concanavalin-a and Ovarian-Cysts

We have isolated an endo-beta-galactosidase designated E-ABase from Clostridium perfringens ATCC 10543 capable of liberating both the A trisaccharide (A-Tri; GalNAcalpha1-->3(Fucalpha1-->2)Gal) and B trisaccharide (B-Tri; Galalpha1-->3(Fucalpha1-->2)Gal) from glycoconjugates containing blood group A and B glycotopes, respectively. We have subsequently cloned the gene (eabC) that encodes E-ABase from this organism. This gene was found to be identical to the CPE0329 gene of C. perfringens strain 13, whose product was labeled as a hypothetical protein (Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S., and Hayashi, H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 996-1001). Since the amino acid sequence of E-ABase does not bear detectable similarity to any of the 97 existing families of glycoside hydrolases, we have proposed to assign this unusual enzyme to a new family, GH98. We also expressed eabC in Escherichia coli BL21(DE3) and obtained 27 mg of fully active recombinant E-ABase from 1 liter of culture. Recombinant E-ABase not only destroyed the blood group A and B antigenicity of human type A and B erythrocytes, but also released A-Tri and B-Tri from blood group A(+)- and B(+)- containing glycoconjugates. The structures of A-Tri and B-Tri liberated from A(+) porcine gastric mucin and B(+) human ovarian cyst glycoprotein were established by NMR spectroscopy. The unique specificity of E-ABase should make it useful for studying the structure and function of blood group A- and B-containing glycoconju-gates as well as for identifying other glycosidases belonging to the new GH98 family.

Topics: ABO Blood-Group System; Amino Acid Sequence; Animals; Base Sequence; Cell Separation; Chromatography; Chromatography, Gel; Chromatography, Thin Layer; Cloning, Molecular; Clostridium perfringens; Concanavalin A; Databases as Topic; DNA Primers; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Escherichia coli; Female; Flow Cytometry; Glycoproteins; Glycoside Hydrolases; Humans; Hydrolysis; Lectins; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Ovarian Cysts; Peptides; Plasmids; Polysaccharides; Recombinant Proteins; Swine; Time Factors

Mucinous tumors of the ovary have been thought to originate in two ways: by müllerian-type metaplasia of surface epithelium, and as monodermal teratomas. To gain a better understanding of their pathogenesis, the authors analyzed these tumors for their expression of estrogen receptors (ER) and progesterone receptors (PR) as markers of müllerian-type differentiation and for their content of gastric-type mucin as a marker of gastric differentiation.. The histochemical expression of ER, PR, and gastric-type mucin was studied in 10 specimens of the cervix with normal endocervical glands (as a representative of müllerian-derived mucin-containing cells), 3 ovary specimens with surface epithelial inclusion cysts that contained endocervical-like mucin-containing cells (representing müllerian-type metaplasia), and 47 mucinous tumors of the ovary (29 benign, 8 with low malignant potential, and 10 malignant).. Normal endocervical glands expressed ER and PR and rarely expressed gastric-type mucin. Ovarian inclusion cysts showed strong expression of ER and PR in the cuboidal cells and drastically reduced expression in the endocervical-like mucin-containing cells. The cuboidal cells were negative for gastric-type mucin, but the endocervical-like mucin-containing cells expressed gastric-type mucin. Endocervical-like mucinous cells in benign and borderline mucinous tumors showed expression of PR and/or gastric-type mucin in all cases.. The staining results for the inclusion cysts support the thesis that the endocervical-like mucinous cells encountered in the ones that express ER and PR weakly or not at all and have histochemical properties of normal gastric epithelium have their origin in metaplasia of müllerian-type epithelium. Application of the same staining methods to benign ovarian tumors and those with low malignant potential suggests strongly that similar müllerian-type metaplasia is a major pathway in their pathogenesis.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Cell Differentiation; Cell Transformation, Neoplastic; Cervix Uteri; Concanavalin A; Cystadenoma, Mucinous; Female; Galactose Oxidase; Gastric Mucins; Humans; Immunohistochemistry; Metaplasia; Middle Aged; Mullerian Ducts; Ovarian Cysts; Ovarian Neoplasms; Receptors, Estrogen; Receptors, Progesterone; Staining and Labeling

In view of the reported disagreement in the physicochemical properties of ovarian carcinoembryonic antigen (CEA), this study was undertaken to compare the properties of CEA obtained from extracts of ovarian tumour tissue, ascitic fluid and cyst fluid. On the basis of molecular weight estimation and binding properties with Concanavalin A and wheat germ lectin, ovarian CEA from these three sources appeared similar, and also possessed similar properties to those of colonic CEA. On isoelectric focusing, however, it was found that the isoelectric point of CEA from tumour tissue and cyst fluid differed from that from ascitic fluid. It is most likely that this is due to a loss of sialic acid from the CEA released into ascitic fluid.

Topics: Ascites; Carcinoembryonic Antigen; Concanavalin A; Cystadenocarcinoma; Female; Humans; Ovarian Cysts; Ovarian Neoplasms; Radioimmunoassay

Topics: ABO Blood-Group System; Animals; Blood Group Antigens; Carbohydrates; Chemical Phenomena; Chemistry; Concanavalin A; Female; Fractional Precipitation; Hemagglutination Inhibition Tests; Humans; I Blood-Group System; Ovarian Cysts; Precipitin Tests

Topics: ABO Blood-Group System; Acetylgalactosamine; Amino Acids; Blood Group Antigens; Borohydrides; Concanavalin A; Dialysis; Female; Fractional Precipitation; Fucose; Galactose; Hexosamines; Humans; I Blood-Group System; Oligosaccharides; Ovarian Cysts

Topics: ABO Blood-Group System; Acetylgalactosamine; Animals; Blood Group Antigens; Chemical Phenomena; Chemistry; Circular Dichroism; Concanavalin A; Deoxyglucose; Female; Fucose; Galactose; Humans; Oligosaccharides; Optical Rotatory Dispersion; Ovarian Cysts

1. Gastric juice, saliva and ovarian-cyst fluid were fractionated into glycoprotein components by centrifuging to equilibrium in a caesium chloride density gradient. 2. The glycoprotein fractions from the gastric juice of two group O non-secretors, two group O secretors and three group A secretors all formed insoluble complexes with concanavalin A. 3. Fractions showing maximum interaction with concanavalin A had maximum blood-group activity measured by the haemagglutination-inhibition technique. The sulphate content of the gastric glycoproteins was unrelated to the capacity to interact with concanavalin A. 4. No interaction was found between concanavalin A and the glycoprotein fractions from any of the saliva or ovarian-cyst-fluid samples tested, implying that there is a structural difference in blood-group-substance glycoproteins in gastric juice when compared with those in saliva and ovarian-cyst fluid. 5. The protein components of each of the secretions tested, gastric juice, saliva and ovarian-cyst fluid, interacted with concanavalin A.

Topics: ABO Blood-Group System; Body Fluids; Carbohydrates; Centrifugation, Density Gradient; Chemical Precipitation; Concanavalin A; Densitometry; Female; Gastric Juice; Glycoproteins; Hemagglutination Inhibition Tests; Humans; Lectins; Lewis Blood Group Antigens; Ovarian Cysts; Saliva; Sulfates