Radical fringe negatively modulates Notch signaling in postmitotic neurons of the rat brain.

Fringe was originally identified as a novel secreted signaling protein with a key role in wing formation of Drosophila. Three vertebrate fringe homologues, Radical, Lunatic and Manic fringe, were also identified, and have been shown to play major roles in neurogenesis during development. However, the expression and roles of vertebrate fringe homologues in the adult brain remain to be elucidated. We isolated the cDNA encoding rat Radical fringe (334 amino acids) from rat embryos, and found its mRNA to be most abundantly expressed in the adult rat brain by Northern blotting analysis. The localization of Radical fringe mRNA in the adult rat brain was also examined by in situ hybridization. The mRNA was abundantly expressed in most neurons, but not glial cells, throughout the brain. Notch signaling was shown to negatively modulate the stability of neurites and connections in postmitotic primary neurons. Furthermore, genetic evidence indicated that fringe modulated the Notch signaling pathway. Therefore, we examined the effects of Radical fringe on the Notch signaling pathway in primary rat neurons of the cerebral cortex using recombinant rat Radical fringe protein. Radical fringe protein significantly inhibited expression of the Notch effector Hes1 mRNA in primary neurons. These results indicated that Radical fringe functions by inhibiting Notch signaling in postmitotic neurons of the brain.

Brain-specific expression of a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-T9).

We isolated cDNA coding for the ninth of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-T9) from human brain by the polymerase chain reaction. The polypeptide encoded by GalNAc-T9 contained the structural features characteristic of GalNAc transferases, such as a GT1 motif, a Gal/GalNAc transferase motif, (QXW)(3) repeats, and conserved His, Cys, and acidic amino acid residues. Northern blot analysis revealed the mRNA expression of the enzyme to be confined to the brain. The brain-specific expression of GalNAc-T9 suggested that this isozyme catalyzes O-glycosylation in the brain.

An O-acetylated sialyl-Tn is involved in ovarian cancer-associated antigenicity.

We reported previously that a monoclonal antibody, 6G9, raised against bovine submaxillary mucin (BSM) reacted with mucinous ovarian cancer and recognized tumor-associated sialylated carbohydrate antigens. To obtain structural information on the carbohydrate antigens recognized by 6G9, the reactivity of several mucins and carbohydrates with the antibody was determined by ELISA. Exoglycosidase digestion of BSM showed that 6G9 recognized Sia as a nonreducing monosaccharide but neither Gal nor GlcNAc. Reactivity of BSM with 6G9 decreased markedly on de-O-acetylation of BSM in mild alkali, and O-acetyl Sia obtained from BSM reacted with the antibody, indicating the presence of O-acetyl groups on Sia in the epitope. A sialyl-Tn structure located in the epitope was also demonstrated by the findings that de-O-acetylated BSM retained weak but significant reactivity with 6G9 and that ovine submaxillary mucin, major sugar chains of which are sialyl-Tn, reacted with 6G9 stronger than de-O-acetylated BSM. Furthermore, weak reactivity of NeuAcalpha2 --> 6GalNAc prepared from BSM demonstrated that 6G9 recognized the sialyl-Tn structure, but the modification of Sia with O-acetyl groups was essential for the recognition. The failure of 9-O-acetyl NeuAc, synthesized chemically, to react with the antibody implied that 6G9 recognized sialyl-Tn with O-acetylation on Sia distinct from C-9 O-acetylation.

The acceptor specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases.

The in vitro and in vivo specificity of the family of peptide:N-acetylgalactosaminyltransferases (GalNAcT) is analyzed on the basis of the reactivity and/or inhibitory activity of peptides and protein segments. The transferases appear to be multi-substrate enzymes with extended active sites containing a least nine subsites that interact cooperatively with a linear segment of at least nine amino acid residues on the acceptor polypeptide. Functional acceptor sites are located on the surface of the protein and extended conformations (beta-strand conformation) are preferred. The acceptor specificity of GalNAc-T can be predicted from the primary structure of the acceptor peptide with an accuracy of 70 to 80%. The same GalNAc-T enzymes catalyze the glycosylation of both serine and threonine residues. The higher in vitro catalytic efficiency toward threonine versus serine is the result of enhanced binding as well as increased reaction velocity, both effects being the result of steric interactions between the active site of the enzyme and the methyl group of threonine. Results from substrate binding studies suggest that GalNAc-T catalyzed transfer proceeds via an ordered sequential mechanism.

Carbohydrate antigens recognized by anti-horseradish peroxidase antiserum are expressed on mammalian cells.

Antiserum raised against horseradish peroxidase (HRP) recognizes neural specific carbohydrate antigens in Drosophila and other insects. The occurrence of the antigens in mammalian cells was investigated. Proteins were extracted from rat pheochromocytoma cells, PC12, and various tissue of rat and bovine origin, and further purified by successive chromatography with Con A-Sepharose and anti-HRP glycopeptide antibody-Sepharose. The proteins were separated by SDS-PAGE and subjected to immunoblot analysis of the antigens. When the Con A- and antibody-Sepharose bound fractions were examined, four glycoproteins with carbohydrate antigens recognized by anti-HRP antiserum were identified in all the tissue. They are low molecular weight glycoproteins that are not membrane-bound. We demonstrated that the carbohydrate antigens recognized by the anti-HRP antibodies are not confined to insects but are expressed on mammalian cells in several tissue.

Production of monoclonal antibodies recognizing cancer-associated antigens expressed on mucin-type sugar chains.

To obtain monoclonal antibodies directed to mucin-type sugar chains, mice were immunized with bovine submaxillary mucin (BSM) that had been conjugated with ovalbumin. Conjugation of BSM with ovalbumin enhanced the antigenicity of BSM to about five to ten times that of intact BSM and resulted in the establishment of ten hybridomas, all of which secreted monoclonal antibodies toward BSM. Most of the antibodies secreted by these hybridomas did not react with glycolipids but did react with glycoproteins. Several antibodies lost their reactivity when sialic acid residues were removed from BSM, indicating that these antibodies recognize carbohydrate moieties of mucins. Immunohistochemical studies revealed that three of the antibodies recognized human ovarian cancer-associated carbohydrate antigens. In addition, one of these three antibodies reacted with a human cultured colonic cancer cell line. The protocol described in this paper was effective in producing monoclonal antibodies that recognize mucin-carbohydrates and some of the generated antibodies can be applied to the detection of cancers.

Structural studies on the chondroitinase ABC-resistant sulfated tetrasaccharides isolated from various chondroitin sulfate isomers.

Various commercially available chondroitin sulfates, including an A isomer from whale cartilage, C and D isomers from shark cartilage, and an E isomer from squid cartilage, were exhaustively digested with a commercial highly purified Proteus vulgaris chondroitinase ABC. Gel chromatography of all digests yielded a disaccharide and an oligosaccharide fraction which was resistant to the enzyme digestion and which accounts for 20-31 mol% of the produced total oligosaccharides. Variably sulfated tetrasaccharides were isolated from the oligosaccharide fraction of each chondroitin sulfate isomer by HPLC, then characterized chemically and enzymatically. One disulfated and three trisulfated components were also characterized by 500-MHz one- and two-dimensional 1H NMR spectroscopy. The structures of one tetrasulfated, four trisulfated, and five disulfated tetrasaccharides with the common core structure, alpha-L-delta 4,5HexpA-(1-->3)-beta-D-GalpNAc-(1-->4)-beta-D-GlcpA-(1-->3) -D-GalpNAc, were determined. All isolated tetrasaccharides were resistant to the highly purified enzyme, but susceptible to the conventional, commercial chondroitinase ABC. The former was also inactive towards alpha-L-delta 4,5HexpA-(1-->3)-beta-D-GalpNAc-(1-->4)-beta-D-GlcpA-(1-->3) -D-GalpNAc isolated from chondroitin, beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->4)-beta-D-GlcpA-(1- ->3)-D-GlcpNAc from hyaluronan, and alpha-L-delta 4,5HexpA-(1-->3)-beta-D-GalpNAc4SO3(-)-(1-->4)-alpha-L-Id opA-(1-->3)-D- GalpNAc4SO3- from dermatan sulfate. These results indicate that, unlike the conventional enzyme, highly purified chondroitinase ABC cannot degrade tetrasaccharides irrespective of their sulfation profiles. The enzymatic action is size-dependent.

The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish peroxidase antiserum.

Antiserum raised against horseradish peroxidase (HRP) recognizes a neural specific carbohydrate antigen in Drosophila and other insects. The epitopic activity of the carbohydrate moiety of HRP recognized by anti-HRP antiserum was measured by a newly developed enzyme-linked immunosorbent assay, in which HRP glycopeptides conjugated with bovine serum albumin were coated onto the wells and then reacted with goat anti-HRP antiserum. HRP sugar moieties released by almond glycopeptidase A digestion of HRP pepsin digests were subjected to pyridylamination. Pyridylamino oligosaccharides were separated into seven fractions by reverse-phase high performance liquid chromatography. The major fraction, which comprised about 80% of the total sugars, reacted strongly with anti-HRP antiserum. The carbohydrate structure of this fraction was determined by sugar composition analysis and 600-MHz 1H NMR spectroscopy as follows: Man alpha 1----6(Man alpha 1----3)(Xyl beta 1----2)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc. Analyses of reactivity with anti-HRP antiserum of various oligosaccharide derivatives obtained from the major fraction by exoglycosidase digestion and partial acid hydrolysis indicated that alpha 1----6-linked mannose and alpha 1----3-linked fucose are predominantly involved in the epitopic structure.

Occurrence of tetra- and pentasaccharides with the sialyl-Le(a) structure in human milk.

The occurrence of two novel oligosaccharides in human milk was investigated. These oligosaccharides were purified by affinity chromatography on a column of an immobilized monoclonal antibody, MSW 113. Structural studies, involving 500-MHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry, indicated the structures of these compounds to be NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNAc and NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNac beta 1----3Gal. This constitutes the first evidence for the occurrence of N-acetylglucosamine or galactose as the reducing-end residue of human milk oligosaccharides. These two oligosaccharides bound MSW 113 to nearly the same extent as sialyl-Lea hexasaccharide but to another sialyl-Le(a) structure-directed monoclonal antibody, NS-19-9, only weakly.

Three novel oligosaccharides with the sialyl-Lea structure in human milk: isolation by immunoaffinity chromatography.

We have determined the structures of three novel oligosaccharides isolated from human milk using the monoclonal antibody MSW 113. These oligosaccharides were purified by affinity chromatography on a column of the immobilized monoclonal antibody and by high-performance liquid chromatography. From the results of 500-MHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry, their structures were deduced to be (formula; see text) These oligosaccharides bound to MSW 113 to nearly the same extent as sialyl-Lea hexasaccharide but bound to another sialyl-Lea structure-directed monoclonal antibody, NS 19-9, only weakly.

A monoclonal antibody that recognizes sialyl-Lea oligosaccharide, but is distinct from NS 19-9 as to epitope recognition.

A murine monoclonal antibody, designated as MSW 113, was generated using a human colonic cancer cell line, SW 1116, as the immunogen. MSW 113 was shown to be directed mainly to mucin-type oligosaccharide with sialyl-Lea antigens. The reactivity of MSW 113 to sialyl-Lea was stronger than that of NS 19-9, which is believed to be raised against the same determinant group. MSW 113 binds to sialyl-Lea-ol, LS-tetrasaccharide a, and disialyllacto-N-tetraose with higher affinities, compared to NS 19-9. These two antibodies could clearly be distinguished in that MSW 113 bound to sialic acid but not to fucose, whereas NS 19-9 bound to fucose but not to sialic acid. Thus, MSW 113 is directed more toward sialic acid-containing terminal structures while NS 19-9 is directed toward fucose-containing internal structures. MSW 113 was found to be useful for detecting antigens in the bloodstream of patients, especially those with pancreas cancer. Even NS 19-9 negative patient sera were positive for MSW 113.

Immunoaffinity isolation of a sialyl-Le(a) oligosaccharide from human milk.

A cancer-associated antigen, sialyl-Le(a) oligosaccharide, was isolated from human milk using a monoclonal antibody recognizing carbohydrate moieties of mucin-type glycoproteins. The structure was identified as: (Formula: see text) based on 500-MHz 1H-NMR spectroscopy. This oligosaccharide comprises 0.07% of sialyloligosaccharides in human milk. The NMR spectra of two fellow oligosaccharides, Le(a) oligosaccharide (or lacto-N-fucopentaose II) and LS-tetrasaccharide a, are also given.

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 glycopeptides 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 mucinous 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.

A monoclonal antibody that recognizes a cluster of a disaccharide, NeuAc alpha(2----6)GalNAc, in mucin-type glycoproteins.

The structure of an epitopic carbohydrate recognized by a monoclonal antibody, MLS 102, was determined. A disaccharide, NeuAc alpha (2----6)GalNAc, the major prosthetic group of ovine submaxillary mucin (OSM) and related synthetic glycosides, NeuAc alpha(2----6)GalNAc alpha----Ser, NeuAc alpha(2----6)GalNAc beta----Ser, and NeuAc alpha (2----6)GalNAc beta----propyl, reacted with MLS 102 to similar extents, but the reaction was considerably weaker compared to that of OSM. This difference in reactivity could be ascribed to the occurrence of a cluster of the disaccharide on OSM. Purification of MLS 102-reactive antigens from a Triton X-100 extract of LS 180 cells by means of immunoaffinity chromatography gave mucin fractions (cMLS 102 antigen) with an OSM-like domain. Correlation between the content of the disaccharide, NeuAc alpha(2----6)GalNAc, in mucins and their reactivity with MLS 102 was observed.

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). NeuAc alpha 2----6Ga1NAc seemed to be involved in the epitope.

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

GalNAc transferase activities of 6 human intestinal cancerous tissues were examined using bovine submaxillary gland mucin and its desialylated derivative, asialomucin, as acceptors. A Triton X-100 extract of these tissues was used an an enzyme source. All the tissues examined had GalNAc transferase that catalyzes the transfer of GalNAc from UDP-GalNAc to serine or threonine residues of the polypeptide chain. One of 6 specimens showed in addition UDP-GalNAc:GalNAc-mucin alpha-GalNAc transferase activity, synthesizing a disaccharide unit, GalNAc alpha----GalNAc, when asialomucin was used as an acceptor. This carbohydrate structure was deduced on the basis of results of gel filtration, exoglycosidase digestion, and high-voltage paper electrophoresis.

Structures of the major oligosaccharides from a human rectal adenocarcinoma glycoprotein.

Four oligosaccharides in the reduced form were isolated from RMG (a mucin-type glycoprotein from a human rectal adenocarcinoma). They were 1) Sia alpha s2 leads to 6GalNAc-ol; 2) Sia alpha 2 leads to 6(Gal beta 1 leads to 3)GalNAc-ol; 3) Sia alpha 2 leads to 6(GlcNAc beta 1 leads to 3)GalNAc-ol; and 4) Sia alpha 2 leads to 6(GalNAc alpha 1 leads to 3)GalNAc-ol. The amounts of oligosaccharides 1, 2, 3, and 4 corresponded to 27, 5, 11, and 8% of the total N-acetylgalactosaminitol produced on alkaline borohydride treatment of RMG. To determine the structures of oligosaccharides 2, 3, and 4, a mixture of the three was subjected to methylation analysis which revealed that the N-acetylgalactosaminitol was substituted at both C-3 and C-6 and other sugars at the nonreducing ends. Desialized oligosaccharides were prepared, and the structures were deduced by analysis of the permethylated sugars on gas-liquid chromatography/mass spectrometry. Anomeric configurations were determined by exoglycosidase digestions except for galactose which was analyzed by chromium trioxide oxidation.

Isolation and characterization of a glycoprotein from a human rectal adenocarcinoma.

A mucin-type glycoprotein was isolated from a human rectal adenocarcinoma, mainly be gel filtration and hydroxyapatite treatment. The glycoprotein, designated as rectal mucin-type glycoprotein (RMG), was great in amount, accounting for about 1% of the wet tissue weight. From a non-cancerous area of the patient's intestine, a similar glycoprotein reacting with anti-RMG antibodies was obtained, but the tissue content was less than 10% of the RMG content. Purified RMG contained about 70% carbohydrate in mass, and is composed of about equimolar amounts of sialic acid, galactose, N-acetylglucosamine and N-acetylgalactosamine. The polypeptide core was characterized by high contents of threonine, serine, and proline. A marked difference between RMG and the normal glycoproteins was that the sialic acid content was much higher in RMG. Of the total N-acetylgalactosamine convertible to N-acetylgalactosaminitol by reductive cleavage with alkaline borohydride, about 15% was free and the rest occupied the reducing ends of acidic oligosaccharides. The acidic oligosaccharides were fractionated into a fraction of high molecular weight and a series of oligosaccharides in which di- and trisaccharides containing sialic acid were dominant. The high molecular weight fraction contained esterified sulfate.