ABSTRACT
Four polysaccharides Pc-1, Pc-2, Pc-3 and Pc-4 were isolated from water and alkali extracts of the lichen Peltigera canina using ethanol fractionation, gel filtration and preparative HP-GPC. The monosaccharide composition was determined by methanolysis and GC and showed mannose and galactose as the predominating structural units. The mean M(r) was determined by HP-GPC. The heteroglycans were tested for in vitro immunomodulating activities and showed mitogenic activity in rat spleen cell proliferation assay and stimulated IL-10 secretion. In rat peritoneal macrophages, the heteroglycans stimulated TNF-alpha secretion, but not IL-10 secretion. These results indicate that the polysaccharides influence cells of the immune system both from the innate and the adaptive systems.
Subject(s)
Immunologic Factors/pharmacology , Lichens , Phytotherapy , Plant Extracts/pharmacology , Spleen/drug effects , Tumor Necrosis Factor-alpha/drug effects , Animals , Cell Proliferation/drug effects , Female , Immunologic Factors/administration & dosage , Immunologic Factors/therapeutic use , Interleukin-10/biosynthesis , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/metabolism , Plant Extracts/administration & dosage , Plant Extracts/therapeutic use , Rats , Rats, Inbred Lew , Spleen/cytology , Spleen/metabolism , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
The specificity of a new anti-epiglycanin antibody (AE-3) which recognizes a mucin-type glycoprotein, the Human Carcinoma Antigen, found in the blood of patients with carcinomas, was studied. Information regarding the chemical nature of the antibody binding site was obtained by altering the structure of epiglycanin by chemical or enzymic means and testing the product in a competitive binding assay for inhibition of the binding of AE-3 to epiglycanin. The need for a high molecular weight antigen containing clustered T disaccharide, Gal,1-3GalNAc, was demonstrated. The specificity was further explored by inhibition studies with glycopeptides having one to three mono- to disaccharides. The results were interpreted using computer graphics molecular modeling which predicted the specific recognition of hydroxyl groups on oligosaccharides on adjacent amino acids. Thus T antigen O-linked glycopeptide tumour markers can be designed to be distinguished by antibodies by the amount of clustering of their oligosaccharides.
Subject(s)
Antibodies, Monoclonal/chemistry , Membrane Glycoproteins/chemistry , Neoplasm Proteins/chemistry , Amino Acid Sequence , Animals , Antibody Specificity , Binding, Competitive , Glycopeptides/chemistry , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin M/chemistry , Membrane Glycoproteins/immunology , Mice , Models, Molecular , Molecular Sequence Data , Neoplasm Proteins/immunologyABSTRACT
A polysaccharide, Ci-3, resembling isolichenan except with a much higher degree of polymerization, has been isolated from the water extract, as well as from the alkali extract, of the lichen Cetraria islandica (L.) using ethanol fractionation, dialysis, ion-exchange chromatography and gel filtration. The mean M(r) of Ci-3 was determined to be 2000 kD, compared to 6-8 kD reported for isolichenan. The structure of Ci-3 was elucidated and found to be composed of (1-->3)- and (1-->4)-alpha-D-glucopyranosyl units in the ratio of 2:1, using methanolysis, methylation analysis, optical rotation and NMR spectroscopy. The immunomodulating activity of Ci-3 was tested in an in vitro phagocytosis assay and anti-complementary, and proved to be active in both tests.
Subject(s)
Adjuvants, Immunologic/pharmacology , Glucans/pharmacology , Lichens/chemistry , Adjuvants, Immunologic/isolation & purification , Carbohydrates/analysis , Chromatography, Gel , Chromatography, Ion Exchange , Chromatography, Thin Layer , Complement Inactivator Proteins/pharmacology , Glucans/isolation & purification , Humans , In Vitro Techniques , Methanol , Methylation , Phagocytosis/drug effectsABSTRACT
Human carcinoma-associated antigen (HCA), detected by the mouse monoclonal anti-epiglycanin antibody, AE-3, has been isolated from ascitic fluid taken from a patient with metastatic ovarian adenocarcinoma and from spent medium of the human endometrial carcinoma cell line KLE-1 and compared with epiglycanin. The ascitic fluid and the spent medium were concentrated by a Filtron Ultrasette 100 K Omega membrane and fractionated by gel filtration on Sepharose CL-2B. The active fractions which consisted mainly of glycoproteins having relative molecular weights in the range 1000-2000 kDa compared to dextrans, were further purified by affinity chromatography on a column of immobilized AE-3. The active fraction was subjected to SDS-PAGE and blotted onto a PVDF membrane. The amino acid composition of HCA isolated from the two sources, were related but not identical and both showed some differences from the amino acid composition of epiglycanin. They all have, however, compositions typical of mucin-type glycoproteins. The isoelectric point for HCA from both KLE-1 and ascitic fluid were determined to be at pH 1.8 and the buoyant densities were about 1.4 g/ml as determined by cesium trifluoroacetate gradient centrifugation.
