Ikuo Funakoshi

A monoclonal antibody directed to Tn antigen

A murine monoclonal antibody, MLS 128, that was assigned to an anti-Tn antibody has been established by immunizing mice with human colonic cancer cells (LS 180). MLS 128 bound to mucin glycopeptides from LS 180 cells and their asialo forms to the same extent as well as to ovine submaxillary mucin (OSM) and asialo OSM. Special non-sialylated GalNAc residue(s) attached to a certain peptide region in the antigens seems to be involved in the binding since N-acetylgalactosaminidase treatment of the antigen abolished the binding and pronase digestion diminished the binding markedly.

Mucin-carbohydrate directed monoclonal antibody

To raise monoclonal antibodies recognizing cancer‐associated alterations of the carbohydrate structure of glycoproteins, Balb/c mice were immunized with human colonic cancer cells (LS 180 from ATCC). One of the generated hybridomas produced a monoclonal antibody that bound to the carbohydrate moiety of mucin‐type glycoproteins from LS 180. The antibody did not bind to glycoproteins from another colonic cancer cell line, SW 1116, or to glycolipids from any of the colonic cancer cell lines. The antibody bound to ovine and bovine submaxillary mucins (OSM and BSM). NeuAcα2→6Ga1NAc seemed to be involved in the epitope.

Structural characterization of proteoheparan sulfate isolated from plasma membranes of an ascites hepatoma, AH 66☆

Abstract A proteoglycan isolated from plasma membranes of an ascites hepatoma, AH 66, was characterized structurally. The glycosaminoglycan was obtained by alkali treatment and was identified as heparan sulfate. It was essentially the only type of carbohydrate chain attached to the core protein. The identification was based on chemical analysis, electrophoresis, and digestibility with heparitinase from Flavobacterium heparinum . Analysis of neutral sugars of the proteoglycan by mass fragmentography indicated the presence of xylose and galactose which should be involved in the linkage region between a heparan sulfate chain and the core protein. The weight-average molecular weights of the proteoglycan and its heparan sulfate chain were determined to be 71,000 and 21,000, respectively, by meniscus depletion equilibrium centrifugation. The latter value was in good agreement with those obtained by chemical analysis and by gel filtration. From these values for molecular weight and the protein content of the proteoglycan (10.6%), the molecular weight of the core protein was estimated to be 7500. On the basis of these molecular parameters, it was proposed that three heparan sulfate chains on average are linked to the core protein.

Quantitative determination of partially methylated alditol acetate of amino sugar by gas chromatography-mass spectrometry☆

Abstract Using doubly labeled N -acetyllactosamine. Hakomoris procedures to prepare partially methylated alditol acetates and their subsequent analysis by gas chromatography-mass spectrometry were followed from a quantitative aspect. It was found that both galactose and glucosamine were nearly quantitatively converted to PMAAs. Preferential loss of PMAA of glucosamine occurred on a column for gas chromatography when the amount of the PMAA injected onto a column was less than a certain level. Above this level, PMAAs of both galactose and glucosamine were eluted from the column with equal yields. However, in mass spectrometry with monitoring of total ions, the molar response factor of PMAA of glucosamine was found to be about 25% higher than that of PMAA of galactose. Based on these findings, methylation analysis of an oligosaccharide from Taka-amylase A composed of one glucosamine and five mannose residues could be carried out quantitatively.

Production of Monoclonal Antibodies Directed against Carbohydrate Moieties of Cell Surface Glycoproteins

Through the use of a technique for raising monoclonal antibodies, coupled with a solid‐phase radioimmunoassay utilizing immobilized glyeopeptides prepared from the surface membranes of the colorectal cancer cells (LS 180) used for the immunization, carbohydrate‐directed monoclonal antibodies were obtained. One of the monoclonal antibodies, MLS 102, reacted immunohistochemically intensely with the colorectal cancer cell surface and the mutinous glycoproteins secreted by the cancer cells, but only weakly with normal colon tissue. The antigenic determinant recognized by MLS 102 was the carbohydrate moiety of glycoproteins with terminal sialic acid. The antigens defined by other monoclonal antibodies, MLS 103 and 104, were immunohistochemically detected in both normal colonic epithelial and cancer cells. These antibodies seemed to recognize the carbohydrate moieties of both glycoproteins and glycolipids. The method described in this report can be generally applied to raise cell surface carbohydrate‐directed antibodies.

Immunoaffinity purification and characterization of nucleotide pyrophosphatase from human placenta

Nucleotide pyrophosphatase [EC 3.6.1.9] was purified to homogeneity from human placenta using a monoclonal antibody affinity column. By sodium dodecylsulfate--polyacrylamide gel electrophoresis, the purified enzyme showed a major band at a molecular size of 130 K. The enzyme was a glycoprotein with N-linked oligosaccharides consisting of both complex- and oligomannoside-types. Substrate specificity to hydrolyze phosphodiester and phosphosulfate linkages as well as other properties were similar to those of nucleotide pyrophosphatase and phosphodiesterase from other sources.

UDP-GalNAc : GalNAc-mucin α-N-acetylgalactosamine transferase activity in human intestinal cancerous tissues

GalNAc transferase Human intestinal cancerous tissue Bovine submaxillary gland mucin O‐Glycosidically linked sugar chain

Structural studies of glycoasparagines from urine of a patient with aspartylglycosylaminuria (AGU).

Patients with aspartylglycosylaminuria (AGU) excrete in their urine two isomeric glycoasparagines composed of two moles each of galactose and N-acetyl. glucosamine and one mole each of N-acetylneuraminic acid and asparagine, in addition to glycoasparagines with smaller carbohydrate moleties including 2-acetamido-N-(4’-Laspartyl)-2-deoxy-P-Dglucopyranosylamine (GlcNAc-Asn) [l-3] . Isolation and characterization of these two isomeric glycoasparagines have been reported previously [3]. This paper describes their complete structure based on the results of periodate oxidation and methylation studies.

Changes in the lectin binding capacities of hepatoma cells after treatment with chondroitinase.

Abstract Binding capacities of cells of ascites hepatoma, AH 130 FN, towards lectins were examined before and after treatment with chondroitinase AC. Chondroitin sulfate A was removed from the cells by the enzyme treatment, and the binding capacity of the cells towards Ricinus communis agglutinin (RCA) increased remarkably while the binding constant was unchanged, whereas that towards Concanavalin A (ConA) remained practically unchanged.

Cancer-associated glycoproteins defined by a monoclonal antibody, MLS 128, recognizing the Tn antigen

A murine monoclonal antibody, MLS 128, recognizing the Tn antigen, was established and used for characterization of glycoproteins expressing the Tn antigen. The Tn antigen was expressed on three polypeptide chains with molecular weights of 250k, 210k and 150k daltons. LS 180 cells were labeled with 3H-glucosamine or 35S-sulfate metabolically, and then the immunoprecipitate derived from the cell lysate was subjected to SDS-PAGE followed by fluorography. It was revealed that these Tn antigen glycoproteins were produced through the processing of a high molecular weight precursor. The carbohydrate moieties of the Tn antigen glycoproteins labeled with 3H-glucosamine were released with alkaline-borohydride, and the released sugars were examined by gel filtration and paper chromatography. The carbohydrates predominantly consisted of GalNAc and sialyl GalNAc (greater than 90%), with a nearly equal distribution.