 1E3 (Figure 2). Serous cells and some serous demilune cells from secretor donors, but not from nonsecretor donors, were stained intensely by anti-H MBr1, irrespective of ABO phenotypes (Figure 2A and Figure 3A). These results suggested that the production of H Type 3/4 antigens in serous cells of human submandibular glands was under the control of the Se gene. In contrast, mucous acini, vascular endothelia, and erythrocytes showed no reaction with anti-H MBr1, although these cells showed a good reaction with anti-H 1E3 (Figure 2), anti-A (Figure 3), or anti-B (not shown) in tissues from corresponding ABO group individuals. Positive staining by MBr1 was observed in the luminal surface and luminal contents of many striated ducts, but only a few epithelial cells (less than 1%) of striated ducts showed a weak reaction with MBr1. Staining by 1E3 appeared to localize on basolateral plasma membranes of striated duct cells, and this mode of distribution obviously differed from that in mucous cells (Figure 2B and Figure 2D). Epithelial cells of interlobular ducts demonstrated a negative reaction with MBr1 (not shown). In the submandibular gland of A secretors, we also observed a strong reaction with anti-A in mucous acini (Figure 3) and a weak reaction in some ducts, but no reaction in serous acini despite the strong staining by anti-H MBr1 in serous cells, suggesting the expression of the ABO gene in mucous cells and duct cells but not in serous cells. It appeared that all serous cells from the A secretors showed a positive reaction with MBr1 (Figure 3A), whereas only a portion of serous cells from the O secretors reacted with the antibody (Figure 2A). The difference in staining of serous cells between A and O secretors was due to individual variation. All results are summarized in Table 1.
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COS7 cells transfected by the H gene (FUT1) or the Se gene (FUT2) showed strong fluorescence with both anti-H MBr1 and 1E3 (not shown), suggesting that both (1,2)fucosyltransferases (H enzyme and Se enzyme) can fucosylate a precursor of Type 3 or Type 4 to form H Type 3 chain or H Type 4 chain in vitro, consistent with a previous report (
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Discussion |
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In this study we examined immunohistochemically the location of H Type 3/4 antigens in cell types in the human submandibular gland using anti-H MBr1, and we identified cell types expressing H Type 3/4 chains as serous cells. Our results extended previous observations (
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The H Type 3 has been found in ovarian cyst fluid and saliva (1-2Galß1-3GalNAc
1-3[Fuc
1-2]Galß1-4GlcNAcß1-3Galß1-4GlcNAc-Cer) was constructed by adding a Gal residue and then a Fuc residue sequentially to the terminal GalNAc of the A Type 2 structure. Alternatively, the formation of the H Type 3 chain needs both the H Type 2 chain as a precursor and the activity of A glycosyltransferase. The other Type 3 chain-based H antigen is a fucosylated T antigen (Fuc
1-2Galß1-3GalNAc
1-O-Ser/Thr), which has been found in the mucosa of the stomach and colon (
1-2Galß1- 3GalNAc
1-O-Ser/Thr) was produced in serous cells and that its fucosylation was under the control of the Se gene in human submandibular glands.
-O-Ser/Thr) and fucosylated T antigen (H Type 3) in human gastric surface epithelial cells and the dependency of its fucosylation on the Se gene, using anti-H MBr1 and PNA as probes. However, the specificity of PNA has been questioned because of different distributions of PNA-positive and anti-T-positive substances before and after neuraminidase treatment and different reactivities of PNA and anti-T antibody after galactose oxidation (
In this study we have demonstrated that reactivity of anti-H MBr1 was found in the serous cells of the submandibular gland from O individuals, and that anti-A- and anti-H MBr1-positive substances were present in different cell types of the submandibular gland from A individuals, suggesting that an MBr1-reactive substance appeared to be not a precursor of repetitive A (H Type 3 glycosphingolipid). Moreover, no A, B, or H Type 2 antigens were detected in serous acini (Figure 3) (1-2Galß1-3GalNAcß1-3Gal
1-R) is the MBr1-defined epitope and that the terminal fucose is essential for MBr1 recognition. However, because anti-H MBr1 cannot discriminate H Type 3 from H Type 4, it is not known whether MBr1-reactive material in serous cells is H Type 3, H Type 4, or both. Because the A type 4 antigen was isolated from human kidney (
It is known that the UEA-I-positive substances, which are regulated by the Se gene, are present in the serous cells of human submandibular gland (
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Acknowledgments |
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Supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan.
We are grateful to Drs Ken Furukawa and Shin Yazawa (Department of Legal Medicine, Gunma University School of Medicine) and to Dr Maria I. Colnaghi (Division of Experimental Oncology E, National Institute for Cancer Research, Milan) for the kind gifts of MAb anti-H 1E3 and anti-H MBr1, respectively. We thank Mr Shigeo Kamimura and Ms Yasuko Noguchi for making serial thin sections.
Received for publication September 8, 1998; accepted January 12, 1999.
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