Biosynthesis of truncated O-glycans in the T cell line Jurkat. Localization of O-glycan initiation

Glycoproteins from the human T leukemia cells Jurkat were found to bind to the GalNAc alpha 1----Ser/Thr-specific lectin from Salvia sclarea seeds. The analysis of the O-linked saccharides of immunopurified leukosialin, the major [3H]glucosamine-labeled glycoprotein in Jurkat cell lysate, revealed the presence of mainly GalNAc alpha 1----Ser/Thr with only minor amounts (approximately 17%) of more complex O-glycans. A comparison between Jurkat and K562 cell glycosyltransferase involved in the biosynthesis of O-linked carbohydrates showed that a markedly lower activity of UDP-Gal:GalNAc alpha 1----Ser/Thr beta 1----3galactosyltransferase is apparently responsible for the presence of truncated O-glycans in the Jurkat cell line. The O-glycosylation defect makes Jurkat cells an ideal model to study the initiation of O-linked saccharides. Pulse-chase experiments with [35S] methionine showed that the addition of GalNAc to leukosialin is responsible for the decreased mobility of the mature glycoprotein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, no biosynthetic intermediates between the O-glycan-free precursor and the fully O-glycosylated form could be detected either with an anti-leukosialin antiserum or with the GalNAc-specific lectin. Lowering the chase temperature to 15 degrees C completely inhibited the transfer of GalNAc to the peptide core indicating that O-glycan initiation takes place in the first Golgi elements and not in transitional vesicles between endoplasmic reticulum and Golgi. In addition, treatment of the cells with monensin did not inhibit GalNAc transfer to leukosialin apoprotein. These results indicate that the initiation of O-glycosylation in Jurkat cells starts in the cis-Golgi stacks.     

Leukosialin, a major O-glycan-containing sialoglycoprotein defining leukocyte differentiation and malignancy.

Leukosialin, also called CD43 or sialophorin, is a major sialoglycoprotein expressed widely in various leukocytes (granulocytes, monocytes/macrophages and T-lymphocytes). Leukosialin is heavily glycosylated by O-linked oligosaccharides (70-80 oligosaccharides/molecule) and the structures of those O-glycans are characteristic to each cell lineage and differentiation stage. In particular, the branched hexasaccharide, NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc is specifically expressed in activated T-lymphocytes as well as in thymocytes and T-lymphocytes from patients with leukaemia, and immuno-deficiency syndromes. A portion of these O-glycans are attached to a domain with tandem repeats in the polypeptide of leukosialin. However, the entire translation product, including such tandem repeats, is coded by one exon and a short novel promoter sequence confers the expression of the leukosialin gene. Leukosialin is apparently involved in T-cell-B-cell interaction during immune reaction and binds to ligands on antigen-presenting B-cells. These results imply that leukosialin plays critical roles in immune cell interaction and differences in attached O-glycans most likely influence the interaction of leukosialin with ligands.     

Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines

Structures of O-linked oligosaccharides of leukosialin isolated from K562 erythroid, HL-60 promyelocytic, and HSB-2 T-lymphoid cell lines were examined. Leukosialin was isolated by specific immunoprecipitation from cells which were metabolically labeled with [3H]glucosamine, and glycopeptides were isolated after Pronase digestion. O-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were elucidated by specific exoglycosidase digestion, Smith degradation, and methylation anaylsis. Oligosaccharides from K562 cells were found to be GalNAcOH, Gal beta 1----3GalNAcOH, NeuNAc alpha 2----6GalNAcOH, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH. On the other hand, oligosaccharides from HL-60 and HSB-2 cells were found to be NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAcOH, Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3)Gal beta 1----3)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3Gal beta 1----3)GalNAcOH. These results clearly indicate that leukosialin can be differently glycosylated with O-linked chains, and each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of O-linked oligosaccharides which differ in core structures as well as in sialylation.     

Presence of an O-glycosidically linked hexasaccharide in fetuin

Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. While Diplococcus pneumoniae endo-alpha-DN-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra- and trisaccharides (Gal beta 1----3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal beta 1----3 [Gal beta 1----4GlcNAc beta 1----6] GalNAc). The number of O-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of O-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggest that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal beta 1----3GalNAc to form the NeuAc alpha 2----3Gal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.     

Poly-N-acetyllactosaminyl O-glycans attached to leukosialin. The presence of sialyl Le(x) structures in O-glycans.

Poly-N-acetyllactosamine extension has been found in O-glycans in addition to N-glycans and glycosphingolipids. Attempts were made in HL-60 and K562 cells to determine the amount of poly-N-acetyllactosaminyl O-glycans in the major sialoglycoprotein, leukosialin. Leukosialin was immunoprecipitated from [3H]glucosamine-labeled HL-60 and K562 cells. Glycopeptides were prepared by Pronase digestion, and O-glycan-containing glycopeptides were isolated by affinity chromatography using Jacalin-agarose. The glycopeptides bound to Jacalin-agarose and those unbound were treated with alkaline borohydride, and the released O-glycans were fractionated by Bio-Gel P-4 filtration. Sequential glycosidase digestion of the O-glycans, with or without pretreatment by fucosidase or neuraminidase, revealed the following conclusions. 1) Leukosialin from HL-60 cells contains about 1-2 poly-N-acetyllactosaminyl O-glycan chains/molecule. 2) About 50% of these poly-N-acetyllactosaminyl O-glycans contain sialyl Le(x) termini, NeuNAc alpha 2-->3Gal beta 1-->4 (Fuc alpha 1-->3)GlcNAc beta 1-->R. The amount of sialyl Le(x) structure in leukosialin is roughly equivalent to that on cell surfaces of HL-60 cells. 3) Leukosialin from K562 cells, on the other hand, contains no detectable amount of poly-N-acetyllactosaminyl O-glycans. 4) The presence of poly-N-acetyllactosamine in O-glycans is dependent on the core 2 beta 1,6-N-acetylglucosaminyl transferase. 5) Jacalin-agarose binds to sialylated small oligosaccharides such as NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->6) GalNAc but not the hexasaccharide NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->6) GalNAc. These results indicate that the formation of polylactosaminyl O-glycans and sialyl Le(x) structure in O-glycans is dependent on the core 2 formation.     

Localization of penultimate carbohydrate residues in zona pellucida and acrosomes by means of lectin cytochemistry and enzymatic treatments

Lectins from peanuts (PNA) and soy beans (SBA) bind terminal residues of galactose (Gal) and N-acetyl-galactosamine (GalNAc) respectively. Galactose oxidase oxidizes the hydroxyl group at C-6 of terminal Gal and GalNAc blocking the binding of PNA and SBA. Binding of these lectins to sugar residues is also severely limited by the existence of terminal residues of sialic acid. In the present study, lectin cytochemistry in combination with enzymatic treatments and quantitative analysis has been applied at light and electron microscopical levels to develop a simple methodology allowing the in situ discrimination between penultimate and terminal Gal/GalNAc residues. The areas selected for the demonstration of the method included rat zona pellucida and acrosomes of rat spermatids, which contain abundant glycoproteins with terminal Gal/GalNAc residues. Zona pellucida was labelled by LFA, PNA and SBA. After galactose oxidase treatment, terminal Gal/GalNAc residues are oxidized, and reactivity to PNA/SBA is abolished. The sequential application of galactose oxidase, neuraminidase and PNA/ SBA has the following effects: (i) oxidation of terminal Gal/GalNAc residues; (ii) elimination of terminal sialic acid residues rendering accessible to the lectins preterminal Gal/GalNAc residues; and (iii) binding of the lectins to the sugar residues. Acrosomes were reactive to PNA and SBA. No LFA reactivity was detected, thus indicating the absence of terminal sialic acid residues. Therefore, no labelling was observed after both galactose oxidase--PNA/SBA and galactose oxidase--neuraminidase--PNA/SBA sequences. In conclusion, the combined application of galactose oxidase, neuraminidase and PNA/SBA cytochemistry is a useful technique for the demonstration of penultimate carbohydrate residues with affinity for these lectins. This revised version was published online in November 2006 with corrections to the Cover Date.     

Comparison of the carbohydrate-binding specificities of seven N-acetyl-D-galactosamine-recognizing lectins

Seven plant lectins, Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin (GSA, isolectin A4), Helix pomatia agglutinin (HPA), soybean (Glycine max) agglutinin (SBA), Salvia sclarea agglutinin (SSA), Vicia villosa agglutinin (VVA, isolectin B4) and Wistaria floribunda agglutinin (WFA), known to be specific for N-acetyl-D-galactosamine-(GalNAc) bearing glycoconjugates, have been compared by the binding of their radiolabelled derivatives, to eight well-characterized synthetic oligosaccharides immobilized via a spacer on an inert silica matrix (Synsorb). The eight oligosaccharides included the Forssman, the blood group A and the T antigens, as well as alpha GalNAc coupled directly to the support (Tn antigen) and also structures with GalNAc linked alpha or beta to positions 3 or 4 of an unsubstituted Gal. The binding studies clearly distinguished the lectins into alpha GalNAc-specific agglutinins like DBA, GSA and SSA, and lectins which recognize alpha- as well as beta-linked GalNAc residues like HPA, VVA, WFA and SBA. HPA was the only lectin which bound to the beta Gal1----3 alpha GalNAc-Synsorb adsorbent (T antigen) indicating that it also recognizes internal GalNAc residues. Among the alpha GalNAc-specific lectins, DBA strongly recognized blood group A structures while GSA displayed weaker recognition, and SSA bound only slightly to this affinity matrix. In addition, DBA and SSA were able to distinguish between GalNAc linked alpha 1----3 and GalNAc linked alpha 1----4, to the support, the latter being a much weaker ligand. These results were corroborated by the binding of the lectins to biological substrates as determined by their hemagglutination titers with native and enzyme-treated red blood cells carrying known GalNAc determinants, e.g. blood group A, and the Cad and Tn antigens. For SSA, the binding to the alpha GalNAc matrix was inhibited by a number of glycopeptides and glycoproteins confirming the strong preference of this lectin for alpha GalNAc-Ser/Thr-bearing glycoproteins.     

Carbohydrate moieties of peanut agglutinin receptors isolated from human gastric cancer cells

Receptors for peanut agglutinin (PNA) were isolated from Kato III human gastric cancer cells by affinity chromatography on PNA agarose, and were labeled by the galactose oxidase-NaB3H4 method. Alkaline NaBH4 treatment of the labeled receptors released two small oligosaccharide alcohols, which were identified as Gal beta 1----3GalNAc-ol and Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAc-ol. Higher oligosaccharides and glycopeptides of both N- and O-linked type were also detected, but they did not appear to bear PNA binding sites. The presence of oligo-N-acetyllactosamine units in the N-linked type sugars was indicated by endo-beta-galactosidase digestion.     

Expression of the Tn antigen on T-lymphoid cell line Jurkat.

Expression of the Tn antigen on a T-lymphoid cell line, Jurkat, was investigated using an anti-Tn monoclonal antibody, MLS 128. Immunoprecipitation or immunoaffinity chromatography of a lysate of Jurkat cells led to the isolation of a 120 kDa glycoprotein carrying the Tn antigen. This glycoprotein and leukosialin (CD43) were indistinguishable on SDS-PAGE and as to immunoreactivity with MLS 128. Leukosialin from an erythroid cell line, K562, exhibited no reactivity with MLS 128 despite that this leukosialin has several GalNAc alpha-Ser(Thr) structures. Pulse-chase experiments with the Jurkat leukosialin showed that newly synthesized leukosialin acquired the antigenecity after a lag of about 30 min, whereas incorporation of GalNAc into the leukosialin occurred earlier. These results indicate that the Tn antigen is expressed on leukosialin and that its epitopic structure is more complex than GalNAc alpha-Ser(Thr).     

Polysialoglycoproteins of Salmonidae fish eggs. Complete structure of 200-kDa polysialoglycoprotein from the unfertilized eggs of rainbow trout (Salmo gairdneri)

Polysialoglycoproteins (PSGP) we first isolated from the unfertilized eggs of rainbow trout (Salmo gairderi) and now found to be a ubiquitous component of Salmonidae fish eggs are a novel type of glycoprotein. PSGP from rainbow trout has a molecular weight of 200 X 10(3), a low protein content (about 15% w/w), and a high sialic acid (N-glycolylneuraminic acid (NeuGc] content (about 60%, w/w). In any evaluation of the biological functions of PSGP, information about the complete structure of this unique macromolecular component is relevant. We have now completed the determination of the overall structural organization of the 200-kDa PSGP, and this is the first report of the complete structural analysis of this novel class of glycoprotein: (Asp)0-2-Ala-Thr*-Ser*-Glu-(Ala-Ala-Thr*-Gly-Pro-Ser-Gly-Asp-Asp-Ala-Thr *-Ser*- Glu)n-Ala-Ala-Thr*-Gly-Pro-Ser-Gly where * indicates the amino acid residues to which oligo- and/or polysialylglycan units are attached and n = 25. Thus the most outstanding structural features of PSGP isolated from the unfertilized eggs of rainbow trout are now the occurrence of (a) tandem repeats of a tridecapeptide and (b) an alpha-2----8-linked oligo(poly)sialyl group on each of the core oligosaccharide chains, i.e. GalNAc- beta 1----4(NeuGc alpha 2----3)GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Fuc alpha 1----3GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n ----6)GalNAc alpha 1----Ser (or Thr), GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), and Gal beta 1----3[----8NeuGc alpha 2)n----6) GalNAc alpha 1----Ser (or Thr).     

O-glycosylation of the coronavirus M protein. Differential localization of sialyltransferases in N- and O-linked glycosylation.

It has previously been shown that the M (E1) glycoprotein of mouse hepatitis virus strain A59 (MHV-A59) contains only O-linked oligosaccharides and localizes to the Golgi region when expressed independently. A detailed pulse-chase analysis was made of the addition of O-linked sugars to the M protein; upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three different electrophoretic forms could be distinguished that corresponded to the sequential acquisition of N-acetylgalactosamine (GalNAc), galactose (Gal), and sialic acid (SA). A fourth and fifth form could also be detected which we were unable to identify. Following Brefeldin A treatment, the M protein still acquired GalNAc, Gal, and SA, but the fourth and fifth forms were absent, suggesting that these modifications occur in the trans-Golgi network (TGN). In contrast, in the presence of BFA, the G protein of vesicular stomatitis virus (VSV), which contains N-linked oligosaccharides, acquired Gal and fucose but not SA. These results are consistent with earlier published data showing that Golgi compartments proximal to the TGN, but not the TGN itself, relocate to the endoplasmatic reticulum/intermediate compartment. More importantly, our data argue that, whereas addition of SA to N-linked sugars occurs in the TGN the acquisition of both SA on O-linked sugars and the addition of fucose to N-linked oligosaccharides must occur in Golgi compartments proximal to the TGN. The glycosylation of the M protein moreover indicates that it is transported to trans-Golgi and TGN. This was confirmed by electron microscopy immunocytochemistry, showing that the protein is targeted to cisternae on the trans side of the Golgi and co-localizes, at least in part, with TGN 38, a marker of the TGN, as well as with a lectin specific for sialic acid.     

Delineation of the minimal catalytic domain of human Galbeta1-3GalNAc alpha2,3-sialyltransferase (hST3Gal I).

The CMP-Neu5Ac:Galbeta1-3GalNAc alpha2,3-sialyltransferase (ST3Gal I, EC 2.4.99.4) is a Golgi membrane-bound type II glycoprotein that catalyses the transfer of sialic acid residues to Galbeta1-3GalNAc disaccharide structures found on O-glycans and glycolipids. In order to gain further insight into the structure/function of this sialyltransferase, we studied protein expression, N-glycan processing and enzymatic activity upon transient expression in the COS-7 cell line of various constructs deleted in the N-terminal portion of the protein sequence. The expressed soluble polypeptides were detected within the cell and in the cell culture media using a specific hST3Gal I monoclonal antibody. The soluble forms of the protein consisting of amino acids 26-340 (hST3-Delta25) and 57-340 (hST3-Delta56) were efficiently secreted and active. In contrast, further deletion of the N-terminal region leading to hST3-Delta76 and hST3-Delta105 gave also rise to various polypeptides that were not active within the transfected cells and not secreted in the cell culture media. The kinetic parameters of the active secreted forms were determined and shown to be in close agreement with those of the recombinant enzyme already described (H. Kitagawa, J.C. Paulson, J. Biol. Chem. 269 (1994)). In addition, the present study demonstrates that the recombinant hST3Gal I polypeptides transiently expressed in COS-7 cells are glycosylated with complex and high mannose type glycans on each of the five potential N-glycosylation sites.     

The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked oligosaccharides of glycoprotein E1

Two size classes of O-glycosidically linked oligosaccharides were liberated from glycoprotein E1 of mouse hepatitis virus (MHV) A59 by reductive beta-elimination and separated by h.p.l.c. The structures of the reduced oligosaccharides were determined by successive exoglycosidase digestions and by methylation analyses involving combined capillary gas chromatography-mass spectrometry and mass fragmentography after chemical ionization with ammonia. Oligosaccharide A (Neu5Ac alpha 2----3 Gal beta 1----3 GalNAc) comprised 35% of the total carbohydrate side chains, while the remaining 65% of the oligosaccharides of E1 had the branched structure B: Neu5Ac alpha 2----3 Gal beta 1----3 (Neu5Ac alpha 2----6) GalNAc. Both oligosaccharides were linked to the E1 polypeptide via N-acetylgalactosamine, and 20% of the sialic acids present in E1 glycopeptides were found to consist of N-acetyl-9-mono-O-acetylneuraminic acid. The reported structures of the O-linked glycans are discussed in the context of the amino acid sequence of E1, which exhibits a cluster of four hydroxyamino acids (Ser-Ser-Thr-Thr) as potential O-glycosylation sites at the amino terminus. Oligosaccharides with identical structures and an identical O-glycosylated tetrapeptide sequence are present in the blood group M-active glycophorin A of the human erythrocyte membrane.     

Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor

In human fibroblasts, the receptor for low density lipoprotein (LDL) is synthesized as a precursor of apparent Mr = 120,000 which is converted to a mature form of apparent Mr = 160,000, as determined by migration in sodium dodecyl sulfate (SDS)-polyacrylamide gels (Tolleshaug, H., Goldstein, J. L., Schneider, W. J., and Brown, M. S. (1982) Cell 30, 715-724). The current paper describes the relationship of N- and O-glycosylation to this post-translational modification. Oligosaccharides were analyzed from precursor and mature forms of LDL receptors that had been immunoprecipitated from cells grown in media containing radioactive sugars. In human epidermoid carcinoma A-431 cells, the receptor precursor appears to contain one N-linked high mannose oligosaccharide and approximately 6-9 N-acetylgalactosamine residues linked O-glycosidically to Ser/Thr residues. In the mature receptor, the O-linked oligosaccharides are mono- and disialylated species having the core structure of galactose leads to N-acetylgalactosamine leads to Ser/Thr. The single N-linked oligosaccharide of the mature receptor can either be a tri- or tetraantennary complex-type species. Similar results were obtained with normal human fibroblast receptor except that the O-linked oligosaccharides on the precursor are neutral disaccharides, of which one component is GalNAc and the N-linked complex type unit on the mature receptor is less branched. Since the addition of GalNAc residues to Ser/Thr residues precedes the conversion of N-linked high mannose-type oligosaccharides to complex-type structures, the transfer of N-acetylgalactosamine must occur prior to the entry of glycoproteins into the region of the Golgi containing the processing enzyme alpha-mannosidase I. We also studied the receptor from tunicamycin-treated cells and after treatment with neuraminidase. In addition, we analyzed the receptor synthesized by a lectin-resistant clone of Chinese hamster ovary cells that is deficient in adding galactose residues to both N- and O-linked oligosaccharides. These studies suggest that the apparent differences in molecular weight between the precursor and mature forms of the LDL receptor are largely, if not entirely, due to the addition of sialic acid and galactose residues to the O-linked GalNAc residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Beta-linked N-acetylgalactosamine residues present at the nonreducing termini of O-linked oligosaccharides of a cloned murine cytotoxic T lymphocyte line are absent in a Vicia villosa lectin-resistant mutant cell line

The O-linked oligosaccharides of the cloned, murine cytotoxic T cell line B6.1.SF.1 were compared with the corresponding oligosaccharides from a Vicia villosa lectin-resistant mutant of B6.1.SF.1 called VV6 (Conzelmann, A., Pink, R., Acuto, O., Mach, J.-P., Dolivo, S., and Nabholz, M. (1980) Eur. J. Immunol. 10, 860-868). The VV6 mutant cells are deficient in binding sites for this GalNAc-specific lectin. Cells were grown in the presence of [3H]glucosamine and [3H] galactose to label the glycoproteins, and the desialyzed, alkaline borohydride-released oligosaccharides were isolated and characterized. The VV6 cells contained a series of O-linked oligosaccharides ranging in size from a disaccharide to a pentasaccharide. These were composed of galactose, N-acetylglucosamine, and N-acetylhexosaminitol, the latter sugar being derived from the reducing terminus. The predominant oligosaccharide had the partial structure Gal beta GlcNAc beta-(Gal beta)N-acetylhexosaminitol. In contrast, the analogous oligosaccharides of the parental cells contained additional beta-linked GalNAc residues located at nonreducing termini. The smallest of these had the structure GalNAc beta 1,4Gal beta-N-acetylhexosaminitol. Neither cell line contained significant amounts of terminal GalNAc linked to Ser/Thr which is the main binding site for the V. villosa B4 lectin on Tn erythrocytes (Tollefsen, S. R., and Kornfeld, R. (1983) J. Biol. Chem. 258, 5172-5176). These findings suggest that the major binding sites for the V. villosa lectin on the parental cytotoxic T cell line consist of structures containing beta 1,4-linked GalNAc residues at the nonreducing ends of conventional O-linked structures. The VV6 cells lack these beta-linked GalNAc residues, and this may account for their deficiency of V. villosa lectin-binding sites. In the following paper (Conzelmann, A., and Kornfeld, S. (1984) J. Biol. Chem. 259, 12536-12542), we demonstrate that the VV6 cells are missing the N-acetylgalactosaminyltransferase that is responsible for the synthesis of these unusual oligosaccharides.     

Galactosyl transferases of baby hamster kidney (BHK) cells. Characterization of two oligosaccharide products synthesised using bovine asialo submaxillary-gland mucin as acceptor

Extracts of BHK (baby hamster kidney) cells catalyse incorporation of galactose from UDP-galactose into asialo bovine submaxillary gland mucin. The galactosylated oligosaccharide products were released by alkaline-borohydride treatment and purified by Bio-Gel P2 chromatography and high-performance liquid chromatography. The structures of the oligosaccharide sequences synthesised have been identified unequivocally by high resolution 500 MHz 1H-NMR as galactosyl-(beta 1----3) N-acetylgalactosamine and galactosyl (beta 1----4) N-acetylglucosaminyl (beta 1----3)-N-acetylgalactosamine. Characterization of the latter sequence shows the presence in bovine mucin of the type III core sequence N-acetylglucosamine-(beta 1----3) N-acetylgalactosamine. Fractionation of BHK cell extracts on alpha-lactalbumin-Agarose has shown that the (beta 1----4)-galactosyl transferase responsible for synthesis of the trisaccharide binds to alpha-lactalbumin, a modulator of the (beta 1----4)-galactosyl transferase involved in N-glycan assembly. The evidence that the same transferase activity may be responsible for galactose transfer to both O-glycans and N-glycans is discussed.     

Occurrence of Gal beta (1----3) GalNAc-Ser/Thr in the linkage region of polygalactosamine containing fungal glycoprotein from Cordyceps ophioglossoides

The nitrous acid deamination liberated the N-acetyl galactosamine containing glycoproteins from galactosminoglycan (CO-N) which has been isolated from Cordyceps ophioglossoides culture. Mild alkaline borohydride degradation of the purified glycoprotein released oligosaccharide alditols. The smallest oligosaccharide alditol was characterized to be Gal beta (1----3)-GalNAcol. This result indicated that polygalactosamine part attached to protein part via Gal beta (1----3) GalNAc-Ser/Thr as the linkage region. To our knowledge, this is the first report of Gal-GalNAc-Ser/Thr linkage from fungal glycoprotein.     

Reactivity of mucin-specific lectin from Sambucus sieboldiana with simple sugars, normal mucins and tumor-associated mucins. Comparison with other lectins.

Mucin-specific lectin from Sambucus sieboldiana (SSA-M) reacts in Western blotting and ELISA with mucins from porcine stomach, bovine and ovine submaxillary glands, the human milk fat globule membrane, in vitro human ovarian, breast and colonic tumor cell lines, and mucins produced in vivo in the ascites of patients with endometrial and ovarian tumors, but not with fetal bovine fetuin or human transferrin. Sialidase treatment of these mucins led to an increase in the binding of SSA-M, suggesting that sialic acid is not part of the binding site for this lectin. Furthermore, sialic acid did not inhibit lectin binding. Treatment of asialomucin with O-glycanase decreased the binding of SSA-M, confirming the reactivity of the lectin with an O-linked carbohydrate. Treatment of mucins with trifluoromethanesulfonic acid, which removes all but core carbohydrate, led to an increase in the binding of SSA-M, suggesting that the lectin reacts with O-linked core glycans. Indeed, the increased reactivity after sialidase treatment of ovine submaxillary mucin suggests the lectin reacts with peptide-linked N-acetylgalactosamine (GalNAc), since more than 98% of the glycan chains attached to this mucin are sialylated GalNAc. The binding of SSA-M to sialidase-treated porcine mucin was inhibited strongly by GalNAc and disaccharides containing galactose (lactose, melibiose, and N-acetyllactosamine) but not by free galactose (Gal), suggesting that the glycan for optimum binding is Gal beta(1-3)GalNAc. This pattern of inhibition was different to other core glycan-reactive lectins tested, indicating that SSA-M is distinct, and should be of use in the isolation and characterisation of mucins and O-linked glycans.     

GalNAc beta 1----3 terminated glycosphingolipids of human erythrocytes

Nonacid glycosphingolipids with 4 to 10 sugar residues isolated from pooled erythrocytes of blood group O donors have been efficiently separated as peracetylated derivatives on silicic acid. This procedure enabled a quantitative estimate of individual compounds and also revealed several GalNAc beta 1----3 terminated structures. The structural characterization of these glycolipids with 1H-NMR spectroscopy, direct inlet mass spectrometry, gas chromatography, and gas chromatography-mass spectrometry identified the compounds as GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl sphingosine and GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl phytosphingosine, GalNAc beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1 ceramide, and GalNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 ceramide.     

Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.